FR3037244A1 - COSMETIC, NUTRACEUTICAL, VETERINARY AND PHARMACEUTICAL COMPOSITIONS CONTAINING AN EXTRACT OF HAZELNUT PERICARP - Google Patents

Abstract

The invention relates to the cosmetic, nutraceutical, veterinary and pharmaceutical applications of a hazelnut pericarp extract as a depigmenting agent for the skin or for improving and / or restoring the barrier function of the skin or mucous membranes.

Description

The present invention relates to the use of extracts of a plant which is very widespread, particularly in Europe, in cosmetic, pharmaceutical, nutraceutical or veterinary compositions. More specifically, it relates to new applications of hazelnut pericarp for treating human or animal skin. The Hazel or Coudrier (Corylus avellana L.) is a shrub 3 to 8 meters high and belonging to the family Betulaceae. It is a plant of woods, hedges and gardens that gives a popular edible fruit, hazelnut. He has a soft wood. He is sometimes called Avelinier. The shrub forms a tuft of 10 to 12 branches up to 3 to 4 m high, multi-stemmed (composed of several thin trunks). Its bark is brown and can come off in thin strips depending on the variety. Its deciduous heart-shaped leaves are toothed with a pointed top. The yellow, yellow flowers form pendent ears or 6 cm kittens, and the highly condensed female flowers form erect ears. Hazelnut is an achene endowed with a woody pericarp called "shell" and containing a single seed (the almond) which occupies the entire internal cavity of the pericarp. This nut, more or less ovoid in shape, can reach 3 cm long and 2 in diameter; it is protected before complete maturity by a tubular envelope, the involucre (bract), of foliaceous aspect and divided into irregular lobes at its end, it is more or less enveloping according to the varieties. The pericarp is a waste of the agro-food industry and is found in large quantities. Most of the time, he underwent a roasting step. Hazelnut pericarp has been studied for its antioxidant properties in view of the tannins it contains (Alasalvar et al., Antioxidant activity of haselnut skin phenolics, J. Agric., Food Chem (2009) 57, 4645). Various scientific publications also describe the tannins it contains (Del Rio et al., Phenolic composition of haselnut skin, J. Agric Food Chem (2011) 59, 9935) or sugars (Montella et al, Identification and characterization of The present invention highlights novel applications of extracts of hazelnut pericarp for at least partially depigmenting normal skin or for removing at least a portion of the skin of the present invention. This is partly due to age-related unsightly brown spots or repeated exposure to the sun, but also the hazelnut pericarp version has significantly improved the barrier function of the skin. epidermis to fight against external aggressions and thus plays a role of skin protector.Les extracts of hazelnut pericarp also play a favorable role in the composites anti-aging.

According to the invention, hazelnut pericarp extracts have been found to reduce the expression of tyrosinase, the key enzyme of pigmentation in normal human melanocytes. This result makes it possible to envisage depigmenting actions on naturally pigmented skin or on sun-induced pigmentation spots, aging or any other external aggressions. The present invention therefore aims at providing new cosmetic or pharmaceutical, nutraceutical or veterinary compositions capable of reducing the natural or induced pigmentation of the skin of a living animal and in particular of a human subject. According to the invention it has also been found that hazelnut pericarp extracts stimulate the expression of various essential protein markers in the formation of the stratum comeum or stratum corneum, including the key enzyme, transglutaminase TGK. The stratum comeum is the most superficial part of the skin and represents the culmination of the differentiation process of keratinocytes. During this differentiation, basement-level keratinocytes undergo a series of metabolic and structural changes throughout their migration to the surface of the skin. The last stage is their transformation into corneocytes whose accumulation in a cohesive structure constitutes the stratum comeum which provides a barrier function. Throughout the differentiation process, several proteins intervene. One of them, TGK, of the glutaminase family, catalyzes the establishment of c (c-glutamyl) -lysine covalent peptide bonds between various protein precursors that form the stratum corneum and confers on it this ability to protect skin against external aggressions and limit the diffusion of water from the inside. Transglutaminase forms generally insoluble protein polymers. These biological polymers are essential for the body to create barriers and stable structures. The present invention aims precisely at providing new cosmetic, pharmaceutical, nutraceutical or veterinary compositions capable of stimulating the factors involved in differentiation, called pro-differentiating factors such as TGK, and thus making it possible to improve the conditions of the skin, of the dander and mucous membranes, healthy or injured. According to the invention, said compositions comprise, as active ingredient, at least one hazelnut pericarp extract. "Hazelnut pericarp extract" means an extract or a mixture of plant extracts of the genus Corylus sp of the family Betulaceae. It is more particularly an extract or a mixture of extracts of cells of Corylus sp. This cellular material can be obtained by in vitro culture or in vivo. By in vitro culture is meant all the techniques known to those skilled in the art, which allow, artificially, to obtain a plant or part of a plant. Thus, the extract may be an extract or a mixture of organ extracts (root, stem, leaf, bark), or even organ cells, of at least one plant of the genus Corylus sp from the family of Betulaceae, or an undifferentiated cell extract from at least one such plant. The applications according to the invention extend to the treatment of the skin, superficial body growths and mucous membranes. By dander, include nails and the hair system, especially the hair. Mucous membranes are the covering tissues of the anatomical cavities, which are connected to the skin by natural orifices, such as the mouth, stomach, intestine, as well as those of natural external cavities such as the nostrils or the ears. Thus, the present invention relates to the use of a hazelnut pericarp extract for depigmenting and / or protecting the skin, integuments and mucous membranes of external physical, chemical or biological agents, and / or for improving and / or to reinforce the hydration of the skin and / or to strengthen the integuments and the mucous membranes. It also relates to a cosmetic treatment method for combating the dryness of a skin or the cosmetic treatment of a dry skin, said method comprising a step according to which at least one hazelnut pericarp extract is applied to the skin. to restore flexibility and / or elasticity. The invention also relates to a method of cosmetic anti-aging treatment of the skin or cosmetic treatment of an aged skin, comprising a step 30 according to which at least one hazelnut pericarp extract is applied to the skin. This treatment allows to delay the phenomena of pigmentation and / or aging of the skin, but also to repair an aged skin by restoring flexibility and elasticity. In any cosmetic application of the invention, a hazelnut pericarp extract may be combined with another or other prodrug-producing agents of the skin type, such as calcium, vitamin D, and the like. and derivatives thereof or at least one other complementary depigmenting agent known to those skilled in the art. In another aspect, the invention relates to pharmaceutical or veterinary applications of a hazelnut pericarp extract as hereinafter presented. One of the objects according to the invention is therefore a pharmaceutical or veterinary composition comprising a hazelnut pericarp extract, to restore the barrier function of the epidermis of an injured skin. This state may result from the action of a physical, chemical or biological external agent and / or a disorder or disease, such as psoriasis or atopic dermatosis. Another object according to the invention is a pharmaceutical or veterinary composition comprising a hazelnut pericarp extract, for improving and / or reinforcing the barrier function of a mucosa or for restoring the barrier function of an injured mucosa. For example, it may be the intestinal mucosa. The lesion of the mucosa may result from a disorder or disease; in the case of the intestinal mucosa, the lesion may be caused by irritable bowel syndrome. In the therapeutic aim of the invention, the hazelnut pericarp extract may be administered topically; it can also be administered orally. According to one aspect of the invention, the extract is administered after the inflammatory process is finished, in particular as repair treatment by reconstruction of the stratum comeum. In a composition of the invention, the hazelnut pericarp extract may also be combined with one or more anti-inflammatory and / or immunosuppressive agents used in the treatment of cutaneous inflammation, such as cyclosporins and their derivatives, anti-cytokine biotherapies and corticosteroids. A hazelnut pericarp extract according to the invention can be obtained by any extraction or purification method known to those skilled in the art. In particular, mention may be made of solid-liquid extraction processes in alcoholic (in particular ethanolic) and aqueous media, as well as in media using solvents such as ketones, esters, ethers, polyols and chlorinated solvents. and mixtures of at least two of the aforementioned solvents, such as hydroalcoholic media. Advantageously, an extract is obtained by extraction of hazelnut pericarp, milled or not, in an aqueous-alcoholic or alcoholic aqueous medium, in particular Version n'-09/06/15 3037244 5 with an aqueous solution of ethanol. The concentration of ethanol can vary from 10 to 90% (v / v). Alternative methods to solvents can also be considered as supercritical CO2 or microwaves ...

These extracts can be used as such, in liquid or powder form, unpurified or purified. If the extract is powdered, its drying can be conducted by any technique well known to those skilled in the art. The extract can be spray dried, evaporated or lyophilized. The powder thus obtained can be encapsulated in liposomes or other vectors and supports for a better homogeneity of the composition and a better diffusion of the active principle, in particular on the skin. The extract is preferably obtained from the pericarp of hazelnuts crushed or not and roasted or not.

It has been observed that the extracts obtained by hydroalcoholic maceration of the hazelnut pericarps exhibit a high biological activity regardless of the percentage of ethanol in the extraction solvent. Similar results are observed in water or ethanol. The hazelnut pericarp extracts may be combined in the compositions according to the invention with substances which increase the skin protection. By way of example but in a nonlimiting manner, mention may be made of associations with mucopolysaccharides, vitamins, ceramides, antiradical substances or UV-filters. In the same way, the reparative activity of the extracts according to the invention is particularly interesting. when they are associated with substances having a healing effect like proteins, hyaluronic acid, amino acids, and / or with anti-inflammatory, anti-aging, after-sun or with anti-acne and anti-active ingredients -dermatoses. The protective activity of the extracts of the invention with respect to free radicals and UV is also very interesting in the hair field, especially in the case of combination with substances facilitating the good condition of the scalp and that of the hair, such as minerals, vitamins, ceramides, protein extracts, mucopolysaccharides, flowers or fruit acids. The compositions of the invention are particularly suitable for topical application to prevent and / or treat many skin disorders. The invention therefore also relates to the dermo-cosmetic use of a composition containing at least one hazelnut pericarp extract, as a depigmenting agent for a naturally pigmented or partially pigmented skin. SUMMARY OF THE INVENTION pigmented, in order to homogenize the complexion, or to reduce brown spots related to age or external agents such as the sun. The subject of the invention is therefore the dermocosmetic use of a hazelnut pericarp extract, as a repairing and / or protective agent for the skin and the hair system, such as the hair, in particular for combating external aggressions, such as those related to pollution, sun, oxidative stress, aging and skin pathologies causing dysfunction of the homeostasis of the epidermis or hair. Therefore, a direct application of the compositions of the invention relates to the fight against aging and cell death induced by UV For cosmetic use, the compositions of the invention can be in the form of creams, gels, lotions, milks, O / W and W / O emulsions, 15 solutions, ointments, sprays, body oils, hair lotions, shampoos, aftershave lotions, soaps, lip sticks, makeup sticks and pencils. The compositions may comprise any excipients necessary for their formulation. Thus, when in the gel form, they include suitable excipients such as cellulose esters or other gelling agents, such as Carbopol®, guar gum, or the like. These cosmetic compositions may also take the form of a lotion or solution in which the extracts and / or molecules are in encapsulated form, for example in microspheres. These microspheres may for example consist of fat, agar-agar and water. The active agents can also be incorporated into liposome, glycosphere-type vectors in chylomicrons, macro-, micro-, nanoparticles as well as macro-, micro- and nano-capsules and can also be adsorbed onto organic powdery polymers, talcs, bentonites and other mineral supports. These emulsions have good stability and can be stored for the time necessary for use at temperatures between 0 and 50 ° C without sedimentation of components or separation of phases. The cosmetic compositions of the invention comprise, on the order of 0.01 to 5% by weight, preferably between 0.1 and 2.5%, of hazelnut pericarp extract when they are in the form of powder and from 0.01 to 25% by weight, preferably from 0.5 to 10%, when in encapsulated form. The percentages given above are expressed by weight of dry extract of hazelnut pericarp relative to the weight of the final composition. For the preparation of these compositions, the hazelnut pericarp extracts are mixed with the excipients generally used in cosmetics.

The cosmetic compositions of the invention may also contain additives or adjuvants customary in cosmetology, such as, for example, antibacterial agents or perfumes, but also extraction and / or synthetic lipids, gelling and viscosity polymers, tensio and emulsifiers, hydro or liposoluble active ingredients, plant extracts, tissue extracts, marine extracts and synthetic actives. The cosmetic or dermocosmetic use of extracts includes all body and skin care products including sun, protective and tanning products, anti-aging products, anti-seborrheic products, tonics, products that improve the skin. skin appearance including acne treatment, skin rash, scalp treatment and hair loss. The cosmetic compositions of the present invention may also comprise other complementary active ingredients chosen for their action, for example for sun protection, the anti-wrinkle effect, the antiradical and antioxidant activity, the anti-irritant activity, the cellular nutrition, cellular respiration, hydration and cell regeneration, anti-seborrhoeic treatments, as well as other active ingredients having an effect on skin tone, protection of the hair. The cosmetic compositions of the present invention are preferably used daily by applying them one or more times per day.

The cosmetic compositions of the present invention are very well tolerated, exhibit no phototoxicity and their application to the skin for prolonged periods of time does not imply any systemic effects. The invention also relates to the use of hazelnut pericarp extracts for the preparation of dermocosmetic compositions having anti-inflammatory and dermoprotective activity. These compositions are useful for preventing and / or treating in particular dermatological diseases related to seborrheic, acneic and / or inflammatory activities. These dermocosmetic compositions are in liquid form, powder, paste or emulsion, alone or in combination with other substances. They comprise from about 0.01 to 25% by weight of hazelnut pericarp extract. The percentages are expressed by weight of dry extract of hazelnut pericarp relative to the weight of the final composition. For the version No. -09 / 06/15 3037244 8 preparation of these dermocosmetic compositions, extracts of hazelnut pericarp are mixed with excipients. Consequently, in general, the invention relates in particular to the use of a hazelnut pericarp extract, for the preparation of a pharmaceutical, veterinary, nutraceutical, dermatological or cosmetic composition intended to prevent and / or treat the pathologies and disorders resulting from: - natural or premature aging of the skin or hair, - dermatoses, such as psoriasis, atopic dermatitis ..., 5 - inflammatory conditions of the cutaneous or hair systems, inducing itching, irritation , chronic redness, irritation and persistent itching and functional disorders of capillary fragility, - cutaneous pigmentation.

A pharmaceutical composition according to the invention comprises, in addition to at least one active agent as defined above, a pharmaceutically acceptable carrier or excipient. The present invention is now illustrated, without limitation, by the following Examples 1 to 4.

Example 1 illustrates processes for preparing hazelnut pericarp extracts according to the invention. Examples 2 and 3 illustrate the biological properties claimed according to the invention. Example 2 shows the depigmenting effects of certain extracts according to the invention. Example 3 illustrates the properties of the extracts of Example 2, which have been evaluated by the ability of these extracts to stimulate the expression of key markers of epidermal differentiation. The selected markers, all involved in the arrangement of keratin filaments and in the formation of the horny layer envelope, are transglutaminase K (TGK), filaggrin and cytokeratin (KRT10). TGK catalyzes the establishment of e (g-glutamyl) -lysine type peptide bonds between various protein precursors. Filaggrin and KRT10 are poorly expressed in primary keratinocyte monolayers and are considered markers of terminal differentiation of the epidermis. These results testify to the ability of extracts to act on aged or injured skin. These properties can be extended to other organs of the human or animal body.

EXAMPLE 1 Preparation of extracts of hazelnut pericarp A series of extractions was carried out with the following solvents: hexane; 5 - ethyl acetate; - ethanol / water: 90/10; - ethanol / water: 60/40; - ethanol / water: 30/70; - water.

The extractions were carried out at room temperature (except for water at 50 ° C.), after a preliminary grinding of the plant material. The plant / solvent ratio is 1/10. The maceration time (with stirring) is 24 hours. For each extract, one part was treated on activated charcoal with a coal / plant ratio of 1/10, the other was kept raw. The extraction yields are summarized in the table below: Solvent extraction Raw extract Yield Extract on coal Extraction extraction yield (%) (%) Hexane EX5MF1173 9.1 EX12MF1173 7.8 Ethyl acetate EX6MF1173 12 EX13MF1173 8.1 Ethanol 90% EX1MF1173 12.9 EX8MF1173 11; 5 Ethanol 60% EX2M F1173 16.1 EX9MF1173 12.8 Ethanol 30% H2O 50 ° C EX3M F1173 EX4M F1173 7.3 EX1OMF1173 2.4 3.1 EX11MF1173 1,1 The appearance of the extracts obtained is described in the table below: 20 Extract crude Solvent extraction Appearance Color Extract on charcoal Appearance Color D6M F1173 Hexane oil brown green green EX12MF1173 oil yellow yellow EX6MF1173 Acetate ethyl oil brown brown EX13MF1173 oil yellow yellow EX1MF1173 Ethanol 90%, fine powder brown chestnut EX9MF1173 fine powder brown chestnut DQMF1173 Ethanol 60% fine powder brown dark brown EX9MF1173 fine powder brown chestnut EX3MF1173 Ethanol 30% fine powder brown dark brown EX1OMF1173 fine powder chestnut brown O It is found first of all that the hexane and ethyl acetate extracts have a yield close to 10%, which seems to indicate the presence of a high proportion of fat. This is confirmed by the oily nature of these extracts. It should be noted that aqueous and 30% ethanol extracts were very difficult to filter. It is possible that these difficulties led to losses that artificially reduce yields. The yield seems optimum for 60% ethanol: 16% crude and 13% treated on charcoal. As the amount of ethanol is decreased, the yield reduction caused by the carbon treatment increases sharply. This is consistent with the high levels of phenolic compounds described in the next section. It is found that the treatment on charcoal is, apart from the oils, not very effective in decreasing the color of the extracts. The phytochemical analyzes of these extracts are as follows: Total phenolics and tannins were assayed by the Folin-Ciocalteu method in chlorogenic acid equivalent. The results are collated in the table below: Extracts Crude Extracts Charcoal Solvent Extracts% Total Phenolic Compounds% Tannins Only% Compounds% Total Phenolic Compounds% Tannins Only% Phenolic Compounds Phenolics non-tanic non-tannic H20 50 ° C EX4MF1173 20, 12 6.67 13.45 EX11MF1173 42.1 42.1 0.0 Eth / Water 30/70 EX3MF1173 58.7 4.6 54.1 EX1OMF1173 45.2 8.2 37.0 Eth / Water 60/40 EX2MF1173 87.9 11.3 76.6 EX9MF1173 95.0 24.5 70.6 Eth / Water 90/10 EX1MF1173 83.3 12.0 71.3 EX8MF1173 98.5 25.4 73.1 Ethyl Acetate EX6MF1173 8.9 8.2 0.7 EX13MF1173 2.4 2.3 0.1 Hexane EX5MF1173 7.6 6.9 0.8 EX12MF1173 2.1 2.2 -0.1 It can be seen that: extraction is rich in ethanol, the more the extract is rich in phenolic compounds; Among the phenolic compounds, the tannins are a minority; from 60% of ethanol, the charcoal treatment makes it possible to enrich the extract with phenol compounds and with tannins; the 90% and 60% ethanol extracts, treated on carbon, contain respectively 98% and 95% of phenolic compounds; of which respectively 25% and 24% tannins. Version No. -09 / 06/15 3037244 11 EXAMPLE 2 Evaluation of Cytotoxicity and Depigmenting Activity of Extracts We evaluated the inhibitory activity of tyrosinase for 30% ethanol (Ex3MF1173) and ethanol 90% extracts (Ex1MF1173) not treated with charcoal. Cytotoxicity We first evaluated the cytotoxicity of extracts on melanocytes. Cell type: NHEM in culture medium. - Incubation time: 72 hours. - Evaluation parameters: reduction of MTT and morphological observations under the microscope. At the end of the treatment, the cells were incubated in the presence of MTT (tetrazolium salt) whose transformation into blue crystals of formazan is proportional to the activity of succinate dehydrogenase (mitochondrial enzyme). After dissociation of the cells and solubilization of formazan by addition of DMSO, the optical density (OD), representative of the number of living cells and their metabolic activity, was measured with a 540 nm microplate reader (VERSAmax, Molecular Devices).

From the results presented in the table below it is concluded that the maximum non-cytotoxic dose is 31.1g / ml for Ex1MF1173 and 101..tg / ml for Ex3MF1173. Version n0-09 / 06/15 3037244 12 Control EX11.11F1173 Unit: gfml Solution st at 100 mg + ml in DAIS ° 0.3 10 30 100 1000 101 97 89 89 95 87 76 36 70 Viability (%) 100 99 99 95 91 87 81 47 101 O 101 102 98 93 93 90 82 43 66 O Mean 100 95 92 88 80 42 79 o esm 2 2 11 O Morphological observations + g op Control indicator EX3MF1173 Unit: pg1m1 Stock solution prepared at 50 mg / ml in DfASO 0.1 0.3 1 5 10 50 150 500 100 102 103 104 102 99 95 83 50 131 Sustainability (%) 99 97 105 102 102 96 93 86 44 159 101 102 101 106 100 98 94 80 38 90 Average 100 103 104 102 98 94 83 44 127 osai 1 1 1 1 1 0 2 3 20 Morphological observations g, 9 Legends +: normal population; : growth reduction; : toxicity; 0: cell death Q: grains of compound: opacity due to the compound; morphological changes; Agglutinated cells my standard error of the mean (standard deviation divided by the square root of the sample tiller). Tnrrosinase activity The melanocytes were inoculated and cultured in culture medium for 24 hours. The medium was then replaced with medium containing or not (control) the compounds or the reference (lipoic acid at 5 μg / ml) and the cells were incubated for 72 hours. All the experimental conditions were realized in n = 3. At the end of the incubation, the culture medium was removed and the cells washed with PBS solution. For each condition, the tyrosinase enzyme was extracted into PBS-Triton X-100 solution, and then incubated with the substrate (2 mM L-DOPA) for 1 hour at 37 ° C. The enzymatic activity of the various cell extracts was evaluated by measuring the optical density (OD) at 540 nm (Versamax Microplate Reader, Molecular Devices) and using a standard range of mushroom tyrosinase (0.39 to 400 WH). The tyrosinase activity was evaluated after 72 hours of NHEM culture in the presence of the reference or test compounds. In this type of protocol, the measurement of the enzymatic activity makes it possible to account for the amount of tyrosinase present in the melanocytes. However, it can not be excluded that compounds, incorporated into cells or covalently bound to the enzyme, can directly inhibit the enzymatic activity of tyrosinase. After 72 hours of culture, tyrosinase activity was detected in the NHEMs of the control condition. The treatment of NHEM with lipoic acid, tested at 5 mg / ml, induced a very strong inhibition of tyrosinase activity (90% inhibition).

These results were expected and validated the test. Extract EX3MF1173, tested at 10 μg / ml, inhibited the tyrosinase activity (25% inhibition). At the lowest concentrations tested, this compound had no effect. The aqueous effect also showed depigmenting activity. Extract EX1MF1173, tested at 3 concentrations, did not modulate tyrosinase activity. However, it should be noted that the Ex1MF1173 extract, because of its greater cytotoxicity, was tested at a concentration three times lower than Ex3MF1173. At this same concentration Ex3MF1173 also has no effect. It is therefore possible that the only difference between Ex1MF1173 and Ex3MF1173 lies only in their non-cytotoxic maximum levels. Version no. -09 / 06/15 3037244 14 Processing Basic data Standardized data Compound tested Concentration Tyrosinase TMyrclosYeinnnasee esm T '% oin esm P (1) Inhilion esm Fie (u / mi) (u / mi) (Lump (%) 1%) 222.8 Control - 246.1 228.5 9.0 100 4 - 0 4 - 216.7 25.4 Lipoic acid 5 μg / ml 23.9 23.3 1.4 10 1 .- 90 1 20.7 171.0 IBM 200 pM 173.8 170.4 2.2 75 1 25 1 166.3 86.5 Dibutyryl cAMP 5 mg / m1 108.9 104.6 9.4 46 4 54 4 118.3 220.2 0.333 μg / ml 234.5 225.9 4.4 99 2 ns 1 2 ns 223.0 231.3 EX1MF1173 1 μg / ml 229.1 228.5 1.8 100 1 ns 0 1 ns 225, 2 221.0 3 mg / mi 216.5 214.2 4.8 94 2 ns 6 2 ns 205.0 280.4 1.1 pg / ml 193.4 230.6 25.9 101 11 ns -1 -Il ns 217.9 253.8 EX3MF1173 3.3 pg / m1 191.3 213.6 20.2 93 9 ns 7 9 ns 195.5 178.0 10 pg / ml 160.2 170.8 5.4 75 2 25 2 174 , 1 (1): Threshold of statistical significance ns:> 0.05, Not significant *: 0.01 to 0.05, Significant **: 0.001 to 0.01, Very significant ***: <0.001, Extremely significant Two extracts of hazelnut pericarp from these first tests. 1er is the ethanol extract 30% crude (Ex3MF1173) which has a significant inhibitory effect of tyrosinase (25%). It contains about 60% of phenolic compounds (chlorogenic acid equivalent), about 5% of which are tannins (chlorogenic acid equivalent). The extraction yield is about 7%. In order to evaluate the color impact in a cosmetic formula, the two extracts will be diluted to 10% in maltodextrin and then formulated in a 1% cream. The formulas as well as the actives will be accelerated aging to evaluate their stability in terms of color and phenol content. Example 3: Evaluation in normal human epidermal keratinocyte (NHEK) primocultures 1) Materials and methods Normal human epidermal keratinocyte (NHEK) cells; reference BlOalternatives K341, used in the third pass. Culture conditions: 37 ° C, 5% CO2 Culture medium: KeratinocyteSFM supplemented with epidermal growth factor (EGF) 0.25neml, pituitary extract (PE) 251..teml and gentamycin 25geml. Test medium: KeratinocyteSFM supplemented with gentamycin 25pg / ml.

Extracts Tested In the initial step of selecting an extract for further testing, and after prior cytotoxicity evaluation, extracts EX8MF1113 and EX9MF1113 are tested at the following concentrations: EX8MF1113, 100 gm / ml EX9MF1113, 100 pig / The above step led to these two extracts being retained at non-cytotoxic concentrations of 10 μg / ml; dose used for the following experiments.

In Situ Immunolabeling The keratinocytes were seeded and grown to confluency in 96-well plates and then the medium was replaced with test medium containing or not (control) the test extract or reference (1.5 mM CaCl 2). and the cells were incubated for 72 hours. All experimental conditions were performed in n = 3, for each marker. Version No. -09 / 06/15 3037244 16 After incubation, the test medium is removed and the cells are rinsed, fixed and permeabilized. Cells are labeled with primary antibodies against the proteins of interest (TGK, filaggrin and KRT10) according to Mcheik JN et al. Foreskin-isolated keratinocytes provide successful extemporaneous 5 autologous pediatric skin grafts. J Tissue Eng Regen Med.

2013 Mar 14. These antibodies were revealed by a secondary antibody coupled to a fluorochrome (GAMAlexa 488). In parallel, the nuclei of the cells are stained with Hoechst 33258 (bisbenzimide). Acquisition of the images is performed with a high-resolution imaging system, INCell Analyzer 1000 (GE Healthcare), for each well 5 digitized images are taken 10. Markings are quantified by intensity measurement protein fluorescence relative to the number of nuclei identified by Hoechst (integration of digital data by Developer Toolbox 1.5 software, GE Healthcare) Extraction screening was performed only on the TGK parameter (Table 2). transcripts, RT-qPCR The keratinocytes were seeded and grown to confluency in 24-well plates and then the medium was replaced with test medium containing or not (control) the test compound or reference (CaCl 2 1 5 mM) and the cells were incubated for 48 hours All the experimental conditions were performed in n = 3 for each marker After incubation, the test medium was removed and the cells were rinsed and frozen at -80 ° C. The expression of the markers was evaluated by RT-qPCR on the messenger RNAs (mRNAs) extracted from the cell mats of each treatment (the replicates were pooled before the extraction of RNA). The steps of RNA extraction, reverse transcription, primers and PCR conditions have been previously described, according to Mcheik JN et al. Foreskin-isolated keratinocytes provide successful extemporaneous autologous pediatric skin grafts. J Tissue Eng Regen Med.

2013 Mar 30 14; Boniface et al. IL-22 inhibitory epidermal differentiation and proinflammatory induces gene expression and migration of human keratinocytes. J Immunol.

2005 Mar 15; 174 (6): 3695-702; Boniface et al. Oncostatin M secreted by skin infiltrating T lymphocytes is a potent keratinocyte activator involved in skin inflammation. J Immunol.

2007 Apr 1; 178 (7): 4615-22.

2) Screening Results The results are shown in Table 2 below which illustrates TGK expression level via fluorescent labeling of NHEKs, fluorescence intensity. NHEKs being proportional to TGK expression. Table 2 Compound tested EX8MF1113 control EX9MF1113 0.001mg / m1 0.001mg / m1 Expression rate of TGK +++ +++ ++++ The extracts are active on stimulating the expression of TGK under these conditions. Influence of EX3MF1046 Extract at Different Concentrations on TGK Expression Table 3 illustrates the level of expression of TGK via fluorescent labeling of NHEKs for different concentrations of EX9MF1113 extract. Treatment Table 3 Control Expression TGK 'Compound tested Concentration Average ESKA% Control 22042 3399 100 CaC12 1.5mM 69610 681 316 0.0016mg / m1 41326 3696 187 0.001mg / m1 133494 4753 606 EX9MF1113 0.005mg / m1 201936 7097 916 0.01mg (a) The expression of TGK is given in fluorescence intensity / number of cells (UA) These results show that the EX9MF1113 extract induced in a dose-5 manner. dependent TGK expression in NHEK cultures. The activity is greater than that of the reference calcium chloride, from the concentration of 1; the maximum effect, which is 4 times greater than that of CaCl2, is observed at the non-cytotoxic concentration of 10 μg / ml.

Influence of EX9MF1113 Extract on Expression of TKG, Filaggrin and KRT10 The same methodology used to evaluate the effect of an extract on TGK expression was applied for the filaggrin and KRT10. Table 4 provides an assessment of the level of expression of the three above markers via fluorescent labeling of NHEKs. Table 4 TKG Expression Expression Filaggrin Expression Test Compound CaCb Indicator EX9MF1113 1.5mM Concentration 101, tg / m1 +++ ++ In this experiment, the inducing effect of TGK was found and there was a very strong overexpression of KRT10 protein. Unlike calcium, which focuses the effect on a few cells by transforming them into very large differentiated cells (corneocytes), EX9MF1113 induces a very strong overexpression of KRT10 in many cells but does not significantly increase cell size. The mechanism of action of calcium and EX9MF1113 are therefore different and could be additional or synergistic. This is confirmed by the analysis of filaggrin, which increases (more moderately) following treatment with EX9MF1113, but without inducing the characteristic production of filaggrin granules in calcium-responsive cells. . Example 4: Dermo-cosmetic formulation of a hazelnut pericarp extract Non-limitingly, we present below an example of formula in emulsion form. PURIFIED WATER Aqua 79.09 GLYCERINE Glycerin 3.00 humectant EMULIUM 22 Tribehenin PEG-20 esters 4.00 emulsifier LIPOCIRE A SG C10-18 Triglycerides 2.00 emollient MIGLYOL 810 N Ca prylic / capric tryglycerides 5.00 emollient DUB ININ Isononyl isononanoate 5,00 emollient SEPIMAX ZEN Polyacrylate Crosspolymer-6 1,00 stabilizer EXTRAIT EX1 MF1173 0,10 Active PHENOXETOL Phenoxyethanol 0,70 preservative TRISAMINO ULTRA PC Tromethamine 0,01 pH adjuster GERMALL II Diazolidinyl urea 0,10 stabilizer 15 20 Version No. -09 / 06/15

Claims (14)

REVENDICATIONS1. A composition for at least partially depigmenting and / or protecting the skin, mucous membranes and integuments of a living animal, said composition containing at least one active agent containing at least one hazelnut pericarp extract. 2. Composition according to claim 1, characterized in that it protects the skin or the mucous membranes by improving and / or reinforcing their barrier function. 10 3. Composition according to claim 2, characterized in that it improves and / or enhances the hydration of the skin. 4. Composition according to one of claims 1 to 3, used for the treatment of the skin of a human subject, characterized in that it depigments the skin by decreasing the expression of tyrosinase in normal human melanocytes. 5. Composition according to claim 4, characterized in that it is used for the treatment of a naturally pigmented skin or for the treatment of induced pigmentation spots. 6. Composition according to claim 3, characterized in that it is used for the treatment of dry or aged skin by application (s) on said skin to increase its flexibility and / or its elasticity. 7. Composition according to claim 2, characterized in that it is used to restore the barrier function of a skin damaged by a physical, chemical or biological external agent and / or by a disease such as dermatitis, dermatitis or dermatitis. atopic or psoriasis. 8. Composition according to claim 2, characterized in that it is used to improve and / or strengthen the barrier function of a mucosa by topical application or orally. Version No. -09 / 06/15 3037244 21 9. Composition according to claim 8, characterized in that it is used orally to improve or enhance the barrier function of an injured intestinal mucosa. 10. Composition according to Claim 1, characterized in that the hazelnut pericardium extract is combined with at least one other skin-producing agent, in particular calcium, vitamin D and their derivatives and / or at least one other depigmenting agent of the skin. 11. Composition according to one of claims 1 to 10, characterized in that it comprises at least one anti-inflammatory and / or immunosuppressive agent (s) used (s) in the treatment of cutaneous inflammation, including cyclosporins and their derivatives, anti-cytokine biotherapies and corticosteroids and / or at least one depigmenting agent other than a hazelnut pericardium extract. 12. Composition according to one of claims 1 to 11, characterized in that the hazelnut pericardium extract is obtained by solid / liquid extraction of said hazel pericardium in an aqueous, hydroalcoholic or alcoholic medium. 13. Composition according to claim 12, characterized in that the solid / liquid extraction is carried out in a water / alcohol mixture in a proportion ranging from 10% to 90% by volume. 20 14. Composition according to one of claims 1 to 11, characterized in that the hazelnut pericardium extract is obtained by solid / liquid extraction in a solvent medium comprising at least one solvent selected from the group consisting of ketones, ethers, polyols, chlorinated solvents and supercritical CO2. 25 Version No. -09 / 06/15