FR2962904A1 - Cosmetic active ingredient, useful e.g. as anti-aging cosmetic active ingredient on the dermis and the epidermis and in cosmetic composition for preserving barrier function, comprises peptides from Verbena officinalis - Google Patents

Abstract

Cosmetic active ingredient (I) comprises peptides having a molecular weight of less than 2000 Da from Verbena officinalis. Independent claims are included for: (1) process for obtaining (I) comprising: solubilization of powder of aerial parts of Verbena officinalisin water; enzymatic hydrolysis in the presence of at least one enzyme; separation of soluble and insoluble phases by decantation; inactivation by heat treatment; filtration; concentration of the active fraction; and sterilization filtration; and (2) cosmetic composition for topical application, comprising (I) (0.01-20 wt.%). ACTIVITY : Dermatological. MECHANISM OF ACTION : Alpha 2 beta 1 integrin synthesis stimulator; Collagen I synthesis stimulator; Collagen III synthesis stimulator; Fibrillin-1 synthesis stimulator. The ability of (I) to stimulate collagen I synthesis was tested in normal human fibroblast. The result showed that the collagen I synthesis was 2091 pg/mu g protein at 2%.

Description

USE OF VERBENA OFFICINALIS PEPTIDES TO COMBAT SKIN AGING

The present invention relates to the use of specific molecules derived from Verbena off icinalis, as well as to a particular active ingredient containing such molecules, for a global and complete anti-aging cutaneous action at the level of the different compartments of the skin.

The invention also relates to a process for obtaining this active ingredient, the compositions including it, and a method of anti-aging cosmetic treatment. There are many cosmetic products to combat the effects of time on the skin. In recent years, the dynamics of the cosmetics market is under the impetus of highly sophisticated skincare products: highly segmented offer, hyper-targeted care with sophisticated technologies and claims, which respond to a particular problem of skin aging. However, these products must be combined with each other to achieve an overall anti-aging effect. Users need to multiply applications and costs incurred. This is why we are now looking for multifunctional active ingredients, presenting a global and complete anti-aging action at the different compartments of the skin, guaranteeing cutaneous homeostasis. In particular, there remains a need for a multi-action anti-aging cosmetic active ingredient, which effectively strengthens the vital functions of the skin in order to fight against the various visible signs of aging at the same time: dehydration, lack of radiance, loss of firmness and wrinkles.

To meet this need, the invention proposes to use particular molecules from Verbena officinalis. Verbena officinalis or verbena officinalis, is a medicinal plant called still sacred grass or grass of all evils, used for its virtues in infusion or external use. The use of Verbena officinalis in cosmetic compositions is poorly known. We can nevertheless cite the application RU-2297844 which describes an anti-cellulite cosmetic composition containing verbena oil, or the application CN-101336876 which relates to a moisturizing skin care made from essences of 16 plants of which verbena. Furthermore, the application KR-20080061079 describes compositions containing extracts of Verbena Officinalis particular very targeted action. The products are obtained by solvent extraction, and are used to prevent skin aging by blocking the reactive oxygen species and promoting the activity of fibroblasts. However, the objective of the present invention is to provide a different specific cosmetic active ingredient, with particular molecules, which has overall anti-aging performance through multiple efficacy on both the epidermis and the dermis.

For this purpose, the invention is directed to the use of peptides derived from Verbena officinalis with a molecular weight of less than 2000ba, or from a hydrolyzate of Verbena officinalis containing peptides with a molecular weight of less than 2000ba, as a cosmetic active ingredient. By "hydrolyzate" is meant any extract obtained from Verbena off icinalis, comprising at least one enzymatic or chemical hydrolysis step, preferably enzymatic. The invention also relates to a cosmetic active ingredient comprising peptides with a molecular weight of less than 2000ba, derived from Verbena off icinalis, as well as a process for obtaining such an active principle. Advantageously, the method of obtaining according to the invention is simple to implement. According to another aspect, the invention also relates to cosmetic compositions including between 0.01 and 20% of a cosmetic active ingredient comprising peptides with a molecular weight of less than 2000 Da of Verbena officinalis. These compositions may be in any form allowing the application topically. Finally, the subject of the invention is also a cosmetic skin care method, intended to globally regulate the imbalances associated with cutaneous aging, comprising the topical application of a composition containing peptides with a molecular weight of less than 2000 Da derived from of Verbena off icinalis or a hydrolyzate from Verbena off icinalis containing peptides with a molecular weight of less than 2000Da. Advantageously, the present invention makes it possible to ensure cutaneous homeostasis by reinforcing the vital and essential functions of the skin: it strengthens the cutaneous barrier function and boosts the dermal metabolism. The compositions according to the invention then constitute a unique treatment that saves time and economic gain, while maintaining pleasure and efficiency. The present invention is now described in detail.

USE According to a first aspect, the invention is directed to the use of peptides with a molecular weight of less than 2000 Da derived from Verbena off icinalis, or a hydrolyzate of Verbena off icinalis containing peptides with a molecular weight of less than 2000 Da, as a principle. anti-aging cosmetic active having multiple efficacy on both the epidermis and the dermis.

With age, the skin is the seat of profound changes that affect all of its compartments and alter its essential and fundamental functions. In the epidermis, keratinocytes lose their vitality and dynamism. The renewal of the epidermis slows down, the keratinocyte differentiation is altered, the hydro-lipidic film is impoverished: the skin barrier is weakened and the skin is prone to dehydration. At the level of the dermis, a general slowing of fibroblast activity is observed, both in terms of their proliferative and metabolic capacity.

The narrowing and disorganization of the three-dimensional matrix network is reflected in the skin surface by loosening, loss of firmness and the appearance of wrinkles. According to the invention, peptides with a molecular weight of less than 2000 Da derived from Verbena off icinalis, or a hydrolyzate obtained from Verbena off icinalis containing peptides with a molecular weight of less than 2000 Da, can be used as a cosmetic agent acting both on the dermis and the epidermis to regulate imbalances related to skin aging. The invention therefore relates to the use of peptides with a molecular weight of less than 2000Da derived from Verbena off icinalis, or a hydrolyzate derived from Verbena officinalis containing peptides with a molecular weight of less than 2000Da, as an anti-aging cosmetic active ingredient. global. It can be used in a global anti-aging cosmetic composition intended to act both on the level of the dermis and the epidermis. Especially at the level of the epidermis, peptides with a molecular weight of less than 2000Da from Verbena officinalis, or hydrolysates obtained from Verbena off icinalis containing peptides with a molecular weight below 2000 Da, can be used to enhance the function. skin barrier, in particular: to stimulate the differentiation of keratinocytes at different stages of maturation by increasing the synthesis of cytokeratin 10, involucrine and loricrin, and to act on the synthesis mechanisms of intercellular lipid cement at a time by increasing on the one hand the expression of the glucosylceramide synthase involved in the formation of lipid precursors and the p-glucocerebrosidase responsible for their maturation into functional ceramides, and on the other hand increasing the level of lipids bound to the proteins of the horny envelope.

By stimulating the differentiation of keratinocytes and promoting the synthesis of intercellular lipids, the invention makes it possible to preserve the barrier function and to increase the level of hydration of the skin. In the dermis, peptides with a molecular weight of less than 2000Da from Verbena officinalis, or hydrolysates obtained from Verbena officinalis containing peptides with a molecular weight of less than 2000 Da, can be used to boost dermal metabolism, in particular - for to stimulate the growth of fibroblasts in a deficient medium, and - to promote the synthesis of the matrix constituents, namely network proteins (collagen I, collagen III and fibrillin-1), of a hydration molecule (hyaluronic acid) , and a junction protein (the integrin a2p1). By promoting the growth of fibroblasts and by increasing the synthesis of these matrix components, the invention makes it possible to smooth the microrelief and to attenuate wrinkles.

By acting transversely, the invention regenerates the skin, revitalize it, and thus reduce all traces left by time on the skin: hydrated skin gains flexibility, its wrinkles fade.

Advantageously, the use on the skin of peptides with a molecular weight of less than 2000 Da derived from Verbena off icinalis or a hydrolyzate derived from Verbena off icinalis containing peptides with a molecular weight of less than 2000 Da, ensures efficient performance simultaneously over many markers recognized for their importance in anti-aging care. According to a particularly suitable embodiment, the invention relates to the use of an active ingredient derived from Verbena off icinalis containing peptides with a molecular weight of less than 2000Da, as described below.

ACTIVE INGREDIENT The invention also relates to a cosmetic active ingredient comprising peptides with a molecular weight of less than 2000 Da from Verbena officinalis or a cosmetic active ingredient derived from Verbena officinalis comprising peptides with a molecular weight of less than 2000 Da.

The active ingredient according to the invention may also comprise other molecules, in particular carbohydrates. Preferably, the peptides with a molecular weight of less than 2000Da represent at least 70% by weight of the total proteins of the active principle. The active ingredient according to the invention is preferably a hydrolyzate of Verbena off icinalis containing peptides with a molecular weight of less than 2000Da, preferably a hydrolyzate resulting from a process comprising an ensymatic hydrolysis. It can be in the form of limpid liquid of amber color. It may be defined by at least one of the following features, all of which are preferred. Dry matter: The dry matter content of an active ingredient according to the invention (measured by passing in an oven at 105 ° C. in the presence of sand of a sample of initial weight given until a constant weight is obtained ) may be between 10 and 90 g / l, preferably between 21 and 35 g / l. PH measurement: The pH (measured by the potentiometric method at ambient temperature) can be between 3.0 and 6.0, preferably between 3.0 and 4.0. Carbohydrates: Determination of the total sugar content The determination of the total sugar content can be carried out by the DUBOIS method (DUBOIS M. et al., (1956), Analytical Chemistry, 28, No. 3, pp. 350-356. ). In the presence of concentrated sulfuric acid and phenol, the reducing sugars give an orange-yellow compound. From a standard range, the total sugar content of a sample can be determined. The total sugar content of an active ingredient derived from Verbena officinalis according to the invention is preferably between 2.3 and 26 g / l, more preferably between 5 and 10 g / l. The total sugar content can also be expressed as a percentage relative to the dry matter. It is preferably between 14% and 48%, even more preferably between 23 and 29% by weight relative to the dry matter.

Characterization of Carbohydrates The molecular weights of the carbohydrates present in the active principle according to the invention are determined by high performance liquid chromatography (HPLC). Their composition in simple sugars is determined by ionic liquid chromatography.

The carbohydrate fraction of the active principle according to the invention is composed mainly of glucose, fructose and galactose, for 21 to 27% in the form of oligosaccharides with a degree of polymerization of between 2 and 6 (molecular weight between 180 and 1100 Da), and for 68 and 84% as monosaccharides (molecular weight less than 180 Da). Proteins Determination of Protein Content: The protein content assay is performed according to the method of Lowry (Lowry et al., Protein Measurement with the Folin Reagent, J. Biol Chem, 193, 265-275, 1951). The Folin reaction gives a blue color with certain amino acids. The coloration obtained is compared with that of a curve established with a standard serum.

The total protein content is preferably between 1.4 and 21 g / l, more preferably between 3.0 and 8.0 g / l. The protein content can also be expressed as a percentage relative to the dry matter. It therefore preferably represents between 8 and 38% by weight relative to the dry matter, more preferably between 14 and 23% relative to the dry matter. Characterization of the Protein Fraction: The characterization of the protein fraction of the active principle according to the invention is carried out by steric exclusion F.P.L.0 (Fast Protein Liquid Chromatography). The calibration of the column is carried out by the passage of defined molecular weight markers (Cytochrome C, Aprotinine, Vitamin B12 and Cytidine). The proteins of the active principle according to the invention are composed of at least 70% of peptides less than 2000Da, preferably at least 80%.

The active ingredient according to the invention may therefore comprise at least 5% of peptides less than 2000Da, by weight relative to the total dry matter.

Crude ash content: The raw ash content is determined by the weighing of residues from incineration at 550 ° C in an electric muffle furnace (VULCANTM 3.550-NDI).

The weight of the residue is calculated by deducting the tare. The mineral content is expressed as a percentage of the dry matter. The ash content is between 26 and 33%. Identification of the active fraction In order to determine the predominantly active fraction of the active ingredient according to the invention, a study has been carried out. This study consists of fractioning the molecular species of the active ingredient according to the invention: a fraction A consisting of neutral carbohydrates, obtained by purification by successive adsorption of the cationic and then anionic compounds on ionic resins, - a fraction B consisting of ash, obtained by resolubilization of the residues resulting from the incineration at 550 ° C in an electronic muffle furnace, taken up in distilled and filtered water, - a fraction C consisting of a peptide fraction containing peptides and free sugars, obtained by hydrolysis of oligosaccharides to simple sugars. A study is then carried out on the effect of this different fractions on the synthesis of collagen I in comparison with the result obtained for the active ingredient according to the invention at 1%. The test is carried out on normal human fibroblasts, according to the operating protocol described below. At 0, human fibroblasts are inoculated in complete medium. The cells are then incubated at 37 ° C.

At day 2, the culture medium is removed and replaced with medium containing the active ingredient at 1% (V / V) or one of the fractions A, B or C. The cells are then incubated at 37 ° C. for 24 hours. On D3, the supernatants are recovered for assaying Collagen I. The assay is performed using an ELISA kit. The results obtained are given in the table below: Collagen I / Control (%) 1% active ingredient +205 Fraction A 0 Fraction B 0 Fraction C +223 Fractions A and B are not effective. It is the fraction C which confers on the active principle its activity. Analysis of fraction C by HPLC (High Performance Liquid Chromatography) consists of peptides with a molecular weight of less than 2000 Da.

PROCESS OF OBTAINING The active ingredient according to the invention as described above can be obtained preferentially by a process comprising an enzymatic hydrolysis. A particularly suitable process comprises at least the succession of the following steps: solubilization of powder of aerial parts of Verbena officinalis in water, enzymatic hydrolysis in the presence of at least one enzyme, preferably a protease, soluble phase separation and insoluble by decantation, - inactivation by heat treatment, - filtration, - concentration of the active fraction, and - filtration and sterilizing filtration. Decolorization and deodorization stages of the soluble phase can be envisaged without modifying the active fraction of the active principles. The parameters of the various steps must be adjusted in order to obtain extracts comprising peptides with a molecular weight of less than 2000 Da, in particular an extract comprising proteins of which at least 70% are peptides with a molecular weight of less than 2000 Da.

COSMETIC COMPOSITIONS AND COSMETIC SKIN CARE PROCESS The present invention also covers cosmetic compositions including Verbena officinalis peptides with a molecular weight of less than 2000Da or at least one active ingredient derived from Verbena off icinalis comprising peptides with a molecular weight of less than 2000Da. , in different galenic forms, adapted for topical administration to the skin. These compositions may in particular be in the form of oil-in-water emulsions, water-in-oil emulsions, multiple emulsions (Water / Oil / Water or Oil / Water / Oil) which may be optionally microemulsions or nanoemulsions, or in the form of solutions, suspensions, hydrodispersions, aqueous gels or powders. They can be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam, or in solid form. These compositions are preferably compositions containing between 0.01 and 20%, by weight of active principle (s) derived from Verbena off icinalis comprising peptides with a molecular weight of less than 2000Da according to the present invention, preferentially between 0.1% and 2%. These compositions comprise, in addition to the active agent, a physiologically acceptable and preferably cosmetically acceptable medium, that is to say which does not cause unacceptable sensations of discomfort for the user such as redness, tightness or tingling. The compositions according to the invention may contain as adjuvant at least one compound chosen from: - oils, which may be chosen in particular from silicone oils, linear or cyclic, volatile or non-volatile; waxes, such as ozokerite, polyethylene wax, beeswax or carnauba wax; silicone elastomers; surfactants, preferably emulsifiers, whether they are nonionic, anionic, cationic or amphoteric; co-surfactants, such as linear fatty alcohols; thickeners and / or gelling agents, humectants, such as polyols such as glycerine; organic filters, inorganic filters, dyes, preservatives, fillers, tensors, sequestering agents, perfumes, and mixtures thereof, without this list being limiting. Examples of such adjuvants are cited in particular in the CTFA Dictionary (International Cosmetic Ingredient Dictionary and Handbook published by the Personal Railway Product Council).

Of course, those skilled in the art will take care to choose any additional compounds, active or non-active, and / or their quantity, so that the advantageous properties of the mixture are not, or not substantially, altered by the addition considered.

These compositions are especially intended for the fight against skin aging by acting on all the major causes of aging of the skin. To this end, the invention aims at a cosmetic process for the care of human skin, intended to preserve the barrier function of the skin, to increase the level of hydration of the skin, to smooth the cutaneous microrelief and to attenuate wrinkles, comprising the topical application of a composition containing peptides of Verbena off icinalis with a molecular weight of less than 2000ba or an active ingredient containing them, in particular a composition containing between 0.01 and 20% by weight of active principle (s) (s) derived from Verbena off icinalis according to the present invention. The invention specifically aims at a cosmetic process for the care of human skin, intended to stimulate the differentiation of keratinocytes, to promote the synthesis of intercellular lipids of the epidermis, to promote the growth of fibroblasts and to increase the synthesis of major matrix components dermis, comprising the topical application of a composition containing Verbena off icinalis peptides of molecular weight less than 2000ba or an active ingredient containing it, in particular a composition containing between 0.01 and 20% by weight of principle (s) active (s) from Verbena off icinalis according to the present invention.

EXAMPLES A non-limiting example of a process for obtaining an active ingredient obtained from Verbena off icinalis comprising peptides with a molecular weight of less than 2000ba is presented following, as well as examples of compositions including such an active principle.

EXAMPLE 1 Process for Obtaining the Active Ingredient According to the Invention An example of a process for obtaining an active ingredient according to the invention comprises the following steps: powder solubilization of Verbena aerial parts icinalis in water at a rate of 50 g / l, - hydrolysis using a protease at initial pH, - separation of the soluble and insoluble phases by decantation, - inactivation by heat treatment at 80 ° C for at least 30 minutes , - cooling, - purification in order to select the molecules lower than 5000ba, - concentration of this soluble fraction, - successive filtrations and sterilizing filtration.

The active ingredient obtained has the following characteristics: - appearance: clear liquid - color: amber - dry matter content: 29.4g / l - pH: 3.5 - total sugar content: 8.35g / l, ie 28, 4% by weight relative to the dry matter, - total protein content: 4.63 g / I, ie 15.7% by weight relative to the dry matter.

The percentage of peptides with a molecular weight of less than 2000 Da is about 88% of the total proteins. The results of the distribution of the proteins are presented according to: Molecular weight Mm (Da) Distribution in (%) Mm> 5000 3% 2000 <Mm 5000 9% 243 <Mm 2000 54% 0 <Mm243 34% Example 2: use of an active ingredient according to the invention in a day fluid Phase A A. Water QSP 100% Butylene Glycol 5% Phase B. CUTINA E24 (Cognis) 5% DUB STG AE30 (Stéarinerie Dubois) 5% Cetyl Palmitate (Gattefossé) 2 % SOPHIDERM (Sophim) 4% OCTOCARE 5M2030 (Cornelius) 3% MARCOL 82 (Exxon) 2% ARLAMOL E (Novéon) 2% Phase C. Carbopol ETD2020 (Novéon) 0.53% Phase D. Preservative 0.7% Phase E Active principle according to the invention (example 1) 3% NaOH QSP pH 6 The amounts indicated are given as a percentage of weight. This fluid emulsion has a pH of 6. It can be obtained by carrying out the following steps: - mixing A - mixing B, - heating A and B separately at 80 ° C., with slight mechanical stirring, - emulsifying B in At a rotor stator underflow emulsifier at 2100 rpm, at 50 ° C, add C, and ensure that the gel is well dispersed in the emulsion, at 30 ° C. add D, in the order indicated under rotor stator stirring, - then adjust the pH with E, and - leave under foam concentrate until complete cooling. Example 3 Use of an Active Principle According to the Invention in an Evanescent Cream Phase A. Water QSP 100% Glycerol 6.67% Phase B. DUB SEG (Stéarinerie Dubois) 4 ° / G Simulsol CS (Seppic) 2% Eumulgin B1 (Cognis) 1% DUB340 (Stéarinerie Dubois) 4 ° / G Montanov S (Seppic) 1% GLYCEROX 767HC (Croda) 1 ° / G DUB LAHE (Stéarinerie Dubois) 2% DUB PIS (Stéarinerie Dubois) 2% Phase C. Preservative 0.7% Active ingredient according to the invention (example 1) 3% 15 20

The quantities indicated are given as a percentage of weight. This firm emulsion application "wet effect" has a pH of 4.5. It can be obtained by carrying out the following steps: - mix A, - mix B, - heat A and B separately at 80 ° C. with magnetic stirring, - emulsify B in A under a rotor stator emulsifier at 1500 rpm at 40 ° C., add C in order, leave stirring until completely homogenized.

EXAMPLE 4 Use of an Active Principle According to the Invention in a Body Milk Phase A. Water QSP 100% Propylene Glycol 4% Phase B. DUB PULPIE (Stéarinerie Dubois) 4 ° / G Cetyl Alcohol 1% DUB ININ (Stéarnerie Dubois) 2% LANOL 2681 (Seppic) 4% DUB ZENOAT (Dubois Stéarinerie) 2% Montanox 60 (Seppic) 2% Montane 60 (Seppic) 4% C. Phase Preservative 0.7% Active principle according to the invention (Example 1 ) 3% The quantities indicated are given as a percentage of weight. This fluid emulsion has a pH of 6.5. It can be obtained by carrying out the following steps: - mixing A, - mixing B, - heating separately A and B at 80 ° C. in a water bath, - emulsifying B in A under an emulsifier, - at 40 ° C. C, - continue the homogenization under an emulsifier until complete cooling.

EXAMPLE 5 Use of an Active Principle According to the Invention in a Cold Cream "Cold Cream" Phase A. Water QSP 100% Phase B. Propylene Glycol 8% DUB MCT5545 (Stéarinerie Dubois) 7 ° / G Iso Propyl Myristate DUB MDIS (Stéarinerie Dubois) 4%, DUB GMS AE (Stéarinerie Dubois) 4%, Simulsol 165 (Seppic) 2% DUB BA (Stéarinerie Dubois) 3%, Isopropylpalmitate 5% DUB OLEINE V2 (Stéarinerie Dubois) 4% Stearyl alcohol 4% APIFIL (Gattefossé) 3% ARLATONE 2121 (Croda) 2% Phase C. Preservative 0.7% Active ingredient according to the invention (Example 1) 3% 10 15

The quantities indicated are given as a percentage of weight. This thick and creamy emulsion has a pH of 6. It can be obtained by carrying out the following steps: - mixing A, - mixing B, - separately heating A and B at 80 ° C. in a water bath, with stirring mechanical, 25 - emulsify B in A under stator rotor emulsifier at 1800 rpm, - at 40 ° C add C, in the order indicated, - continue homogenization in the emulsifier until complete cooling.

Example 6: Use of an active ingredient according to the invention in a night cream Phase A. Water QSP 100% Propylene Glycol 10% Phase B. PHOENATE GC7 (Cornelius) 1.3% DUB GMS AE (Stéarinerie Dubois) 2, 3% RITA PEO 27 (Rita) 1% DUB Wax A (Stéarinerie Dubois) 3% Cetearyl alcohol 1% Isopropylmyristate 2% DUB MICROLUB 50 (Stéarinerie Dubois) 4% Phase C. Preservative 0.7% Active ingredient according to the invention ( example 1) 3% Talc 1% The quantities indicated are given as a percentage of weight. This thick, creamy emulsion has a pH of 5. It can be obtained by carrying out the following steps: - mixing A, - mixing B, - separately heating A and B at 80 ° C. in a water bath, with stirring Mechanically, - emulsify B in A under a stator rotor emulsifier at 2000 rpm, - at 30 ° C slowly add C, in the order indicated, - continue homogenization in an emulsifier until complete cooling. Example 7: Use of an active ingredient according to the invention in a bath oil Phase A. Water QSP 100% Carbopol Ultrez 10 (Novéon) 0.1% 10 Phase B. MARCOL 82 20% DUB RG AE30 (Stéarinerie Dubois ) 4.5% AAB2 (Aiglon) 20% EMEREST 2380 (Chemical Station) 107.

SOPHIDERM (Sophim) 6% MONOMULS 900 18 (Cognis) 8% PHELEMOL DP 144 (Cornelius) 8% Isopropylmyristate 107. Phase C. Preservative 0.7% Active ingredient according to the invention (Example 1) The amounts indicated are given in percentage weight. This two-phase fatty gel has a pH of 6. The two phases of disperse easily and the mixture becomes milky and gives a silky touch. It can be obtained by carrying out the following steps: - mixing A, - mixing B, - separately heating A and B at 60 ° C in a water bath, with mechanical stirring, - emulsifying B in A under a rotor stator emulsifier to 1200 rpm, 20 - 50 ° C add C, and increase the agitation of the emulsifier around 2000 cpm, - leave under foam concentrate until complete cooling.

These tests are intended to illustrate the invention, by showing the anti-aging efficacy of the active ingredient according to the invention, through its action on both the epidermis and the epidermis. and on the dermis. The active ingredient used for these studies is that of Example 1.

The cosmetic composition used for these studies is as follows: Active ingredient according to Example 1 3.0% PEG-7 glyceryl cocoate (PHOENATE GC-7, Phoenix Chemical) 1.2% Caprilic / capric triglycerides (DUB MCT5545, Stéarinerie Dubois ) 0.8% Preservatives 0.7% Acrylate / C10-30 alkyl acrylate crosspolymer (Carbopol EDT2020, 0.2% Noveon) Water QSP 100% NaOH QSP pH 6.3 A. Studies on the epidermis I. Effect on the keratinocyte differentiation This study was conducted with the aim of demonstrating the ability of an active principle according to the invention to stimulate the differentiation of keratinocytes at different stages of maturation, in particular its ability to stimulate the synthesis of: cytokeratin 10, which is a cytoskeleton protein expressed in the spinous layer of the epidermis, - involucrin, which is a protein of the horny envelope expressed from the spinous layer to the stratum corneum, and - loricrin, which is a protein of the envelope co expressed from the granular layer of the epidermis to the stratum corneum.

The study was performed on normal human keratinocytes by Western Blot. The operating protocol is described following. At Jo, normal human keratinocytes are inoculated and incubated at 37 ° C. Between J1 and J3 the cells are treated with the active ingredient according to the invention at 0.25%, 0.50% or 1% (V / V) or with a positive control, calcium (Ca2 +) at 1.5 mM.

The cells are then incubated at 37 ° C. On D4, the cell extracts are recovered to assay for cytokeratin (K10), involucrine and loricrin by Western blotting. The results obtained are presented as follows: Rate Rate of Rate Rate Rate K10 / K10 / Involucin Rate of Involucin Loricrin / Loricrin / Actin Control / Actin / Control Tubulin Control (%) (AU) (%) (AU) (%) (UA) Control 0.064 / 4.55 / 0.501 / Ca '1.5mM 0.318 +395 9.52 +109 1.130 +125 Active ingredient 0.095 +49.76 +55 0.736 +47 according to the invention 0.25% Principle active agent 0.109 +70 9.57 +110 0.922 +84 according to the invention 0.5% active principle 0.178 +177 10.71 +135 1.029 +105 according to the invention It is found that the active ingredient according to the invention stimulates the synthesis of cytokeratin 10, involucrine and loricrin by normal human keratinocytes. These results show in particular that tested at 1%, the active ingredient according to the invention increases the synthesis by keratinocytes, cytokeratin of 177%, involucrin of 135% and loricrin of 105%. The active ingredient according to the invention therefore stimulates the differentiation of keratinocytes.

II. Effect on epidermal lipid synthesis a. Study of the Expression of the Ceramide Synthesis Enzymes This study was conducted with the aim of demonstrating the ability of an active ingredient according to the invention to increase the expression of the mRNAs encoding enzymes involved in the synthesis of ceramides: glucosylceramide synthase, involved in the synthesis of the major precursors of ceramides, glucosylceramides, p-glucocerebrosidase, responsible for the maturation of glucosylceramides into functional ceramides. The study was performed on normal human keratinocytes by quantitative PCR. The operating protocol is described following. At Jo, normal human keratinocytes are inoculated and incubated at 37 ° C. Between J1 and J3 the cells are treated with the active ingredient according to the invention at 0.25%, 0.50% or 1% (V / V) or with a positive control, calcium (Ca2 +) at 1.5 mM. The cells are then incubated at 37 ° C. At day 4, the cell extracts are recovered and the total RNA extracted. The RNAs are reverse transcribed and the complementary DNAs obtained are analyzed by quantitative PCR (Chain Reaction to Polymerase). The results obtained are presented as follows: Expression (%) Glucosylceramide p-glucocerebrosidase synthase (%) (%) Control 100 100 Ca 2 + 1.5 mM 149 128 Active ingredient according to the invention 0.25% 107 115 Active principle according to the invention The active ingredient according to the invention increases the expression of glucosylceramide synthase and p-glucocerebrosidase on normal human keratinocytes. These results show in particular that, tested at 1%, the active ingredient according to the invention increases the expression of glucosylceramide synthase by 59% and the expression of p-glucocerebrosidase, enzymes responsible for the synthesis of ceramides, by 41%. The active ingredient according to the invention thus promotes the formation of the intercellular lipid cement of the epidermis. b. In vivo study of the lipid synthesis of the horny envelope This study aims to quantify in vivo, on volunteers, the effect of the active ingredient according to the invention formulated at 3% in emulsified gel on the synthesis of the lipids bound by covalently to the proteins of the horny envelope.

These lipids are strongly involved in the architecture of the horny envelope as well as in the organization of the lamellar layers of intercellular lipids, thus actively participating in maintaining the qualities of the barrier function. In this study, the effect of the active ingredient was observed after 14 days of twice-daily application and compared to placebo after a chronic artificial disruption of the barrier function using a detergent, Sodium Lauryl Sulfate (SLS). at 10% (aggression that induces disruption of the lipid structure of the stratum corneum). The study is performed on 25 healthy female volunteers with normal skin in the arms.

Stratum corneum samples are collected using adhesives. At each measurement time, 2 samples are taken. The measurements are performed on symmetrical areas at the arms. The operating protocol is as follows. 7 days before the start of the study, volunteers stop applying any cream to the upper arms. At Jo, three measurement zones are determined at the level of the arms: - an untreated zone - a placebo zone - a zone treated with the active ingredient in emulsion Samples are taken on each zone. Between Jo and J13, twice a day volunteers: - wash the areas studied with an irritating soap (SLS) - apply the active ingredient according to the invention or placebo. At D14, samples are taken again on each zone. From the samples, the labeling of the lipids is carried out using the red Nile dye. This marking makes it possible to evaluate the hydrophobic nature of the corneocytes characteristic of a mature horny envelope. The labeled hydrophobic material mainly corresponds to the lipids covalently attached to the proteins. The fluorescence of each sample is then observed by means of a fluorescence microscope coupled to an image analysis software. The results are calculated as a percentage of variation observed under the effect of the active ingredient according to the invention versus placebo. They are presented in the following table: Variation / OJ (%) Variation / placebo (%) Placebo -4.0% / Active ingredient according to the invention + 11.3% + 16.0% It is found that the active ingredient according to the invention makes it possible to increase the level of corneocyte lipids. In particular, under the conditions of this study, after 14 days of twice-daily applications and in comparison with placebo, the active ingredient according to the invention formulated with 3% gel-emulsified, increases by 16% the lipid level of the horny envelope of stratum corneum after repeated aggression at SLS. By stimulating the synthesis of corneocyte lipids, the active ingredient according to the invention contributes to preserving the lipid architectural organization of the horny envelope responsible for maintaining the qualities of the barrier function.

III. Effect on the barrier function of the skin The objective of this study is to quantify in vivo, on volunteers, the effect of the active ingredient according to the invention formulated at 3% gel-emulsified on the insensible loss of water (PIE) skin.

This effect was observed after 14 days of twice-daily application and compared to placebo after chronic artificial disruption of the barrier function using a sodium lauryl sulphate (SLS) detergent. The cutaneous barrier plays a regulating role in the water balance of the skin. When this is damaged, it appears disturbances in the regulation of water exchanges. The water then migrates more easily to the outside environment which increases the insensible loss of water. On the other hand, if the state of the cutaneous barrier improves, the values of water loss will decrease because the regulation of water exchanges will be ensured in a correct way. The study was performed on 25 healthy female volunteers aged between 42 and 72 years with normal skin in the arms. The measurement is carried out using a Tewameter® TM 210 (Courage & Khazaka, Germany). The standardized protocol is as follows.

Between J_7 and Jo, the volunteers stop the application of cream on the level of the upper arms. At Jo, 3 measurement zones are determined at the level of the upper arms: - an untreated zone - a zone treated with the placebo - a zone treated with the active principle according to the invention formulated at 3% A Tewameter® measurement of the PIE is carried out on each zone. Between Jo and J13, the volunteers wash the zones studied daily with an irritating soap (SLS) and apply the products (active ingredient according to the invention to 3% and placebo). At D14, after marking the measurement zones, a new Tewameter® measurement of the PIE is made on each zone. Under the conditions of this study, after 14 days of twice-daily application and in comparison with placebo, the active ingredient according to the invention formulated at 3% gel-emulsified, decreases the insensitive loss in water after repeated aggression with SLS of 7, 4%. The active ingredient according to the invention formulated at 3% therefore limits the alteration of the barrier function and thus preserves the integrity of the stratum corneum B. Studies on the dermis 1. Effect on the growth of fibroblasts in a deficient medium This study was carried out conducted in order to evaluate the effect of an active ingredient according to the invention on the growth of normal human fibroblasts in a nutrient and calcium deficient medium. The study is based on the measurement of cell viability by spectrophotometry. The operating protocol is described following. At Jo, normal human fibroblasts are seeded in complete medium.

After 7 hours, the cells are treated with the active principle according to the invention at 0.5%, 1% and 2% or retinoic acid at 10-6M, used as a positive control. The cells are then incubated at 37 ° C. in an oven containing 5% CO2 in a nutrient and calcium deficient medium.

At day 3, cell growth is determined by measuring cell viability with M.T.T. (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide). The results in the table below, represent the optical density and the growth capacity of the treated cells compared to the control. Optical density (AU) Growth capacity / control (%) Control 0.252 / Retinoc acid 0.349 +39 10-6M Principle active ingredient 0.356 +42 according to the invention 0.5% Active ingredient 0.363 +44 according to the invention 1% Active ingredient 0.395 +57 according to the invention 2% It is therefore found that an active ingredient according to the invention makes it possible to stimulate the growth of human fibroblasts. In particular, tested at 2%, an active ingredient according to the invention stimulates the growth of normal human fibroblasts in a deficient medium of 57%.

II. Effect on the synthesis of the major components of the dermis a. Evaluation of the synthesis of collagens I and III and hyaluronic acid The purpose of this study is to evaluate the effect of an active ingredient according to the invention on its ability to increase the synthesis of collagens I and III and hyaluronic acid. 'hyaluronic acid. Collagens I and II are the major fibrillar collagens of the dermis, which assemble with collagen V to form collagen fibers. Hyaluronic acid is the main glycosaminoglycan of the dermis, ensuring the water reservoir of the skin. The test is carried out on normal human fibroblasts, according to the operating protocol described below.

At Jo, human fibroblasts are inoculated in complete medium. The cells are then incubated at 37 ° C. At D2, the culture medium is removed and replaced with medium containing: the active principle at 0.5%, 1% or 2% (V / V), vitamin C at 0.10pg / ml or 0, 25 μg / ml, or TGF-β at 10 μg / ml. Vitamin C and TGF-β are used as a positive control. The cells are then incubated at 37 ° C, 24 or 48 hours. On D3, the supernatants are recovered for assaying Collagens I and III. On D4, the supernatants are recovered to determine the hyaluronic acid. The assay is performed using ELISA kits. The results obtained are shown in the following table: Synthesis Synthesis Rate Synthesis Rate Collagen Collagen I Collagen / Collagen III / Hyaluronic Hyaluronic Acid Collagen I (pg / μg Thread Control (μg / μg Control) (pg / μg / Protein (%) Protein) (%) Protein (%)) Control 423/97/1330 / Vitamin C 1207 +185 / / / 0.1 μg / ml Vitamin C / / 1058 +991 0.25 μg / ml TGF-β / / 1757 +32 10 ng / ml Active ingredient 656 +55 185 +91 1516 +14 according to the invention 0.5% active ingredient 1291 +205 586 +504 2095 According to the Invention Active Principle 2091 +394 1316 +1257 2343 +76 According to the Invention It is thus found that an active ingredient according to the invention makes it possible to stimulate the synthesis of collagen I and collagen III and hyaluronic acid. in the dermal cells. In particular, tested at 2%, on normal human fibroblasts, an active ingredient according to the invention stimulates the synthesis of collagen I by 394%, collagen III by 1257% and hyaluronic acid by 76%. b. Evaluation of the Integrin Synthesis a2131 This study aims to evaluate the effect of an active ingredient according to the invention on its ability to increase the synthesis of a junction protein, the integrin a2131.

Α2p1 integrins are transmembrane glycoproteins that play an important role in the assembly of the extracellular matrix by ensuring cell-matrix interaction. The study is carried out on normal human fibroblasts, according to the operating protocol described below. At Jo, human fibroblasts are inoculated in complete medium. The cells are then incubated at 37 ° C. On D1, the culture medium is removed and replaced with medium containing: the active ingredient at 0.5%, 1% or 2% (V / V), or TGF-β at 10 ng / ml, used as a positive control. The cells are then incubated at 37 ° C. At D2, the cells are recovered and an anti-α2p1 immunostaining is performed. Quantification measured using a flow cytometer. The acquisition and analysis are performed using dedicated software.

The results are shown in the table below: Intensity intensity intensity level a2 (51 / fluorescence (AU) Control (/) Control 12.91 / TGF-p 16.35 +27 10 ng / ml Principle active compound according to the invention 0.5% 15.00 +16 Active principle according to the invention 1% 16.96 +31 Active principle according to the invention 2% 20.20 +56 It is therefore found that an active ingredient according to the invention The invention makes it possible to stimulate the synthesis of α2β1 integrin in dermal cells, In particular, tested at 2%, on normal human fibroblasts, an active ingredient according to the invention stimulates the synthesis of α2p1 integrin by 56%. Evaluation of the Formation of the Fibrillin-1 Network The objective of this study is to evaluate the effect of an active principle according to the invention on the fibrillin-1 network Fibrillin-1 is a glycoprotein strongly involved in the formation of the elastic network of the dermis This study is carried out on normal human fibroblasts according to the operative protocol dec laughs at Jo, the human fibroblasts are inoculated in complete medium. The cells are then incubated at 37 ° C. At day 2, the culture medium is removed and replaced with medium containing: - the active ingredient at 0.5%, 1% or 2% (V / V), or - vitamin C at 0.25 μg / ml , used as a positive control. The cells are then incubated at 37 ° C. At D4, the cells are treated in the same way as at D2. On day 7 cells were recovered for immunocytologic labeling of fibrillin-1 followed by immunolabeling. The results are visualized using a microscope coupled to an image analysis system. The fluorescence intensity is proportional to the fibrillin-1 synthesis. The results are shown in the table below: Synthesis of fibrillin-1 / Control (%) Vitamin C 0.25 μg / ml +76 Active principle according to the invention 0.5% +31 Active principle according to the invention 1% +51 active principle according to the invention 2% +76 It is therefore found that an active ingredient according to the invention makes it possible to stimulate the synthesis of fibrillin-1 in dermal cells. In particular, tested at 2%, on normal human fibroblasts, an active ingredient according to the invention stimulates the synthesis of fibrillin-1 by 76%. III. Study of anti-wrinkle properties This study was conducted with the aim of evaluating in vivo the anti-wrinkle effect of the active ingredient according to the invention formulated at 3% in emulsion against placebo at the level of crow's feet by projection fringes. The study is carried out on 25 healthy female volunteers aged between 40 and 75 years old with crow's feet wrinkles. Acquiring data on the crow's feet are made using a fringe projection device dedicated to the 3D measurement of the cutaneous relief before and after 56 days of twice-daily treatment. The measured parameters selected for this study are: - roughness parameters in 3D: * Sq, the root mean square of surface roughness, * Sa, the arithmetic average of surface roughness, - volume parameters: 20 * positive volume that is to say the volume greater than the surface of the skin, * negative volume, that is to say the volume less than the surface of the skin. A decrease in these various parameters reflects an improvement in the relief of the studied surface and a decrease in wrinkles. The operating protocol of the study is described following. 14 days before the start of the study, the volunteers apply a placebo cream on the entire face twice a day.5 At Jo, we make a 3D acquisition of crow's feet by fringe projection. Between Jo and J27, the products are applied twice daily to the face. At D28, a new 3D acquisition of the crow's feet is performed by fringe projection.

Between D28 and D55, the products are applied twice a day on the face. At J56, we make a final 3D acquisition of the crow's feet by fringe projection. The results obtained are presented in the table below: Variation / Placebo (%) J28 J56 Parameter Sq -4,6 -7,3 Parameter Sa -4,3 -7,9 Volume positive -14,3 -21 3 Volume negative -8.5 -15.7 These results show that an active ingredient according to the invention makes it possible to smooth the cutaneous relief at crow's feet and reduce wrinkles. In particular, it is found that under the conditions of this study, after 56 days of twice-daily application and in comparison with placebo, the active ingredient according to the invention formulated at 3% in emulsion makes it possible: to reduce the Sa parameter of 7.9 % and the Sq parameter of 7.3%, and thus to smooth the skin relief at crow's feet, - to reduce the negative volume parameter by 15.7% and the positive volume parameter by 21.3%, and therefore reduce wrinkles.

Claims (18)

REVENDICATIONS1. Cosmetic active ingredient comprising peptides with a molecular weight of less than 2000Da from Verbena off icinalis. 2. Active ingredient according to claim 1, characterized in that it is a hydrolyzate of Verbena officinalis. 3. Cosmetic active ingredient according to claim 1 or 2, characterized in that peptides with a molecular weight of less than 2000Da from Verbena officinalis represent at least 70% by weight of the total proteins of the active principle. 4. Cosmetic active ingredient according to one of the preceding claims, characterized in that the protein content is between 8 and 38% by weight of dry matter. 5. Cosmetic active ingredient according to one of the preceding claims, characterized in that it also comprises carbohydrates. 6. The cosmetic active ingredient according to claim 5, characterized in that the content of carbohydrates is between 14 and 48% by weight of dry matter. 7. Cosmetic active ingredient according to one of the preceding claims, characterized in that it has a solids content of between 10 and 90g / l. 20 8. Process for obtaining an active ingredient according to one of the preceding claims, characterized in that it comprises at least the following stages: solubilization of powder of aerial parts of Verbena officinalis in water, enzymatic hydrolysis in the presence of at least one enzyme, - separation of the soluble and insoluble phases by decantation, - inactivation by heat treatment, - filtration, - concentration of the active fraction, and - filtration and sterilizing filtration. 9. Process for obtaining an active ingredient according to one of claims 1 to 7, characterized in that the enzymatic hydrolysis is carried out in the presence of a protease. 10. Use of peptides with a molecular weight of less than 2000ba derived from Verbena off icinalis, or a hydrolyzate derived from Verbena off icinalis containing peptides with a molecular weight below 2000ba, as a global anti-aging cosmetic active ingredient. 11. Use of an active ingredient according to one of claims 3 to 7, as a global anti-aging cosmetic active ingredient. Use according to claim 10 or 11 as a cosmetic active ingredient in a global anti-aging cosmetic composition intended to act on both the dermis and the epidermis. 13. The use as claimed in one of claims 10 to 12, as a cosmetic active principle in a cosmetic composition intended to preserve the barrier function, to increase the level of hydration of the skin, to smooth the microrelief and to attenuate the wrinkles. 14. Use according to one of claims 10 to 13, as a cosmetic active ingredient in a cosmetic composition intended to stimulate the differentiation of keratinocytes at different stages of maturation, to act on the mechanisms of synthesis of intercellular lipid cement, to favor growth of fibroblasts and to promote the synthesis of matrix constituents of the dermis. 15. Use according to one of claims 10 to 13, as a cosmetic active principle in a cosmetic composition intended to stimulate the differentiation of keratinocytes at the different stages of maturation by increasing the synthesis of cytokeratin 10, of involucrine and loricrin, to Act on the mechanisms of synthesis of intercellular lipid cement by increasing the expression of glucosylceramide synthase and p-glucocerebrosidase and by increasing the level of lipids bound to the proteins of the horny envelope, to promote the growth of fibroblasts and to promote in the dermis the synthesis of collagen I, collagen III, fibrillin-1, hyaluronic acid and integrin a2131. 16. Cosmetic composition for topical application, characterized in that it comprises an active ingredient according to one of claims 1 to 7 present between 0.01 and 20% by total weight of the composition. 17. Cosmetic process for the care of human skin, intended to preserve the barrier function of the skin, to increase the level of hydration of the skin, to smooth the skin microrelief and to attenuate wrinkles, including the topical application of the skin. a composition containing Verbena officinalis peptides with a molecular weight of less than 2000ba or an active ingredient according to one of claims 1 to 7. 18. Cosmetic process for the care of human skin, intended to stimulate the differentiation of keratinocytes, to promote the synthesis of intercellular lipids of the epidermis, to promote the growth of fibroblasts and to increase the synthesis of the major matrix components of the dermis, comprising the topical application of a composition containing Verbena officinalis peptides of molecular weight less than 2000ba or an active ingredient according to one of claims 1 to 7.