WO2023237833A1

WO2023237833A1 - Anti-ageing composition with rose extracts and peptides

Info

Publication number: WO2023237833A1
Application number: PCT/FR2023/050797
Authority: WO
Inventors: Jocelyne Franchi; Carine Nizard; Valérie GORZELANCZYK; Laure VERT
Original assignee: L V M H Recherche
Priority date: 2022-06-07
Filing date: 2023-06-06
Publication date: 2023-12-14
Also published as: FR3136172A1

Abstract

The present invention relates to a cosmetic composition comprising, in a physiologically acceptable medium: (i) an aqueous extract of rose wood of the Evanrat variety, preferably of summer rose wood, obtained by enzymatic extraction by means of a cosmetically acceptable polar solvent, (ii) a hexapeptide with the INCI name: Acetyl Hexapeptide-8, (iii) a tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7, and (iv) optionally also an extract of roses in the form of an isolated bioactive fraction obtained from fresh roses of the Evanrat variety.

Description

Anti-aging composition with rose extracts and peptides

FIELD OF THE INVENTION

The present invention relates to a cosmetic composition, in particular a cosmetic care composition intended to prevent and/or reduce the signs of skin aging, in particular of the skin of the face and/or body.

STATE OF THE ART

The skin is the body's first protective barrier against the environment. It is daily subject to the effects of factors of exogenous origin (e.g.: UV radiation, temperature variations, atmospheric pollution, cigarette smoke, etc.) and/or endogenous (e.g.: hormones, etc.).

Aging leads to changes in the skin of the body and/or face, including the lips, areas particularly subject to the deleterious effects of the environment, such as wrinkles and fine lines, skin having lost its firmness, suppleness and its elasticity, a reduction in the thickness of the epidermis linked in particular to a slowdown in the renewal of skin cells.

A large number of anti-aging active ingredients such as peptides, vitamins, antioxidants and plant extracts are known from the prior art. Mention may in particular be made of the peptides Acetyl Hexapeptide-8 from the company Lipotec and Palmitoyl Tetrapeptide-7 from the company Sederma.

We also know the use of certain rose extracts for anti-aging, such as the aqueous extract of rosewood of the Evanrat variety described in patent application FR3090380.

However, there is a constant need to find new compounds or new combinations of active ingredients with improved properties in the prevention and/or aging of the skin.

Unexpectedly, the Applicant has demonstrated a synergistic anti-aging in vitro effect between the peptides (Acetyl Hexapeptide-8 and Palmitoyl tetrapeptide-7) and an aqueous extract of rosewood of the Evanrat variety. It also demonstrated anti-aging synergy between peptides (Acetyl Hexapeptide-8 and Palmitoyl tetrapeptide-7), an extract aqueous rosewood of the Evanrat variety and a rose extract in the form of an isolated bioactive fraction obtained from fresh roses of the Evanrat variety.

STATEMENT OF THE INVENTION

The present invention therefore relates in particular to a cosmetic composition comprising, in a physiologically acceptable medium:

(i) An aqueous extract of rosewood of the Evanrat variety, preferably summer rosewood, obtained by enzymatic extraction using a cosmetically acceptable polar solvent,

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8, and

(iii) A tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7.

The present invention also relates to a non-therapeutic cosmetic process for caring for and/or making up the skin and/or lips comprising the application to said skin and/or said lips, in particular the skin, of a cosmetic composition such as as defined in the present invention.

The invention also relates to a combination of cosmetic active ingredients comprising:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8,

(iii) A tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7, and

(iv) Optionally also an isolated bioactive fraction obtained from fresh roses of the Evanrat variety and comprising at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose, and/or at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium.

The invention further relates to the non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined in the present invention, as an active association for preventing and/or reducing the signs of skin aging, in particular loss of density. and/or firmness of the skin, reduction in the thickness of the epidermis, reduction differentiation and/or proliferation of epidermal cells, and/or the appearance of wrinkles and/or fine lines.

The invention also relates to a cosmetic set comprising:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8,

(iii) A tetrapeptide with INCI name: Palmitoyl Tetrapeptide-7, and

(iv) Optionally also an isolated bioactive fraction obtained from fresh roses of the Evanrat variety and comprising at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose, and/or at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium, characterized in that at least one of the active ingredients (i) to (iv) is packaged in a composition distinct from the other active ingredients, and in particular each active ingredient is packaged in a distinct composition.

The invention finally relates to a cosmetic process comprising the successive application, to the skin and/or lips, of the compositions of the cosmetic assembly as defined in the present invention.

DESCRIPTION OF FIGURES

[Fig. 1] Effect of the different active ingredients and combinations of active ingredients according to the invention on the protein expression of the KI67 factor in cultures of reconstructed human skin.

[Fig. 2] Effect of the different active ingredients and combinations of active ingredients according to the invention on the thickness of the epidermis in cultures of reconstructed human skin.

[Fig. 3] Effect of the different active ingredients and combinations of active ingredients according to the invention on the protein expression of Collagen VII in cultures of reconstructed human skin.

DETAILED DESCRIPTION OF THE INVENTION

Thus, the present invention therefore relates in particular to a cosmetic composition comprising, in a physiologically acceptable medium: (i) An aqueous extract of rosewood of the Evanrat variety, preferably summer rosewood, obtained by enzymatic extraction using a cosmetically acceptable polar solvent,

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8, and

The roses or rose bushes used as plant material for the preparation of the extracts used according to the invention correspond to the rose bush or rose 'Jardin de Granville®', a hybrid variety offered exclusively by "Roses ancien André Eve S.A.S" and protected by Certificate of Obtention Plant under number 20110345 with the species name Rosa L. and the variety the name EVANRAT. This bush rose belongs to the group of modern hybrids, which, from May to October, is permanently covered with roses, thus showing an excellent repeating character.

Rosewood extract and preparation process

The aqueous extract of rosewood of the Evanrat variety or 'Jardin de Granville®' rose, preferably summer rosewood, used according to the invention is obtained by enzymatic extraction using a cosmetically acceptable polar solvent. , according to the techniques described in application FR3090380.

We will speak indifferently of aqueous extract of rosewood or rosewood or polar extract of rosewood or rosewood or hydrophilic extract of rosewood or rosewood or eco-extract obtained by an eco-extraction process of rosewood or rosewood in the description.

The expression "polar solvent" means that the solvent has a Polarity index value which is equal to or greater than a value of 4. The polarity index is a quantity calculated on the basis of thermodynamic quantities (solubility and change of state) which highlights the more or less polar nature of a molecule. Refer, for the polarity indices of solvents, to the article by L.R. SNYDER: Classification of the solvent properties of common liquids; Journal of Chromatography, 92 (1974), 223-230.

The preferred polar solvents are those consisting of a compound comprising at least one polar covalent bond of the OH type. As a cosmetically acceptable polar solvent, a solvent or a mixture of solvents chosen from water, C1-C4 alcohols, such as ethanol, glycols, such as ethylene glycol, glycerol, butylene glycol and propylene glycol, and mixtures thereof. Preferably, water is used.

According to a particular mode, said extract is characterized in that it comprises water in a content ranging from 94 to 96%, a dry extract content ranging from 3 to 5% and preservatives in a content ranging from 0, 5 to 1% by weight relative to the total weight of the extract.

Plant material

There are two types of rosewood depending on the time of year they are harvested:

- Winter wood, generally harvested between the months of November and April

- Summer wood, generally harvested between the months of May and October.

According to a particular and preferred method, we will use summer wood. We will speak indifferently in the description of rosewood or summer rosewood.

The rosewood according to the invention is obtained from freshly cut whole roses and is isolated from the rest of the plant by manual or mechanical separation.

In a particular embodiment, the rosewoods are directly used for extraction.

In another embodiment, the rosewood is previously dried before extraction.

According to a particular and preferred method, the rosewood is crushed before the extraction stage.

Rosewood includes monosaccharide sugars (fructose, glucose), and polysaccharides in particular disaccharides, polyphenols (catechin), amino acids (mainly aspartic acid, tyrosine and arginine), minerals (ash, sodium, potassium, calcium), flavonoids (mostly glycosylated) and tannins.

In particular, calcium improves epidermal differentiation and strengthens the structure of the skin, while potassium amplifies skin hydration and boosts energy assimilation. Phytosugars (fructose, glucose) are intended to fill skin cells with energy. The aqueous rosewood extract according to the invention is an extract concentrated in polar natural compounds. The aqueous extract of rosewood according to the invention is distinct from rose water. This is a rosewood extract using the extraction of natural rosewood compounds in the presence of aqueous solvents (polar) with or without the addition of a pH modifier, and an implementation, in the formulation, in the form of an aqueous solution or concentrate, the solvent of the concentrate possibly being the extraction solvent and/or an additional solvent.

According to a particular embodiment, the aqueous extract of rosewood according to the invention further comprises in a small proportion an extract of rose flowers representing from 0.01 to 1%, preferably from 0.05 to 0.5%, preferably 0.1 to 0.2% of the total aqueous rose extract according to the invention. In this embodiment, the original plant material comprises rosewood and rose flowers in a proportion 0.01% flowers for 99.99% wood to 1% flowers for 99% wood.

Extraction process

The aqueous extract of rosewood according to the invention can be obtained according to different eco-extraction processes known to those skilled in the art and in particular that described below.

According to a particular and preferred mode, we will use as plant material in the extraction processes adapted to the invention described below, rosewood of the Evanrat variety, preferably the Jardin de Granville® rose, preferably wood of roses previously dried.

The wood can be used fresh, frozen or previously dried using conventional drying tools known to those skilled in the art, such as drying in the open air, in ovens, by freeze-drying or zeodration. Thus, according to one embodiment, and prior to the extraction step itself, the wood may have been dried and/or crushed. According to another embodiment, the wood is used wet then crushed.

According to a particular method, the wood is crushed before extraction, for example by means of a mortar, cryo-grinding, a mixer, a traditional grinder or a centrifuge according to methods known to those skilled in the art. job.

The aqueous extract of rosewood according to the invention can be obtained in particular according to enzymatic extraction processes. Depending on the molecules that we seek to extract, the enzymes will be adapted to the walls to be hydrolyzed to access these said molecules. Advantageously, it is possible in particular to use enzymes of the cellulase, hemicellulase, pectinase or protease type, alone or in combination.

We can thus use a cellulase to release glucose and cellulose; a hemicellulase to release reduced sugar mono- and oligomers; a pectinase to release uronic acids; and advantageously a protease to hydrolyze the structural proteins and improve the lysis of the raw material.

In a preferred embodiment, eco-extraction is carried out using the protocol described in patent application WO2011045387 (corresponding to patent application FR2351461) in the name of the National Polytechnic Institute of Lorraine INPL. This is an alternative technology that meets regulatory and environmental constraints while remaining economically competitive. This innovative process makes it possible to extract from plant material, in a single step, three types of products, namely an oil (rich in polyphenol nutrients, sterols, vitamin E, etc.), an aqueous extract as well as a cake ( solid phase).

Thus, in one embodiment of the invention, the aqueous extract of rosewood used according to the invention is obtained by an enzymatic extraction process comprising the steps comprising the steps: a) addition of water to the wood of previously crushed rose having an appropriate particle size, b) addition of an enzymatic mixture containing at least one cellulase, at least one hemicellulase and at least one pectinase, and advantageously also at least one protease, c) incubation with stirring of the wood of previously crushed rose and the enzymatic mixture to release into the reaction medium oils, proteins and fermentable sugars, for a period depending on the desired yields, d) separation of the reaction medium to obtain free oil, an aqueous phase containing fermentable proteins and sugars, and a solid phase, and e) separation of the aqueous phase and possible addition of preservatives.

Said rosewood extract used according to the invention being obtained after the separation step (e). According to a particular mode, in step a), the appropriate particle size of the plant material is advantageously obtained by grinding said plant material. The grinding must be as fine as possible to promote the action of the enzymes. Ideally, all particles should be close to 50 pm in size, preferably close to 10 pm.

Preferably, the mass of water added to the plant material is equal to 1 to 2 times the mass of said plant material and does not exceed this quantity.

In particular in step b), the ratio between the pectinase activity and the cellulase activity being at least 0.14, preferably between 0.3 and 2.5, and more preferably between 0.35 and 0.45, and the ratio between the pectinase activity and the hemicellulase activity being at least 7.10 ^-3 ; preferably between 1.10 ^-2 and 0.5, and more preferably between 1.10 ^-2 and 2.10 ^-2 .

More particularly, the enzymatic mixture used contains at least one cellulase, at least one hemicellulase, at least one pectinase, and advantageously also at least one protease. According to a particular mode, the cellulase(s), hemicellulase(s) and pectinase(s) represent 75% of the enzymatic mixture and the protease(s) represent 25% of said enzymatic mixture. According to a particular mode, the cellulases are present in a content ranging from 1 to 50%, preferably from 20 to 30% by weight of the enzymatic mixture, the hemicellulases are present in a content ranging from 1 to 50%, preferably from 20 to 30% by weight of the enzymatic mixture and the pectinases are present in a content ranging from 1 to 50%, preferably from 20 to 30% by weight of the enzymatic mixture. And advantageously the proteases are present in a content ranging from 1 to 50%, preferably from 20 to 30% by weight of the enzymatic mixture.

In a preferred embodiment, the enzymatic mixture used contains 25% cellulases, 25% hemicellulases, 25% pectinases and 25% proteases.

Preferably, the quantity used of the enzymatic mixture as defined above is between 0.25% and 10%, preferably between 1% and 6%, by volume of the water/plant material mixture.

Advantageously, the incubation according to step c) is carried out for 2 to 20 hours, preferably between 4 and 12 hours, at a temperature between 25°C and 75°C, preferably between 40°C and 60°C. , and preferably around 50°C. The hydrolysis reaction is then stopped by deactivation of the enzymes by heating preferably between 80°C and 105°C for for example 5 to 20 minutes.

Then the reaction medium is separated, according to step d), using all suitable known separation techniques, such as centrifugation or decantation.

According to a particular mode, the mixture is pre-filtered through a 0.8mm then 0.5mm sieve in order to remove the most important part of the solid phase, then sterilizing filtration is advantageously carried out on clarifying plates with a pore size of 0.2pm, at a pressure of 2 Bars.

The aqueous phase obtained after sterilizing filtration is stabilized with preservatives (citric acid, potassium sorbate, sodium benzoate).

According to a particular and preferred embodiment, the cosmetic composition according to the invention is characterized in that the aqueous extract of rosewood is obtained by an enzymatic extraction process comprising the steps: a) addition of water to the wood of previously crushed rose having an appropriate particle size, b) addition of an enzymatic mixture containing at least one cellulase, at least one hemicellulase and at least one pectinase, and advantageously also at least one protease, c) incubation with stirring of the previously crushed rosewood and the enzymatic mixture to release fermentable oils, proteins and sugars into the reaction medium, for a period depending on the desired yields, d) separation of the reaction medium to obtain free oil, an aqueous phase containing fermentable proteins and sugars, and a solid phase, e) separation of the aqueous phase and possible addition of preservatives.

The aqueous extract of rosewood used according to the invention is characterized in particular by the presence of amino acids (aspartic acid, tyrosine, arginine) and total sugars (glucose, fructose).

The aqueous rosewood extract used according to the invention is also characterized by the fact that it is not a fermented extract. According to a particular mode, the aqueous extract of rosewood used according to the invention comprises water in a content ranging from 94 to 96%, a dry extract content ranging from 1 to 6% and preferably from 3 to 5 % and preservatives in a content ranging from 0.5 to 1% by weight relative to the total weight of the extract. This rosewood extract according to the invention is called INCI water (and) rose extract (and) citric acid (and) potassium sorbate (and) sodium benzoate.

The aqueous extract of rosewood used according to the invention will generally be present in a content ranging from 0.1 to 1%, in particular from 0.2 to 0.5% by weight relative to the total weight of the composition.

Peptides

The compositions of the invention also comprise an acetyl hexapeptide and a palmitoyl tetrapeptide, in particular the following peptides:

Acetyl hexapeptide 8 from the company LIPOTEC marketed under the name ARGIRELINE® Amplified peptide with INCI name: WATER and ACETYL HEXAPEPTIDE-8 and SODIUM BENZOATE.

According to a particular mode, the commercial product ARGIRELINE® Amplified peptide containing acetyl hexapeptide-8 is present in the composition of the invention in a content ranging from 0.1% to 10% by weight relative to the total weight of the composition , preferably from 1% to 5% by weight relative to the total weight of the composition.

In particular, acetyl hexapeptide-8 is present in the composition of the invention in a peptide content ranging from 0.005% to 0.5% by weight relative to the total weight of the composition, preferably from 0.05% to 0.25% by weight relative to the total weight of the composition.

Palmitoyl tetrapeptide-7 which is a synthetic peptide with the sequence Palmitoyl-Gly-GIn-Pro-Arg (Palmitoyl-GQPR) from the company SEDERMA marketed under the name RIGIN® with the INCI name: Glycerin, Lactic Acid and Palmitoyl Tetrapeptide-7.

According to a particular mode, the commercial product RIGIN® containing palmitoyl tetrapeptide-7 is present in the composition of the invention in a content ranging from 0.1% to 10% by weight relative to the total weight of the composition, preferably from 2% to 5% by weight relative to the total weight of the composition. In particular palmitoyl tetrapeptide-7 is present in the composition of the invention in a peptide content ranging from 0.005% to 0.5% by weight relative to the total weight of the composition, preferably from 0.1% to 0. 25% by weight relative to the total weight of the composition.

According to a particular and preferred embodiment, the composition of the invention further comprises a bioactive fraction isolated from roses as described below.

Thus, the composition of the invention will generally include:

(i) The aqueous extract of rosewood of the Evanrat variety in a content ranging from 0.1 to 1%, in particular from 0.2 to 0.5% by weight relative to the total weight of the composition,

(ii) The INCI name hexapeptide: Acetyl Hexapeptide-8 in a content ranging from 0.05% to 0.25% by weight (of peptide) relative to the total weight of the composition, and

(iii) The tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7 in a content ranging from 0.1% to 0.25% by weight (of peptide) per relative to the total weight of the composition, and

(iv) The bioactive fraction isolated from fresh roses of the Evanrat variety, in a content ranging from 0 to 1%, in particular from 0.1 to 1% and in particular from 0.2 to 0.5% by weight relative to the total weight of the composition.

Bioactive fraction isolated from roses and method of preparation

By “bioactive fraction” or “bioactive fraction isolated from roses” or “Sérum Rose De Granville®”, “Zêta” or “Zêta fraction”, we mean an extract of roses of the Evanrat variety, or rose 'Jardin de Granville®' , comprising the enzymes, proteins, sugars, ions and other active molecules present in the cytosol of the cells making up the different plant tissues of roses. The extract according to the invention is distinct from a cryoextract of pink petals as described in application FR3066388.

Plant material

The rose extract in the form of an isolated bioactive fraction used according to the invention can be prepared from fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, frozen, freeze-dried, or any mixture thereof. In the context of the invention, fresh roses are preferably used.

According to a particular method, summer roses will be used to prepare the bioactive fraction isolated from roses, in particular summer rose petals. According to another method, we will preferably use winter roses, in particular winter rose petals.

It is particularly advantageous to use the petals of roses of the Evanrat variety because they are rich in monosaccharide sugars (fructose, glucose, sucrose), organic acids (citric acid, malic acid), polyphenols (catechin), vitamin C, amino acids (mainly aspartic acid, glutamic acid, asparagine and glutamine), minerals (ash, potassium, calcium), and carotenoids.

By rich in monosaccharide sugars is meant a rose extract in the form of an isolated bioactive fraction, in which said extract comprises at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose.

By rich in minerals, we mean a rose extract in the form of an isolated bioactive fraction, in which said extract comprises at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium.

Thus, according to one embodiment, the composition according to the invention further comprises a rose extract in the form of an isolated bioactive fraction obtained from fresh roses of the Evanrat variety and comprising at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose, and/or at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium.

Advantageously, calcium improves epidermal differentiation and strengthens the structure of the skin, while potassium amplifies skin hydration and boosts energy assimilation. Phyto-sugars (fructose, glucose, sucrose) help fill skin cells with energy.

Extraction process

We use fresh roses of the Evanrat variety or ‘Jardin de Granville®’ roses as plant material. According to a preferred mode, these are fresh rose petals of the Evanrat variety or ‘Jardin de Granville®’ roses.

Advantageously, said process does not require the addition of any exogenous solvent or liquid. The process implemented to obtain the isolated bioactive fraction used according to the invention comprises the main steps of: a) Cleaning of the plant material, maceration, pressing then mechanical separation of the plant material to obtain an intracellular colloidal dispersion (ICD) and a material enriched with fiber (fraction A); b) “Treatment A” then mechanical separation of the Intracellular Colloidal Dispersion (ICD) to obtain Supernatant A and a Membrane Fraction (Fraction B); c) “Treatment B” then mechanical separation of Supernatant A to obtain Supernatant B and Fraction C (Cytoplasmic Fraction); d) “Treatment C” then mechanical separation of Supernatant B to give a “Bioactive Serum Fraction” and a Fraction D (Precipitate); and e) Optionally, mixing the “bioactive serum fraction” with at least one preservative and/or stabilizer.

The extract of fresh roses, preferably fresh rose petals, of the Evanrat variety or 'Jardin de Granville®' rose obtained at the end of this process, is a "bioactive serum fraction" or "bioactive fraction" in the sense of the invention.

“Cleaning” means the removal of debris from fresh Granville® roses, and preferably fresh petals, before further processing, in a manner that avoids injury to the plant, or the removal of valuable components. For example, it can be carried out by low pressure rinsing with potable water under conditions where washing with runoff water would not contain appreciably plant pigments. Excess wash water is then removed from the washed plants.

By “maceration” we mean the act of transforming fresh Granville® roses, and preferably fresh petals, into smaller particles to break their integrity and subsequently facilitate the expulsion of the liquid intracellular colloidal dispersion (ICD). Examples of suitable macerating instruments include, but are not limited to, devices such as a crusher, grinding stone, or grinder (e.g., knife mill, hammer mill, etc.). To avoid temperature-induced degradation of the plant material, the maceration step may include temperature monitoring and selection of maceration parameters ensuring that there is no significant increase in the temperature of the plant material during this step. By “pressing” we mean the separation of the liquid material from fresh Granville® roses, and preferably from fresh petals, by application of mechanical force. This includes, but is not limited to, techniques such as ambient gravity drainage, pressing by a heavy object, centrifugal force from a rotating expeller, piston pressure from a hydraulic press, or rollers or a screw of the appropriate type for a press.

By “fiber-enriched material” or “MEF” or “FEM” (Fiber Enriched Material), we mean a solid and/or semi-solid fraction enriched with fresh Granville® rose fibers, preferably fresh petals, of which the Liquid intracellular colloid dispersion (ICD) was removed by pressing.

By “intracellular colloidal dispersion” or “DCI” or “ICD” (Intracellular Colloidal Dispersion) is meant the liquid material expelled by pressing fresh Granville® roses, preferably fresh petals. The resulting liquid contains dispersed solid and/or semi-solid particles and, potentially, water-immiscible liquid droplets of various sizes (collectively referred to as particles), in a contiguous aqueous medium. The particles mainly consist of plant cell organelles, organelle fragments and fiber-enriched residual materials. The aqueous medium is mainly made up of cytosols and vacuoles.

By “separation” we mean the separation of solid and/or semi-solid particles, and droplets of non-aqueous liquid from an aqueous liquid by exploiting the density and/or size of the particles. This includes, but is not limited to, techniques such as dewatering, filtration (including filtration using a pressure gradient), skimming, ambient gravity sedimentation, decantation, centrifugation, or a combination of the above. Preferably, continuous flow mechanical separation will be used, but this does not exclude batch processing. “Separation 1” and “Separation 2” refer to the respective steps of the process, carried out with respective parameters.

By "supernatant" is meant an aqueous material from which particles have been separated. “Supernatant A” and “Supernatant B” designate the supernatants resulting from the respective separation steps of the process.

By "precipitate" refers to particles from which aqueous material has been separated. “Fraction B” and “Fraction C” designate the precipitates resulting from the respective separation steps of the process. The terms “fresh Granville® rose serum fraction” and “fresh Granville® rose petal serum fraction” designate compositions produced by the process as shown above without preservatives and/or stabilizers added to protect the composition of the ingredient against environmental factors such as temperature, atmosphere (e.g., oxygen), light and microorganisms.

By “preservatives and/or stabilizers” we mean substances which, when added to a “fraction of fresh Granville® rose serum”, preferably a “fraction of fresh Granville® rose petal serum”, protect it against environmental factors such as temperature, atmosphere (e.g. oxygen), light and microorganisms. Particular suitable substances may include, without limitation, a preservative, a stabilizer and/or a mixture thereof.

The term “Granville® rose serum” or “fresh Granville® rose extract” as used herein means a combination of a fraction of fresh Granville® rose serum and preservatives and/or stabilizers.

The term “Granville® rose petal serum” or “Granville® fresh rose petal extract” as used herein means a combination of a fraction of Granville® fresh rose petal serum and preservatives and /or stabilizers. In a preferred embodiment, the extract used according to the invention is obtained by implementing the extraction process disclosed in patents EP2919757, JP 6130924, CN

ZL201380057567.6, EP2491939B1, CN1929851 B, US Patents No. 8,734,861 and No. 7,473,435; US patent application No. 16/078925.

Preferably, rose flowers (Roses de Granville®) and 4 to 5 cm of stem are harvested in such a way as to avoid chopping or crushing the collected biomass to avoid disruption of the cellular structure of the flowers. Collected plants can be tested for viability using an OS5p multi-mode chlorophyll fluorometer (Opti-Sciences Inc, Hudson, NH, USA).

According to a particular embodiment the live fresh flowers, including the petals, the pistil and the stamen, have been removed from the stem, including the sepal and the receptacle, and packed in storage bags and are placed )s immediately at negative temperatures between -20°C and -80°C, such as -20°C, -40°C or -60°C. The flowers can thus be stored for long periods, such as several months, or several years, before being used for the extraction process. According to a preferred embodiment, the live fresh flowers, including the petals, pistil and stamen, are removed from the stem, including the sepal and receptacle, and packed into storage bags and placed in storage at a temperature between 0 and 20°C and preferably at a temperature between 2 and 6°C until the harvest is complete.

Step a

After the harvest is completed, the roses, preferably rose petals, are immediately rinsed by spraying with water at 10°C to 15°C for 0.1 to 0.3 minutes with a flow rate of 5 to 6 liters per minute. Excess water is preferably removed from the rinsed flowers by allowing them to drain for at least 1 minute. The rinsed flowers can then undergo maceration, pressing, and separation by mechanical, roller, hydraulic, or juice extractor pressing to extract the contents of the liquid intercellular colloidal dispersion (DC I) from the fiber-enriched material (“Fraction A ").

At the end of these steps, the yield of fraction A is between 30% and 65%, preferably between 35% and 60% and even more preferably between 40% and 55% (weight/weight).

The DCI typically comprises from 2% to 20% dry matter, preferably from 4% to 16% dry matter, and even more preferably from 6% to 12% dry matter.

At this stage, the ICD can be frozen for storage. Typically, it is frozen at -20°C.

Step b

When the ICD has been frozen for storage, it must first be thawed gently. Typically, it is placed to thaw at 4°C or in ice.

“Treatment A” is carried out by a destabilization treatment of the DCI with electromagnetic waves produced from magnetrons operating at a frequency between 2.45 and 5.8 GHz. The parameters of the destabilization treatment are set to obtain the reduction in the value of the real component of the low frequency dielectric constant (e'o) of approximately 20 Farads per meter (F/m) compared to its value before the treatment . This processing degrades the stability of the ICD by causing agglomeration and/or aggregation of particles (i.e., organelles, organelle fragments, residual fibrous material) into assemblies large and stable enough to allow and/or or improve mechanical separation. Indeed, we consider the intracellular colloidal suspension obtained as a relatively stable colloidal dispersion composed of a continuous phase (cytoplasm and vacuole contents) and a dispersed phase (suspended organelles and their fragments). According to the Derjaguin-Laundau-Verwey-Overbeek (DLVO) theory, this stability is maintained by the sum of the attractive van der Waals forces and the repulsive forces of the electrical double layers. The energy barrier resulting from the repulsive force prevents the particles of the dispersed phase from approaching unless they have enough energy to overcome this barrier, in which case the force of attraction will bring them into contact (they will adhere then irreversibly). DLVO theory describes the interaction and potential energy of particles based on their parameters, their distance from each other and the characteristics of the continuous phase. Changing the values of the variables affecting the repulsive force affects the stability of the dispersion. Under normal conditions of colloidal stability, an increase in potential energy as particles approach each other constitutes a potential energy barrier that cannot be overcome without external energy input. This energy barrier keeps the particles separated and the dispersion stable. The conditions modified during treatment A allow the repulsive force of the double layer to decrease to the point that there is no longer a potential energy barrier and the particles can approach and agglomerate freely.

Restoring the initial conditions does not restore stability, because the particles have irreversibly agglomerated. They are therefore easily removed by mechanical means (Koganov et al., sofw journal 2017).

Preferably, at the end of “treatment A”, the step of mechanical separation of the DCI is carried out by centrifugation in order to produce “Supernatant A” and “Fraction B”.

Typically, "supernatant A" has a turbidity below about 100 NTU.

“Fraction B” typically comprises from 10% to 30% dry matter, preferably from 13% to 27% dry matter, and even more preferably approximately 15.0% to 25.0% dry matter.

Step c

“Treatment B” is carried out by adjusting the pH level in the “supernatant A” by titration with, for example, an alkali until a pH greater than 6 is obtained, preferably a pH ranging from 6.5 to 7.5. Typically, potassium carbonate will be used as the preferred alkali.

At the end of “treatment B”, the step of mechanical separation of “Supernatant A” is preferably carried out by centrifugation in order to produce “Supernatant B” and “Fraction C”.

The “fraction C” typically comprises from 5% to 25% dry matter, preferably from 8% to 22% dry matter, and even more preferably approximately 10.0% to 20.0% dry matter.

Step d

“Treatment C” is carried out by adjusting the pH level in the “supernatant B”, in particular by titration with, for example, acid until a pH lower than 4.5 is obtained. Typically, a solution of citric acid will be used as the preferred acid.

At the end of “treatment C”, the step of mechanical separation of “Supernatant B” is preferably carried out by centrifugation in order to produce the “fresh Granville® rose serum fraction”, preferably the “serum fraction”. of fresh Granville® rose petals” (Unpreserved Extract) and “Fraction D”.

The “Granville® fresh rose petal serum fraction” typically comprises from 2% to 20% dry matter, preferably from 4% to 16% dry matter, and even more preferably from 6.0% to 10.0% dry matter.

Step e

According to certain embodiments, the serum fraction obtained at the end of step d) is mixed with at least one preservative or at least one stabilizer to give a finished ingredient, or with a combination of these to give the fresh Granville® rose extract or “Granville® Rose Serum”, preferably extract of fresh Granville® rose petals or “Granville® Rose Petal Serum”.

Particularly suitable stabilizing agents may include, without limitation, a preservative, a stabilizer and/or mixtures thereof. Suitable preservatives and stabilizers for use in the present invention include, but are not limited to, potassium sorbate, sodium benzoate, sodium metabisulfite, glycerin, propylene glycol, dipropylene glycol, butylene glycol, pentylene glycol, hexylene glycol and caprylyl glycol. In a particular embodiment, the stabilizing agents can comprise at least one preservative, at least one stabilizer, at least one antioxidant or mixtures thereof.

Thus, according to one embodiment, the bioactive fraction isolated from roses and used according to the invention is obtained by the process comprising the steps of: a) cleaning the plant material, maceration, pressing then mechanical separation of the plant material to obtain a dispersion intracellular colloid (DCI) and a fiber-enriched material (fraction A); b) destabilization of the DCI with electromagnetic waves then mechanical separation of the intracellular colloidal dispersion (DCI) to obtain supernatant A and a Membrane Fraction (fraction B); c) adjustment of the pH level in supernatant A until obtaining a pH greater than 6, preferably a pH ranging from 6 to 7, then mechanical separation of supernatant A to obtain supernatant B and fraction C (cytoplasmic fraction) ; d) adjustment of the pH level in the supernatant B until obtaining a pH lower than 4.5 then mechanical separation of the supernatant B to give a “Bioactive serum fraction” and a fraction D (precipitate); and e) Optionally, mixing the “bioactive serum fraction” with at least one preservative and/or stabilizer.

According to a particular mode, the bioactive fraction isolated from roses, preferably from rose petals, used according to the invention comprises water in a content ranging from 90 to 94%, a dry extract content ranging from 6 to 10% . This bioactive fraction isolated from roses used according to the invention is called INCI name Rosa Hybrid Flower Extract.

According to another particular mode, the bioactive fraction isolated from roses, preferably from rose petals, obtained according to the invention comprises water in a content ranging from 89 to 93%, a dry extract content ranging from 6 to 10 % and preservatives and/or stabilizers in a content ranging from 0.3 to 1% by weight relative to the total weight of the extract.

The bioactive fraction isolated from roses may be present in the composition of the invention in a content ranging from 0.1 to 1% by weight, in particular from 0.2 to 0.5% by weight relative to the total weight of the composition.

Thus, in a particular embodiment, the cosmetic composition according to the invention comprises: 1. an aqueous extract of rosewood of the Evanrat variety in a content ranging from 0.1 to 1%, in particular from 0.2 to 0.5% by weight relative to the total weight of the composition,

2. a hexapeptide with the INCI name: Acetyl Hexapeptide-8 in a content ranging from 0.05% to 0.25% by weight (of peptide) relative to the total weight of the composition,

3. a tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7 in a content ranging from 0.1% to 0.25% by weight (of peptide) relative to to the total weight of the composition, and

4. a bioactive fraction isolated from fresh roses of the Evanrat variety, in a content ranging from 0.1 to 1%, in particular from 0.2 to 0.5% by weight relative to the total weight of the composition.

Cosmetic and galenic composition

By “cosmetic composition” is meant any composition for cosmetic purposes, that is to say aesthetic, which can be brought into contact with the superficial parts of the human body and more particularly with keratin materials, in particular the skin and/or the lips, particularly the skin of the face and/or neck.

By “physiologically acceptable medium” is meant any excipient suitable for topical use, in contact with keratin materials, without risk of toxicity, incompatibility, instability and/or allergic response.

The physiologically acceptable medium generally represents 1 to 99% by weight, relative to the total weight of said composition.

By “topical use” or “topical application” is meant a composition intended for application to keratin materials. In fact, an oral composition is not intended for topical application to the skin.

According to a particular embodiment, the cosmetic composition according to the invention is characterized in that it is a care and/or makeup composition for the skin and/or lips, in particular a composition skin care. A composition for topical application according to the invention may for example be in the form of a cream, oil-in-water emulsion, or water-in-oil or multiple emulsion, solution, suspension, gel, milk, lotion, serum , balm, stick, or even powder.

According to a particular embodiment, the cosmetic composition of the invention is in the form of a cream, oil-in-water emulsion, or water-in-oil or multiple emulsion, solution, suspension, gel, milk, lotion, or serum .

In a preferred embodiment, said composition used according to the invention is in the form of a cream or a serum.

The aqueous phase of the composition according to the invention comprises water and optionally a water-soluble solvent.

By 'water-soluble solvent' according to the invention is meant a compound that is liquid at room temperature and miscible with water (miscibility in water greater than 50% by weight at 25°C and atmospheric pressure). We can cite in particular: lower C1 -C5 mono-alcohols such as ethanol, isopropanol and their mixtures; C2-C8 glycols such as ethylene glycol, propylene glycol, 1,3-butylene glycol, dipropylene glycol, and mixtures thereof; C2-C32 polyols such as polyglycerols, polyethylene glycols, and mixtures thereof, and mixtures thereof.

It may also include hydrophilic gelling agents, antioxidants, preservatives and mixtures thereof.

The cosmetic composition according to the invention may also comprise a fatty phase (solid fatty substances) or oily phase.

By “oil phase” is meant an oil or a mixture of oils which may or may not be miscible with each other. By “oil” is meant, within the meaning of the invention, a fatty substance, not soluble in water, liquid at 25°C and atmospheric pressure. These oils can be volatile or non-volatile, vegetable, mineral or synthetic.

An oily phase according to the invention may comprise natural, hydrocarbon, silicone oils, and mixtures thereof. The content of fatty or oily phase in the cosmetic composition of the invention will generally range from 0.2% to 45%, preferably from 0.5% to 30%, and more preferably from 2% to 25% by weight per relative to the total weight of said composition.

The composition of the invention may also comprise any additive usually used in cosmetics such as antioxidants, perfumes, cosmetic active agents, such as for example emollient agents, moisturizing agents, vitamins, anti-aging agents, lifting agents, tightening agents, plumping agents, lightening agents, fillers, pearls and their mixtures.

Thus according to a particular embodiment, the invention relates to a cosmetic composition for topical application to the skin and/or lips, in particular the skin of the face and/or body, comprising in a physiologically acceptable medium, at least one aqueous extract of rosewood, Acetyl Hexapeptide-8, Palmitoyl tetrapeptide-7 and optionally an isolated bioactive fraction obtained from fresh rose, and at least one cosmetic adjuvant chosen from the group consisting of antioxidants, perfumes , emollient agents, moisturizing agents, vitamins, anti-aging agents, lifting agents, tightening agents, plumping agents, lightening agents, fillers, pearls and mixtures thereof.

Cosmetic process

The invention also relates to a non-therapeutic cosmetic process for caring for and/or making up the skin and/or lips comprising the application to the skin and/or lips, in particular the skin, of a composition such as defined according to the invention.

By “skin and/or lips” according to the invention, we mean in particular healthy skin and/or lips, that is to say not presenting disorders or disorders which would relate to a pathological state (subjects ' unhealthy', suffering from a pathology).

According to a particular mode, the application of the composition as defined according to the invention to the skin and/or lips makes it possible to prevent and/or reduce the signs of skin aging, in particular the loss of density and/or firmness. of the skin, the reduction in the thickness of the epidermis, the reduction in the differentiation and/or proliferation of epidermal cells, and/or the appearance of wrinkles and/or fine lines. Association of assets and uses

The present invention also relates to a combination of cosmetic active ingredients comprising:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8,

The invention also relates to the non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined above, as an active association for preventing and/or reducing the signs of skin aging, in particular loss of density and/or or firmness of the skin, the reduction in the thickness of the epidermis, the reduction in the differentiation and/or proliferation of epidermal cells, and/or the appearance of wrinkles and/or fine lines.

Cosmetic set and use

The present invention also relates to a cosmetic set comprising:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8,

(iii) A tetrapeptide with INCI name: Palmitoyl Tetrapeptide-7, and

(iv) Optionally also an isolated bioactive fraction obtained from fresh roses of the Evanrat variety and comprising at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose, and/or at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium, characterized in that at least one of the active ingredients ( i) to (iv) is packaged in a composition distinct from the other active ingredients, and in particular each active ingredient is packaged in a distinct composition.

Another object is a cosmetic process comprising the successive application, to the skin and/or lips, of the compositions of the cosmetic set as defined above.

The invention will now be illustrated in the following non-limiting examples. Unless otherwise indicated, percentages (%) are expressed as percentages (%) by weight relative to the total weight of the composition.

EXAMPLES

Example 1: Materials and methods 1.1 Preparation of cell cultures

The various tests of the ingredients alone or in combination according to the invention were carried out on samples of skin reconstructed by bioprinting by extrusion with cells from a donor of mature age (= 42 years). Cells used

All of the cells used were extracted by LabSkin Creations from surgical residues from donors who underwent plastic or reconstructive surgery. The collection of these operational residues was obtained according to current French regulations and authorized by the Ministry of Research under number DC2020-4346. In order to guarantee their use in tissue engineering, primary cell banks were produced at different passages depending on the cell type considered. The cells have been microbiologically safe and are free of mycoplasma. [Table 1]

Bioprinting and culture of reconstructed skins

Dermis bioprinting

Fibroblasts from elderly donors were trypsinized and counted. The cells were suspended in the bioink consisting of alginate, gelatin and fibrinogen (LabSkin Creations) at a rate of 250,000 cells per mL of ink. The bioinks were transferred into sterile syringes mounted on 10 gauge cannulas.

The printed objects were modeled with the software Slic3r (GNU Affero General Public License) and Repetier (Hot-World GmbH & Co. KG). The dermis were printed using a 3D printer (LabSkin Creations) to a size of 1.5cm x 1.5cm and had two layers of cells extruded into the bioink.

The constructs were then polymerized for 1 hour in a solution of calcium and thrombin (LabSkin Creations) allowing the consolidation of the bioink. After three rinses in a saline buffer, the equivalent dermis were cultured in a container of DMEM supplemented with 10% calf serum and 1% antibiotics and antifungal and placed in the incubator (37°C, 5 % CO2) for 12 days. The culture medium was renewed every 2 days.

Epidermization

Confluent keratinocytes were trypsinized and then counted. The equivalent of 500,000 cells was seeded on the surface of each bioprinted dermis. After 30 minutes of adhesion at 37°C, culture medium containing DMEM supplemented with 10% fetal bovine serum, hydrocortisone and insulin was added to submerge the bioprinted dermo-epidermal constructs.

Maturation and differentiation

The bioprinted dermo-epidermal assemblies were cultured for 7 days in culture medium containing DMEM supplemented with 10% fetal calf serum, hydrocortisone and insulin allowing the proliferation of keratinocytes. They were then raised at the air/liquid interface in order to initiate the differentiation of the epidermis. This final 7-day culture step made it possible to obtain multistratified epidermis.

Treatments

Treatments with active ingredients alone or in combinations were dissolved in the culture media. The final concentrations of active ingredients are specified for each example.

Preparation of rosewood extract:

Rosewood of the Evanrat variety, in particular of the Jardin de Granville® rose variety, possibly with the addition of rose flowers, was reduced to powder in three stages.

Step 1: passage through a garden shredder to obtain sections of two centimeters Step 2: passage through an industrial shredder

Dissolution

The enzymatic extraction was carried out in a reactor with a maximum capacity of 70 I. The reactor is stirred by propeller and is thermostatically controlled by a double jacket.

16Kg of rosewood powder with 0.1% rose flowers are dispersed and suspended in 47.5Kg of water.

To deactivate endogenous enzymes, the mixture was raised to a temperature of 85°C for 1 min and then stabilized at 50°C.

Lysis of plant walls

0.8 Kg of an HEL1 PR1 Enzymatic cocktail comprising a mixture of cellulases, hemicellulases and pectinases at 75% and proteases at 25%, were added to the corresponding reaction mixture (5% w/w). The reaction is continued for 4 hours, the temperature is maintained at 50°C.

Inactivation of enzymes

The mixture was heated to 85°C for 1 min to inactivate the enzymes, then returned to 35°C.

Pre filtration

The mixture is pre-filtered through a 0.8mm then 0.5mm sieve in order to remove the most important part of the solid phase, then on a sheet. Sterilizing filtration

Filtration was carried out on clarifying plates (PALL corporation) with a pore size of 0.2 pm, at a pressure of 2 Bars.

Formulation

The aqueous phase obtained after sterilizing filtration is stabilized with the following preservatives:

- Citric acid,

- Potassium sorbate and

- Sodium benzoate.

After filtration, an aqueous extract of rosewood is obtained with 3.7% by weight of dry matter relative to the total weight of the extract.

Preparation of the active fraction of fresh roses (ZETA fraction):

We use fresh rose petals of the Evanrat variety or “Jardin de Granville®” rose as plant material.

The bioactive fraction of rose petals used according to the invention is obtained according to the following protocol: a1) cleaning the fresh rose petals by spraying with water at a temperature of approximately 12°C, for 0.1 to 0.3 minutes at a flow rate of 5 to 6 liters per minute, a2) maceration, pressing then mechanical separation of the roses using a mechanical screw press (model CP-6 Vincent Corporation, FL) to extract the contents the liquid intercellular colloidal dispersion (DCI) of the fiber-enriched material (“Fraction A”); b1) destabilization of the DCI with electromagnetic waves produced from magnetrons operating at a frequency between 2.45 and 5.8 GHz, b2) centrifugation of the intracellular colloidal dispersion (DCI) to obtain the supernatant A and a Membrane Fraction ( fraction B); c1) adjustment of the pH level in the supernatant A using an alkali (Le., potassium carbonate), in order to obtain a pH ranging from 6 to 7, c2) mechanical separation of the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction); d1) adjustment of the pH level in the supernatant B with citric acid in order to obtain a pH lower than 4.5, d2) mechanical separation of the supernatant B to give a “fraction of fresh Granville® rose petal serum” and a fraction D (precipitate); and e) mixing “Granville® fresh rose petal serum fraction” with potassium sorbate and sodium benzoate.

Granville® rose petal serum (also called “Zeta serum”, or “Zf serum” or “ZETA FRACTION” or “ZETA fraction” in the following examples) contains 8% dry matter (active ingredient), 91.5 % by weight water, 0.15% by weight potassium sorbate and 0.3% by weight sodium benzoate. These contents are by weight relative to the total weight of the extract. The INCI name for this extract is Rosa Hybrid Flower Extract, Potassium Sorbate, and Sodium Benzoate.

Acetyl hexapeptide 8 from the company LIPOTEC is marketed under the name ARGIRELINE® Amplified peptide with INCI name: WATER and ACETYL HEXAPEPTIDE-8 and SODIUM BENZOATE. In the examples, “Acetyl hexapeptide 8” means the commercial product ARGIRELINE® Amplified peptide comprising 0.05% peptide.

The percentages indicated in the examples correspond to the percentages by weight of commercial product in culture media or in cosmetic formulas.

Palmitoyl tetrapeptide-7 is a synthetic peptide with the sequence Palmitoyl-Gly-GIn-Pro-Arg (Palmitoyl-GQPR) from the company SEDERMA marketed under the name RIGIN® with the INCI name: Glycerin, Lactic Acis and Palmitoyl Tetrapeptide-7. In the examples, “Palmitoyl tetrapeptide-7” means the commercial product RIGIN® comprising 0.05% peptide. The percentages indicated in the examples correspond to the percentages by weight of commercial product in culture media or in cosmetic formulas.

The concentrations of active ingredients or combinations were selected based on the pre-performed cytotoxicity results.

The reconstructed skins were treated during dermal reconstruction, from D5 to D 12 and epidermal from D14 to D18 then from D20 to D26.

Stopping cultivation

The culture of samples from the 12 conditions was stopped on day 26. Half of the samples were fixed in 4% buffered formalin solution (Alphapath, France) then embedded in paraffin. 1.2 Methods for analyzing skin samples reconstructed by bioprinting after testing

The different treatments (active ingredients alone or in combination) were tested on the skin samples reconstructed by bioprinting according to Example 1. Different analyzes were carried out on these skin samples reconstructed by bioprinting to determine the effect of each treatment on the skin.

Hematoxylin-Phloxine-Saffron coloring

Paraffin sections of 5 μm were made on the different skin samples reconstructed by bioprinting. After deparaffinization and rehydration, the samples were stained with Hematoxylin, Phloxine and Saffron (HPS) in three successive 5 min baths. After rinsing, the sections were dehydrated before being mounted on coverslips with a hydrophobic xylene-based mounting medium.

Immunofluorescence on paraffin sections

All markings were made on sections 5 pm thick. After unmasking with heat, a saturation step in PBS/BSA 5% made it possible to block the non-specific antigenic sites. The cells were then incubated overnight with a primary antibody specific for the antigen of interest:

- Anti-Ki-67 antibody: 1/50th monoclonal, mouse, Dako;

- Anti-Collagen VII antibody: 1/50th, monoclonal, mouse, Santa Cruz.

The next day, after rinsing steps, the sections were incubated with an anti-mouse Alexa-Fluor 568 secondary antibody diluted 1/1000 (Thermo Fisher Scientific) and Hoechst nuclear dye (Thermo Fisher Scientific). A negative control without primary antibody was carried out in parallel.

Image acquisition

Histological staining and immunohistological markings were observed with the Axio Observer D1 optical microscope / Axiocam high-resolution camera system (Zeiss, Le Pecq, France). Images were acquired using ZEN pro 2012 software and saved in “tif” format (high resolution).

Image analysis

Image analysis was carried out with Imaged software (Rasband, WS, Imaged, US National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2017). For each parameter a statistical analysis was carried out with a Student test, which is significant if its value is less than 0.05. The results are indicated according to: * P < 0.05, ** P < 0.01 and *** P < 0.001.

Thickness measurement

The thickness of the cohesive layers of the epidermis is measured using a Euclidean distance map. The pixels corresponding to the epidermis are segmented from the other pixels of the image. The segmented image is then binarized and converted into a Euclidean distance map. The basement membrane of the epidermis is selected and applied to the distance map to measure at each point the distance between the basement membrane and the terminal layers of the epidermis. The value obtained then corresponds to the average distance between the basement membrane and the terminal layers of the epidermis. 9 images are analyzed for each condition.

Example 2: Effects of the active ingredients and combinations according to the invention on the KI67 factor The effect of the active ingredients alone or in combinations according to the invention on the production of the KI67 factor by the skin samples reconstructed by bioprinting was analyzed. Each test was performed in triplicate.

The skin samples reconstructed by bioprinting were tested respectively with the following active ingredients and combinations (dissolved in the culture media): the aqueous extract of rosewood alone (final concentration 0.07%); a mixture of hexapeptide Acetyl Hexapeptide-8 (final concentration 0.62pg/ml) and tetrapeptide Palmitoyl Tetrapeptide-7 (final concentration 0.19%); a mixture of hexapeptide Acetyl Hexapeptide-8 (final concentration 0.62pg/ml) and tetrapeptide Palmitoyl Tetrapeptide-7 (final concentration 0.19%) + aqueous extract of rosewood (final concentration 0.025%).

An untreated negative control was carried out, without composition according to the invention.

Results

The results are shown in Figure 1. The significance of the differences observed between the different conditions tested is measured by the Student test, which is said to be significant if its value is less than 0.05 (* p<0.05; ** p<0.01; *** p<0.001); ns= not significant. The mixture of hexapeptide Acetyl Hexapeptide-8, tetrapeptide Palmitoyl Tetrapeptide-7, and aqueous extract of rosewood helps stimulate KI67, a marker of cell proliferation, at the level of the epidermis. This result demonstrates that this association stimulates the proliferation of the basal layers of the epidermis and therefore promotes its renewal.

Example 3: Effects of active ingredients and combinations according to the invention on the thickness of the dermis

The effect of the active ingredients alone or in combinations according to the invention on the thickness of the dermis of skin samples reconstructed by bioprinting was analyzed. Each test was performed in triplicate.

Results

The results are represented in Figure 2 The significance of the differences observed between the different conditions tested is measured by the Student test, which is said to be significant if its value is less than 0.05 (* p<0.05; ** p <0.01; ***p<0.001); ns= not significant. These results demonstrate that the mixture containing the hexapeptide Acetyl Hexapeptide-8, the tetrapeptide Palmitoyl Tetrapeptide-7, and the aqueous extract of rosewood increases the thickness of the epidermis. This association is therefore very interesting to combat the thinning of the epidermis observed with age.

All of these results show that the combined use of an aqueous extract of rosewood, an acetyl hexapeptide-8, a palmitoyl tetrapeptide-8 and possibly also a bioactive fraction isolated from petals of roses, allows you to obtain a synergistic anti-aging effect. Example 4: Effects of active ingredients alone or in combinations according to the invention on Collagen VII

The effect of the active ingredients alone or in combinations according to the invention, on the production of Collagen VII by skin samples reconstructed by bioprinting, was analyzed. Each test was performed in triplicate.

The skin samples reconstructed by bioprinting were tested respectively with the following active ingredients and combinations according to the invention (dissolved in the culture media): the bioactive fraction isolated from fresh roses, otherwise called ZETA fraction, alone (final concentration 0.05% ) ; a mixture of hexapeptide Acetyl Hexapeptide-8 (final concentration 0.21 g/ml) and tetrapeptide Palmitoyl Tetrapeptide-7 (final concentration 0.06%) + aqueous extract of rosewood (final concentration 0.005%); a mixture of hexapeptide Acetyl Hexapeptide-8 (final concentration 0.10pg/ml) and tetrapeptide Palmitoyl Tetrapeptide-7 (final concentration 0.003%) + aqueous extract of rosewood (final concentration 0.004%) + ZETA fraction ( final concentration 0.003%).

An untreated negative control was carried out, without active ingredients or combinations of active ingredients according to the invention.

Results

The results are represented in Figure 3. The significance of the differences observed between the different conditions tested is measured by the Student test, which is said to be significant if its value is less than 0.05 (* p<0.05; ** p<0.01; *** p<0.001); ns= not significant. These results show that the mixture containing the hexapeptide Acetyl Hexapeptide-8, the tetrapeptide Palmitoyl Tetrapeptide-7, the aqueous extract of rosewood and the ZETA fraction very significantly and synergistically stimulates the expression of type collagen. VII, essential for the good structure of the skin. Indeed, collagen VII is an essential component of the extra cellular matrix, and gives the skin its firmness. This association is therefore very advantageous for preventing the loss of expression of collagen VII observed with aging, and therefore combating the signs of aging.

Example 5: Cosmetic formulations according to the invention 5.1 Composition in the form of an emulsion

Demineralized water qsp 100.0%

Glycols 20.0%

Preservatives 0.6%

Chelating 0.04%

Carbomer (Carbopol® 981) 0.3%

Sodium polyacrylate (Covacryl® MV60) 0.2%

Sodium Hydroxide 0.15%

Aqueous extract of rosewood * 1%

Acetyl hexapeptide-8 (ARGIRELINE® Amplified peptide comprising 0.05% peptide) 3% (i.e. 0.0015% peptide)

Palmitoyl tetrapeptide-7 (RIGIN® comprising

0.05% peptide) 4% (i.e. 0.002% peptide)

Vegetable oil, esters 16%

Anti-oxidant 0.2%

Perfume concentrate 0.4%

Steareth-2 0.8%

Steareth-21 1.5%

* as described in example 1

Applied to the skin, in particular the skin of the face, this composition gives tone, elasticity and firmness, the features are enhanced, the face is reshaped.

5.2: Composition in the form of a rich facial cream Demineralized water qsp 100.0%

Glycols 13.0%

Carbomer (Carbopol® 981) 0.3%

Sodium polyacrylate (Covacryl® MV60) 0.2%

Sodium Hydroxide 0.15%

Aqueous extract of rosewood * 0.5%

ZETA fraction ** 0.3%

Acetyl hexapeptide-8 (ARGIRELINE® Amplified peptide comprising 0.05% peptide) 2% (i.e. 0.001% peptide) Palmitoyl tetrapeptide-7 (RIGIN® comprising

0.05% peptide) 3% (i.e. 0.0015% peptide)

Vegetable oil, esters 16%

Triglycerides of fatty acids 4%

Shea butter 1%

Antioxidant 0.2%

Perfume concentrate 0.4%

Steareth-2 0.8%

Steareth-21 1.5%

Conservatives qs

* and ** as described in Example 1

Applied to the skin of the face, this composition gives tone, elasticity and firmness, the features are enhanced, the face is reshaped.

5.3: Composition in the form of a gel-serum for the eye contour

Purified water Qsp 100.00%

Glycols 13.0%

Preservatives 0.60%

Carbomer 0.80%

Glyceryl stearate citrate 0.70%

Lecithin and sodium acrylates copolymer (Lecigel PCR negative) 1.20%

Isostearyl isostearate 9.0%

Bis-diglyceryl polyacyladipate-2 (Softisan 649 MB) 1.0%

Silica 2.0%

Mother-of-pearl 1.0%

Centella asiatica extract 0.5%

Horse chestnut extract 0.1%

Aqueous extract of rosewood * 0.5%

Palmitoyl tetrapeptide-7 (RIGIN® comprising 0.05% peptide) 4% (i.e. 0.002% peptide)

ZETA Fraction ** 0.3% Tocopheryl acetate 0.10%

Rose scent 0.20%

* and ** as described in example 1 After application to the eye contour, this gel-serum helps to smooth away wrinkles and fine lines, the eye area is firmed. The youthfulness of the eyes is restored over the course of applications.

Claims

1. Cosmetic composition comprising, in a physiologically acceptable environment:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8, and

2. Cosmetic composition according to claim 1, characterized in that the aqueous extract of rosewood is obtained by an enzymatic extraction process comprising the steps: a) addition of water to the previously crushed rosewood having a size of appropriate particle, b) addition of an enzymatic mixture containing at least one cellulase, at least one hemicellulase and at least one pectinase, and advantageously also at least one protease, c) incubation with stirring of the previously ground rosewood and the mixture enzymatic to release fermentable oils, proteins and sugars into the reaction medium, for a period depending on the desired yields, d) separation of the reaction medium to obtain free oil, an aqueous phase containing fermentable proteins and sugars, and a solid phase, e) separation of the aqueous phase and possible addition of preservatives.

3. Cosmetic composition according to claim 1 or claim 2, characterized in that it further comprises a rose extract in the form of an isolated bioactive fraction obtained from fresh roses of the Evanrat variety and comprising at least 500 pg/pL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose, and/or at least 500 mg/Kg of at least one mineral chosen from potassium, calcium and sodium.

4. Cosmetic composition according to claim 1 or 2, characterized in that: (i) The aqueous extract of rosewood of the Evanrat variety is present in a content ranging from 0.1% to 1%, in particular from 0.2% to 0.5% by weight relative to the total weight of the composition,

(ii) The hexapeptide with INCI name: Acetyl Hexapeptide-8 is present in a content ranging from 0.05% to 0.25% by weight relative to the total weight of the composition, and

(iii) The tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7, is present in a content ranging from 0.1% to 0.25% by weight relative to the total weight of the composition.

5. Cosmetic composition according to claim 3, characterized in that:

(i) The aqueous extract of rosewood of the Evanrat variety is present in a content ranging from 0.1% to 1%, in particular from 0.2% to 0.5% by weight relative to the total weight of the composition,

(ii) The INCI name hexapeptide: Acetyl Hexapeptide-8 is present in a content ranging from 0.05% to 0.25% by weight relative to the total weight of the composition,

(iii) The tetrapeptide Pal-GQPR-NH2 or Pal-GQPR-OH, in particular the tetrapeptide with the INCI name: Palmitoyl Tetrapeptide-7, is present in a content ranging from 0.1% to 0.25% by weight relative to the total weight of the composition, and

(iv) The bioactive fraction isolated from fresh roses of the Evanrat variety is present in a content ranging from 0.1 to 1% and in particular from 0.2 to 0.5% by weight relative to the total weight of the composition .

6. Cosmetic composition according to any one of claims 1 to 5, characterized in that it is a care and/or makeup composition for the skin and/or lips, in particular a composition skin care.

7. Non-therapeutic cosmetic process for caring for and/or making up the skin and/or lips comprising the application to said skin and/or said lips, in particular the skin, of a composition as defined in one any of the claims

8. Cosmetic process according to claim 7, characterized in that the application of the composition to the skin and/or lips makes it possible to prevent and/or reduce the signs of skin aging.

9. Combination of cosmetic active ingredients including:

(ii) A hexapeptide with the INCI name: Acetyl Hexapeptide-8,

10. Non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined in claim 9, as an active association for preventing and/or reducing the signs of skin aging, in particular the loss of density and/or firmness of the skin. skin, the reduction in the thickness of the epidermis, the reduction in the differentiation and/or proliferation of epidermal cells, and/or the appearance of wrinkles and/or fine lines.