FR3143365A1 - ANTI-AGING COMPOSITION WITH EXTRACTS OF ROSE BUDS AND PETALS AND PEPTIDE - Google Patents

Abstract

The present invention relates to a cosmetic composition comprising, in a physiologically acceptable medium: (i) an extract of rose buds of the Evanrat variety, (ii) an extract of rose petals of the Evanrat variety, and (iii) a synthetic dipeptide, in particular the dipeptide of INCI name: dipeptide-4.

Description

ANTI-AGING COMPOSITION WITH EXTRACTS OF ROSE BUDS AND PETALS AND PEPTIDE 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 neck.

STATE OF THE ART

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

Aging causes changes in the skin and/or lips, such as wrinkles and fine lines, skin that has lost its density and elasticity, a decrease in the thickness of the epidermis linked in particular to a slowdown in the renewal of skin cells. Aging is also associated with an alteration of the barrier function of the skin, which is then less well hydrated and can have a dull and uneven appearance.

A large number of anti-aging active ingredients are known from the prior art, such as peptides, vitamins, antioxidants and plant extracts. One example is dipeptide-4 from Ashland.

The use of certain rose extracts in anti-aging is also known, such as the extract of rose buds of the Evanrat variety described in patent application WO2019/20227 or the extract of rose petals of the Evanrat variety described in patent application FR2113885.

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 demonstrated in vitro a synergistic anti-aging effect between dipeptide-4, an extract of rose buds of the Evanrat variety and an extract of rose petals of the Evanrat variety.

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

(i) an extract of rose buds of the Evanrat variety,

(ii) an extract of rose petals of the Evanrat variety, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4.

Preferably, the cosmetic composition according to the invention is characterized in that the rose bud extract is obtained by a cryoextraction process comprising the steps of:

a) crushing the buds

b) extraction in a polar solvent,

c) clarification,

d) addition of glycerin,

e) optional addition of preservatives,

f) freezing, and

g) filtration.

Preferably, the cosmetic composition according to the invention is characterized in that the rose petal extract is in the form of an isolated bioactive fraction comprising at least 500 µg/µL 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.

Preferably, the cosmetic composition according to the invention is characterized in that:

(i) the extract of rose buds of the Evanrat variety is present in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition,

(ii) the extract of rose petals of the Evanrat variety is present in a content ranging from 0.01 to 5% by weight relative to the total weight of the composition, and

(iii) the synthetic dipeptide is present in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition.

Preferably, the cosmetic composition according to the invention is characterized in that it is a composition for caring for and/or making up the skin and/or lips, in particular a skin care composition.

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 composition as defined above.

Preferably, the cosmetic process according to the invention is 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, in particular to promote and/or improve the density and/or elasticity of the skin, to promote and/or improve the skin barrier function, to promote and/or improve the hydration of the epidermis, to preserve the thickness of the epidermis, and/or to prevent and/or avoid the appearance of a dull and uneven complexion.

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

(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,

(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4.

The present invention also relates to the non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined above, as an active combination for preventing and/or reducing the signs of skin aging, in particular promoting and/or improving the density and/or elasticity of the skin, promoting and/or improving the skin barrier function, promoting and/or improving the hydration of the epidermis, preserving the thickness of the epidermis, and/or preventing and/or avoiding the appearance of a dull and uneven complexion.

The present invention further relates to a cosmetic assembly comprising:

(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,

(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4,

characterized in that at least one of the active ingredients (i) to (iii) is packaged in a composition distinct from the other active ingredients, and in particular each active ingredient is packaged in a distinct composition.

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

DESCRIPTION OF FIGURES

protocol for studying the impact of the secretome of senescent keratinocytes treated with glyoxal on untreated keratinocytes.

Morphological analysis on bioprinted skins. (A) Cross-section of bioprinted skins stained with Masson's Trichome. (“CONTROL” = untreated condition; “STRESS” = glyoxal treatment; “STRESS + ROSAPEPTIDE PREMIER” = glyoxal treatment + complex according to the invention). (B) Measurement of epidermal thickness and dermis density under different conditions. (“CONTROL” = untreated condition; “ROSAP P” = treatment with the complex according to the invention; “GO” = glyoxal treatment; “GO-ZETA” = glyoxal treatment + rose petal extract; “GO-ZETA+BUD” = glyoxal treatment + rose petal extract + rose bud extract; “GO-ROSAP P” = glyoxal treatment + complex according to the invention; “GO-REFERENCE” = glyoxal treatment + aminoguanidine).

Immunohistological analysis of markers present in the epidermis on bioprinted skins: Filaggrin, Transglumatinase I, and Aquaporin 3. (“CONTROL” = untreated condition; “GO” = glyoxal treatment; “GO-ZETA” = glyoxal treatment + rose petal extract; “GO-ZETA+BUD” = glyoxal treatment + rose petal extract + rose bud extract; “GO-ROSAP P” = glyoxal treatment + complex according to the invention; “GO-REFERENCE” = glyoxal treatment + aminoguanidine).

Immunohistological analysis of markers present in the dermis on bioprinted skin: Collagen I, Elastin. (“CONTROL” = untreated condition; “GO” = glyoxal treatment; “GO-ZETA” = glyoxal treatment + rose petal extract; “GO-ZETA+BUD” = glyoxal treatment + rose petal extract + rose bud extract; “GO-ROSAP P” = glyoxal treatment + complex according to the invention; “GO-REFERENCE” = glyoxal treatment + aminoguanidine).

Measurement of carbonylated proteins in the dermis and epidermis on bioprinted skins. (“CONTROL” = untreated condition; “GO” = glyoxal treatment; “GO-ZETA” = glyoxal treatment + rose petal extract; “GO-ZETA+BUD” = glyoxal treatment + rose petal extract + rose bud extract; “GO-ROSAP P” = glyoxal treatment + complex according to the invention; “GO-REFERENCE” = glyoxal treatment + aminoguanidine).

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 extract of rose buds of the Evanrat variety,

(ii) an extract of rose petals of the Evanrat variety, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4.

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 anciennes André Eve S.A.S” and protected by Plant Variety Certificate under No. 20110345 with the species name Rosa L. and the variety name EVANRAT. This shrub rose belongs to the group of modern hybrids, which, from May to October, is permanently covered in roses, thus showing an excellent remontant character.

Rose bud extract and preparation process

The buds are particularly rich in phytohormones and concentrate a significant number of molecules essential to the growth and development of the plant.

To take advantage of the unique properties of rose buds, the extract of rose buds of the Evanrat variety, used according to the invention, is obtained by a very low temperature extraction process. This patented process (EP0425391) is particularly suited to the fragile nature of the buds and allows the active molecules to be preserved.

Plant material

The extracts of the invention are extracts of rose buds of the Evanrat variety. Advantageously, it will be an extract of fresh rose buds.

The Rose of Granville blooms twice, in spring and at the end of summer. It is at the end of the winter months that the first blooming buds begin to form. They need several weeks to reach the final stage of their maturation, and it is during the month of May that these first blooming buds reach, just before opening, their maximum concentration of molecular active ingredients.

According to a particular and preferred embodiment, the invention uses “first blooming buds”, or “spring buds”. These buds are therefore preferably collected before the first flowering. In the description, we will speak indifferently of buds, rose buds, first blooming buds or spring buds.

In the context of the present invention, the rose buds may be picked mechanically or manually. In a preferred embodiment, the rose buds used to prepare the extract of the invention are harvested manually.

Advantageously, the development of bud growth is monitored daily so that the buds are harvested when they reach the final stage of their maturation, just before hatching. This harvest takes place over a period of approximately 15 days from the end of May to the beginning of June.

According to a preferred method, the harvested buds are stored immediately at -20°C in order to best preserve their richness in active molecules. Once the harvest is complete, the buds are transported at this same temperature, -20°C, to the place of their transformation where they are then directly used for extraction.

Extraction process

The plant material used is fresh rose buds of the Evanrat variety or “Jardin de Granville®” roses. These are in particular first-flowering rose buds of the Evanrat variety or “Jardin de Granville®” roses. Preferably, the rose buds have been stored directly at -20°C after harvest.

The process used to obtain the rose bud extract is a cryoextraction process, and more particularly the process described in patent application EP0425391.

The process implemented to obtain the rose bud extract also called “rose bud cryoextract” used according to the invention comprises the steps of:

a) crushing the buds

b) extraction in a polar solvent,

c) clarification,

d) Addition of glycerin,

e) optional addition of preservatives,

f) freezing, and

g) filtration.

Prior to the extraction itself, the rose buds may have been dried and/or crushed.

Step a

The bud grinding step is carried out at very low temperature. By “very low temperature” we mean that the temperature is below -20°C, preferably below -50°C and more preferably below -70°C.

According to a particular and preferred method, the rose buds, previously frozen and stored at -20°C, are further cooled to a temperature of -80°C, at which temperature they are then crushed to obtain a “cryo-crushed material”.

Step b

By “rose bud extract” is meant in particular that the polar (hydrophilic) compounds of the rose buds have been solubilized and/or extracted in a polar solvent.

By "polar solvent" we mean that the solvent has a polarity index value that 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 character of a molecule. For the polarity indices of solvents, please refer to the article by L.R. Snyder, 1974.

The preferred polar solvents are those consisting of a compound comprising at least one polar covalent bond of the O-H type. As a particularly preferred 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, is chosen.

The rose bud extract used according to the invention is advantageously obtained by means of a cosmetically acceptable polar solvent. Preferably, this process uses water, ethanol or their mixture, and more preferably water.

According to a particular embodiment, the plant material is extracted using a hydroalcoholic mixture, advantageously a mixture of water and ethanol. In particular, the plant material is extracted using a solvent consisting of water and ethanol, the ethanol representing from 50% v/v to 99% v/v of the water/ethanol mixture. Preferably, the plant material is extracted using a mixture comprising 30% v/v of water and 70% v/v of ethanol. The extraction can be carried out hot by reflux or by maceration at room temperature.

According to a particular and preferred embodiment, the polar solvent used to extract the plant material is water.

For the purposes of extraction, the cryo-ground material obtained in step a) is supplemented with water. A person skilled in the art will easily be able to adjust the amount of polar solvent, in particular water, to be added during the extraction step. Indeed, the amount of solvent may in particular be adjusted depending on whether the buds have been previously dried or not and depending on the desired final concentration of dry extract. This mixture is then kept stirring at a temperature of between 0 and +4°C.

For the purposes of this application, where ranges of values are stated, the extreme values, or limits, are included. Thus, by temperature between 0 and +4°C, it is meant that the temperature can be equal to 0°C or equal to 4°C, as well as any temperature between these two values.

This extraction step is preferably carried out for a duration of between 6 and 24 hours, preferably 8 to 15 hours and more preferably 10 to 12 hours.

According to certain embodiments, the extraction method implemented in the invention uses ultrasound during extraction in order to improve the mass yield of said extraction.

Advantageously, it is possible to reproduce the extraction and filtration cycle several times in order to exhaust the plant material of substances having an affinity for the extraction solvent.

Step c

Preferably, a clarification step is then carried out. This step, which separates the liquid phase from the exhausted plant material, can in particular be carried out by centrifugation, decantation or filtration.

Preferably, clarification is carried out by filtration.

The extraction process can further be supplemented by an optional step of partial or total removal of the extraction solvents. The extract can advantageously be concentrated by removing part of the solvent or the mixture of the extraction solvent. An aqueous concentrate can thus be obtained or, by removing all of the extraction solvent, a dry residue can be obtained.

Alternatively, the extract can, at this stage of the process, be freeze-dried or atomized to present itself in the form of a powder.

At the end of clarification step c), a “volume V” of “aqueous cryoextract” is obtained.

The dry matter content of the aqueous cryoextract is preferably between 0.1% and 5%, preferably between 0.3% and 3%, more preferably between 0.5% and 2%, in particular the “aqueous cryoextract” comprises approximately 1% of dry matter relative to the total weight of said aqueous cryoextract.

By “about” 1% is meant that the dry matter value may vary in small proportions, in particular in proportions of less than 20% and preferably in proportions of less than 10%. Thus, “about” 1% means between 0.8% and 1.2%, preferably between 0.9 and 1.1% of dry matter relative to the total weight of said aqueous cryoextract.

Step d

According to step d) of the extraction process, glycerin is added to the aqueous cryoextract. The volume of glycerin added is variable because it depends on the ‘volume V’ of aqueous cryoextract.

According to a preferred embodiment, the volume of glycerin added is between 0.5 times and 2 times the “volume V”, preferably between 0.7 times and 1.5 times the “volume V” and more preferably between 0.9 times and 1.1 times the “volume V” of aqueous cryoextract.

According to a preferred embodiment, the volume of glycerin added is equal to 1 time the “volume V” of aqueous cryoextract.

Step e

According to a particular embodiment, the extraction method implemented comprises an optional step e) of adding preservatives.

The person skilled in the art knows the preservatives suitable for cosmetic use. In particular, by way of example, and in a non-limiting manner, citric acid and/or sodium benzoate and/or potassium sorbate may be used.

Thus, according to a particular embodiment, said rose bud extract used according to the invention comprises at least one preservative chosen from citric acid and/or sodium benzoate and/or potassium sorbate and their mixtures.

Preferably, the total amount of preservatives is less than 5%, preferably less than 3% and more preferably less than 2% relative to the weight of the extract.

Step f

The extraction process used to obtain the extract also includes a step f) of refrigeration. This step, which aims to precipitate the insoluble residues, is carried out at a temperature between 0°C and 10°C, preferably between 2°C and 6°C and more preferably at 4°C.

Preferably, the freezing step is carried out for a period of between 6 and 72 hours, preferably between 12 and 60 hours and more preferably between 24 and 48 hours.

Step g

At the end of the refrigeration step, the extract is then filtered in order to separate the insoluble materials precipitated during step f). Preferably, the filtration step g) is carried out using a microfiltration membrane with a pore size of the order of 0.2 µm. Advantageously, microfiltration also makes it possible to sterilize the rose bud extract of the invention.

The product obtained from this extraction process is the rose bud extract used according to the invention, otherwise called “Cryoextract” or “Bud cryoextract” or even “BUD” in the illustrative examples presented below.

Preferably, the “bud cryoextract” is stored at low temperature, in particular at a temperature between 0°C and 10°C, preferably between 2°C and 6°C and more preferably at 4°C.

According to a particular and preferred embodiment, the “cryoextract” used according to the invention comprises from 0.1% to 3% by weight of dry matter (active matter) of rose bud extract, and 97 to 99.9% by weight of a 50/50 mixture of water and glycerol. Preferably, the ‘Cryoextract’ comprises 0.3 to 1.0% by weight of dry matter (active matter), and 99 to 99.7% by weight of a 50/50 mixture of water and glycerol.

According to a particular embodiment, the extract of rose buds of the Evanrat variety, used according to the invention, comprises from 0.5% to 1% (corresponding to 75µg/mL to 150µg/mL) of dry matter in a mixture of water/glycerol (50/50). The INCI name (International Nomenclature Cosmetics Ingredients) of this extract of rose buds is: Water, Glycerin, Rose Extract.

This extract of rose buds of the Evanrat variety may be present in the composition of the invention in a content ranging from 0.1% to 10% by weight, preferably from 0.5% to 5% by weight, and in particular from 1% to 3% by weight relative to the total weight of the composition. These percentages are expressed by weight of aqueous or hydroalcoholic extract (in other words by weight of raw material).

If we reason in terms of weight of dry extract, this extract of rose buds of the Evanrat variety may be present in the composition of the invention in a content ranging from 0.0005% to 0.1% by weight of dry extract (in other words by weight of active material), preferably from 0.0025% to 0.05% by weight of dry extract, and in particular from 0.005% to 0.03% by weight of dry extract relative to the total weight of the composition.

Rose petal extract and preparation process

In the context of the present invention, said rose petal extract is in the form of a bioactive fraction.

By “bioactive fraction” or “isolated bioactive fraction of roses” or “Serum Roses De Granville®”, “Zeta” or “Zeta fraction”, is meant an extract of roses of the Evanrat variety, or roses ‘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 rose petals as described in application FR3066388.

Plant material

The rose petal extract in the form of an isolated bioactive fraction used according to the invention can be prepared from fresh rose petals 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 isolated bioactive fraction of roses, preferably summer rose petals, and in particular petals from fresh roses from the first flowering.

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 µg/µL of at least one monosaccharide sugar chosen from fructose, glucose and sucrose.

By “mineral-rich” is meant an extract of roses in the form of an isolated bioactive fraction, wherein said extract comprises at least 500 mg/Kg of at least one mineral selected from potassium, calcium and sodium.

Thus, according to one embodiment, the composition according to the invention comprises an extract of rose petals in the form of an isolated bioactive fraction comprising at least 500 µg/µL 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 to fill skin cells with energy.

Extraction process

As plant material, fresh roses of the Evanrat variety or “Jardin de Granville®” roses are used. In particular, these are fresh petals of roses of the Evanrat variety or “Jardin de Granville®” roses.

Advantageously, said method does not require the addition of any solvent or exogenous 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 in fibers (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®” roses obtained at the end of this process, is a “bioactive serum fraction” or “bioactive fraction” within the meaning of the invention.

“Cleaning” means the removal of debris from fresh Granville® roses, and preferably fresh petals, prior to further processing, in a manner that avoids injury to the plant, or the removal of components of interest. For example, this may be accomplished by low-pressure rinsing with potable water under conditions where the runoff wash would not substantially contain plant pigments. Excess wash water is then removed from the washed plants.

By “maceration” is meant 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 maceration instruments include, but are not limited to, devices such as a crusher, grinder or mill (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 that ensure there is no significant increase in the temperature of the plant material during this step.

“Pressing” means the separation of liquid material from fresh Granville® Roses, and preferably fresh petals, by the 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 a type appropriate for a press.

“Fibre Enriched Material” or “MEF” or “FEM” means a solid and/or semi-solid fraction enriched with fibres from fresh Granville® roses, preferably fresh petals, from which the liquid intracellular colloidal dispersion (ICD) has been removed by pressing.

“Intracellular Colloidal Dispersion” or “ICD” means 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, “particles”), in a contiguous aqueous medium. The particles consist primarily of plant cell organelles, organelle fragments, and fiber-enriched residual material. The aqueous medium consists primarily of cytosols and vacuoles.

By "separation" is meant the separation of solid and/or semi-solid particles, and non-aqueous liquid droplets from an aqueous liquid by exploiting the density and/or particle size. 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. Continuous flow mechanical separation will preferably be used, but this does not preclude batch processing. "Separation 1" and "Separation 2" refer to the respective process steps, carried out with respective parameters.

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

By “precipitate” is meant the particles from which an aqueous material has been separated. “Fraction B” and “Fraction C” mean 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” refer to compositions produced by the process as depicted above without added preservatives and/or stabilizers to protect the ingredient composition from environmental factors such as temperature, atmosphere (e.g., oxygen), light and microorganisms.

By "preservatives and/or stabilizers" are meant substances which, when added to a "fresh Granville® rose serum fraction", preferably a "fresh Granville® rose petal serum fraction", 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 terms “Granville® Rose Serum” or “fresh Granville® Rose Extract” as used herein mean a combination of a fresh Granville® Rose Serum fraction and preservatives and/or stabilizers. This extract is referred to as “ZETA” in the illustrative examples presented below.

The terms “Granville® rose petal serum” or “Granville® fresh rose petal extract” as used herein mean a combination of a Granville® fresh rose petal serum fraction and preservatives and/or stabilizers. In a preferred embodiment, the extract used according to the invention is obtained by implementing the extraction method disclosed in EP2919757, JP 6130924, CN ZL201380057567.6, EP2491939B1, CN1929851B, US Patents Nos. 8,734,861 and 7,473,435; US Patent Application No. 16/078925.

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

In a particular embodiment, the fresh live flowers (i.e., just harvested), including the petals, pistil and stamen, have been removed from the stem, including the sepal and receptacle, and packed in storage bags and are immediately placed 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 purposes of the extraction process.

In a preferred embodiment, the fresh live flowers, including the petals, pistil and stamen, are removed from the stem, including the sepal and receptacle, and packed in 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 harvesting is complete.

Step a

After harvesting, the roses, preferably the rose petals, are immediately rinsed by spraying with 10°C to 15°C water for 0.1 to 0.3 minutes at 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 may then undergo maceration, pressing and separation by mechanical, roller, hydraulic pressing or juice extractor to extract the liquid intercellular colloidal dispersion (ICD) content of 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 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 more preferably from 6% to 12% dry matter.

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

Step b

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

“Treatment A” is performed 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 decrease in the value of the real component of the low-frequency dielectric constant (ε'o) by approximately 20 Farads per meter (F/m) compared to its value before the treatment. This treatment degrades the stability of the DCI by causing the agglomeration and/or aggregation of particles (i.e. organelles, fragments of organelles, residual fibrous material) into sufficiently large and stable assemblies to allow and/or improve mechanical separation.

Indeed, the resulting intracellular colloidal suspension is considered to be 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 electric double layers. The energy barrier resulting from the repulsive force prevents the particles of the dispersed phase from approaching each other unless they have enough energy to overcome this barrier, in which case the attractive force will bring them into contact (they will then adhere irreversibly). The DLVO theory describes the interaction and potential energy of the particles as a function of 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 altered conditions in treatment A allow the repulsive force of the double layer to decrease to the point where 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 and are thus easily removed by mechanical means (Koganov et al., 2017).

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

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

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

Step c

"Treatment B" is carried out by adjusting the pH level in "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 mechanical separation step of “Supernatant A” is preferably carried out by centrifugation in order to produce “Supernatant B” and “Fraction C”.

“Fraction C” typically comprises from 5% to 25% dry matter, preferably from 8% to 22% dry matter, and more preferably about 10.0% to 20.0% dry matter.

Step d

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

At the end of “treatment C”, the mechanical separation step of “Supernatant B” is preferably carried out by centrifugation in order to produce the “fraction of serum from fresh Granville® roses”, preferably the “fraction of serum from 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 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 thereof to give the extract of fresh Granville® roses or “Granville® Rose Serum”, preferably the extract of fresh Granville® roses 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 may include at least one preservative, at least one stabilizer, at least one antioxidant, or mixtures thereof.

Thus, according to one embodiment, the extract of rose petals of the Evanrat variety used according to the invention is obtained by the process comprising the 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 in fibers (fraction A);

b) destabilization of the DCI with electromagnetic waves then mechanical separation of the intracellular colloidal dispersion (DCI) to obtain the supernatant A and a Membrane Fraction (fraction B);

c) adjusting the pH level in supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating supernatant A to obtain supernatant B and fraction C (cytoplasmic fraction);

(d) adjusting the pH level in supernatant B until a pH below 4.5 is obtained, then mechanically separating 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 embodiment, the extract of rose petals of the Evanrat variety, used according to the invention, comprises water in a content ranging from 90 to 94%, and a dry extract content ranging from 6 to 10%. This extract, in the form of a bioactive fraction isolated from roses, used according to the invention is called in the INCI name “Rosa Hybrid Flower Extract”.

According to another particular embodiment, the extract of rose petals of the Evanrat variety, used 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.

This extract of rose petals of the Evanrat variety may be present in the composition of the invention in a content ranging from 0.01% to 5% by weight, preferably from 0.05% to 2% by weight, and in particular from 0.1% to 1% by weight relative to the total weight of the composition. These percentages are expressed by weight of aqueous extract (in other words by weight of raw material).

If we reason in terms of weight of dry extract, this extract of rose petals of the Evanrat variety may be present in the composition of the invention in a content ranging from 0.0006% to 0.5% by weight of dry extract (in other words by weight of active material), preferably from 0.003% to 0.2% by weight of dry extract, and in particular from 0.003% to 0.1% by weight of dry extract relative to the total weight of the composition.

Peptides

The compositions of the invention also comprise a dipeptide, and in particular a synthetic dipeptide, in particular a dipeptide with an INCI name comprising DIPEPTIDE-4. In the remainder of the description, the terms dipeptide and dipeptide-4 will be used interchangeably.

According to a preferred embodiment, this is the dipeptide from the company ASHLAND marketed under the name Quintescine™ IS with the INCI name: WATER and BUTYLENE GLYCOL and DIPEPTIDE-4. Quintescine™ IS comprises 0.01% by weight of dipeptide (active ingredient).

According to a particular embodiment, dipeptide-4 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 0.5% to 5% by weight, and in particular from 1% to 3% by weight relative to the total weight of the composition. These percentages are expressed as the weight of dipeptide solubilized in a solvent (in other words as the weight of raw material).

If we reason in terms of dipeptide weight (active material), this dipeptide may be present in the composition of the invention in a content ranging from 0.00001% to 0.001% by weight of material, preferably from 0.00005% to 0.0005% by weight, and in particular from 0.0001% to 0.0003% by weight of active material relative to the total weight of the composition.

Thus, the composition of the invention will generally comprise:

(i) the extract of rose buds of the Evanrat variety in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition,

(ii) the extract of rose petals of the Evanrat variety in a content ranging from 0.01 to 5% by weight relative to the total weight of the composition, and

(iii) the synthetic dipeptide in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition.

According to a particular and preferred embodiment, the composition of the invention comprises, in a physiologically acceptable medium:

(i) the extract of rose buds of the Evanrat variety described in patent application WO2019/20227,

(ii) the extract of rose petals of the Evanrat variety described in patent application FR2113885, and

(iii) dipeptide-4, in particular Quintescine™ IS marketed by the company ASHLAND.

According to another preferred embodiment, the composition of the invention comprises, in a physiologically acceptable medium:

(i) 0.1 to 10% by weight of the extract of rose buds of the Evanrat variety described in patent application WO2019/20227,

(ii) 0.01 to 5% by weight of the extract of rose petals of the Evanrat variety described in patent application FR2113885, and

(iii) 0.1 to 10% by weight of dipeptide-4, in particular Quintescine™ IS marketed by the company ASHLAND,

the percentages being expressed as a percentage by weight of raw material (respective extracts and dipeptide-4 solubilized in their solvents) relative to the total weight of the composition.

Cosmetic and galenic composition

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

By "physiologically acceptable medium" we mean 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 from 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 composition for caring for and/or making up the skin and/or lips, in particular a skin care composition.

A composition for topical application according to the invention may be, for example, 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.

According to the invention, the term "water-soluble solvent" means 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). In particular, we can cite:

- lower C1-C5 monoalcohols 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 their mixtures,

and their mixtures.

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

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

The term "oily phase" means an oil or a mixture of oils miscible with each other, or not. For the purposes of the invention, the term "oil" means a fatty substance, not soluble in water, liquid at 25°C and atmospheric pressure. These oils may be volatile or non-volatile, vegetable, mineral or synthetic.

An oily phase according to the invention may comprise natural, hydrocarbon, silicone oils, and their mixtures.

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 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, mother-of-pearl and mixtures thereof.

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 neck, comprising in a physiologically acceptable medium, at least one extract of rose buds of the Evanrat variety, an extract of rose petals of the Evanrat variety, and a dipeptide-4, 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, mother-of-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 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 that would be part of a pathological state (“non-healthy” subjects, suffering from a pathology). We will speak indifferently of healthy skin and/or lips or skin and/or lips in the remainder of the description.

According to a particular embodiment, the application of the composition as defined according to the invention to the skin and/or the lips makes it possible to prevent and/or reduce the signs of skin aging, in particular to promote and/or improve the density and/or elasticity of the skin, to promote and/or improve the skin barrier function, to promote and/or improve the hydration of the epidermis, to preserve the thickness of the epidermis, and/or to prevent and/or avoid the appearance of a dull and uneven complexion.

Association of assets and uses

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

(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,

(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4.

The invention also relates to the non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined above, as an active combination for preventing and/or reducing the signs of skin aging, in particular promoting and/or improving the density and/or elasticity of the skin, promoting and/or improving the skin barrier function, promoting and/or improving the hydration of the epidermis, preserving the thickness of the epidermis, and/or preventing and/or avoiding the appearance of a dull and uneven complexion.

Cosmetic set and use

The present invention also relates to a cosmetic assembly comprising

(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,

(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and

(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4,

characterized in that at least one of the active ingredients (i) to (iii) 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 the lips, of the compositions of the cosmetic assembly as defined above.

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

EXAMPLES

Example 1: Plant extracts and peptides

1.1. Preparation of rose bud extract (named “BUD” in the figures)

Rose buds of the Evanrat variety, in particular of the Jardin de Granville® rose variety, preferably first-blooming rose buds, were directly stored at -20°C after harvest.

Then, the rose bud extract also called “rose bud cryoextract” was obtained using the following extraction process:

Crushing the buds

20 kg of rose buds previously frozen and stored at -20°C were cooled to a temperature of -80°C, at which temperature they were crushed to obtain a “cryo-crushed material”.

Extraction

1 kg of this cryo-ground material, with added water, was kept stirring at a temperature between 0 and +4°C overnight.

Clarification

The extract thus obtained was then filtered using a plate filter (AF 21 plates) to separate the liquid phase from the plant material. The aqueous cryoextract obtained at the end of this step comprises an average dry matter of 1%.

Formulation

The aqueous phase obtained after filtration is then mixed with glycerin in a volume ratio of 1 to 1, then stabilized with the following preservatives in combination:

- Citric acid,

- Potassium sorbate and

- Sodium benzoate.

Freezing

A freezing stage is then carried out for a period of 24 to 48 hours at a temperature of 4°C.

Sterilizing filtration

Sterilizing filtration was carried out on a microfiltration membrane with a pore size of 0.2µm.

After filtration, an aqueous extract of rose buds is obtained comprising 0.5% to 1% by weight of dry matter relative to the total weight of the extract.

1.2. Preparation of rose petal extract (named “ZETA” in the figures)

As plant material, fresh petals of roses of the Evanrat variety or “Jardin de Granville®” roses are used.

The bioactive fraction of rose petals according to the invention is obtained according to the following protocol:

a1) cleaning 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 and then mechanical separation of the roses using a mechanical screw press (model CP-6 Vincent Corporation, FL) to extract the contents of the liquid intercellular colloidal dispersion (ICD) from 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 (ICD) to obtain the supernatant A and a Membrane Fraction (fraction B);

c1) adjustment of the pH level in supernatant A by an alkali (i.e., potassium carbonate), in order to obtain a pH ranging from 6 to 7,

c2) mechanical separation of supernatant A to obtain supernatant B and fraction C (cytoplasmic fraction);

d1) adjustment of the pH level in supernatant B with citric acid to obtain a pH lower than 4.5,

d2) mechanical separation of supernatant B to give a “fraction of serum from fresh Granville® rose petals” and a fraction D (precipitate); and

e) mixture of “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” in the following examples) contains 8% dry matter (active matter), 91.5% by weight of water, 0.15% by weight of potassium sorbate and 0.3% by weight of sodium benzoate. These contents are by weight relative to the total weight of the extract. The INCI name of this extract is: Rosa Hybrid Flower Extract, Potassium Sorbate, and Sodium Benzoate.

1.3. Peptide

Dipeptide-4 from the company ASHLAND, marketed under the name Quintescine ^TM IS, with INCI name: AQUA, BUTYLENE GLYCOL, DIPEPTIDE-4, was used in the following examples.

Example 2: Impact of the secretome of senescent keratinocytes treated with Glyoxal on “fresh” keratinocytes (not treated with glyoxal)

2.1. Materials and methods

Preparation of conditioned media from normal human keratinocytes treated with glyoxal

Normal human keratinocytes (NHK) of passage 5 from a 48-year-old female donor were used. Cells were maintained in EpiLife culture medium in the presence of HKGS supplement (a growth supplement used for culturing human keratinocytes). Supplement was omitted in some phases of the experiment (see below).

Cells were seeded on D0 in 6-well plates, and treated on D1 with 200 µM Glyoxal (Sigma) in EpiLife medium with or without HKGS. A second treatment was carried out on D2 under the same conditions to obtain 48 hours of treatment in total. The conditioned medium corresponding to the culture supernatant was collected on D3. The conditioned medium of cells that had not undergone glyoxal treatment was taken as a control.

Treatment of KHN with conditioned medium

Passage 5 HCNs were seeded in 6-well culture plates on D0. On D1, the cells were placed in the presence of the conditioned medium described in paragraph 2.1 above (glyoxal conditioned medium with or without HKGS as well as the conditioned medium without glyoxal) ( ).

RNA extraction was performed after 24 hours of treatment on D2.

RNA extraction

The culture medium was removed and 250 µL of lysis buffer from the Nucleospin RNA trace extraction kit (Macherey-Nagel) was added to the cell lawn. The cells were scraped using a cell scraper to recover the lysate and proceed to the extraction of total RNAs following the supplier's recommendations.

The quality of total RNAs was verified using a microplate reading (spectrostar NANO from BMG Labtech) coupled with the Microlab STAR. The results are analyzed with the MARS software.

Complementary DNA synthesis

The reverse transcription step was performed using the High-Capacity Reverse Transcription kit (Thermo Fisher) and following the supplier's protocol.

TaqMan low density array (TLDA) PCR

Real-time PCR was performed on a thermal cycler (Applied Biosystems) with a TaqMan Gene Expression Master Mix kit (Life technologies) and TaqMan Gene Expression Assays (Life technologies) that consisted of two primers and a sequence-specific probe. The Taqman Gene Expression Master Mix contained DNA polymerase, dNTP, UDG (to avoid DNA contamination), ROX (a passive fluorescent reference), and buffer.

The data were normalized with the expression obtained for the housekeeping gene beta-2-microglobulin, whose expression is constitutive and allows to control for variations of the experiment.

Each sample was analyzed in triplicate.

Quantification was performed by the comparative ΔΔCt method. The relative quantification (RQ) values obtained correspond to the expression level compared to the control. The RQ is obtained by the following calculation, where the control is equal to 1:

RQ = 2–ΔΔCt = 2–(ΔCt treated – ΔCt untreated)

ΔCt treated = Ct target gene treated – Ct housekeeping gene treated

ΔCt untreated = Ct target gene untreated – Ct housekeeping gene untreated

Statistical analysis was performed using Student's t test. Results were considered significant when p<0.05(*) or p<0.01(**).

2.2. Results

The Applicant evaluated the impact of the secretome of senescent keratinocytes treated with the stress factor (Glyoxal), on “fresh” keratinocytes (not treated with glyoxal).

Embarrassed Relative quantification by qPCR Conditioned medium versus “fresh” (untreated) medium Variation after treatment Filaggrin (FLG) 0.14 -86% Transglutaminase (TGM1) 0.48 -52% Transient receptor potential cation channel, subfamily V, member 3
(TRPV3) 1.52 +52% Aryl hydrocarbon receptor (AHR) 1.62 +62%

Changes were observed in the gene expression of keratinocytes exposed to this conditioned medium: an impact on the barrier function with a decrease in the gene expression of filaggrin and transglutaminase 1. A stimulation of pathways linked to inflammation was also observed, with an increased expression of TRPV3 and AHR.

These data confirm that senescent cells secrete deleterious molecules that functionally disrupt surrounding cells.

Example 3: Effect of the complex according to the invention on bio-printed skin stressed with glyoxal

3.1. Materials and methods

Ethical considerations and isolation of human skin cells

Adult skin was collected according to the principles of the Declaration of Helsinki and its use was declared to the French Ministry of Research (declaration no. DC-2020-4346). Written informed consent was obtained from the donor in accordance with the French Bioethics Act of 2014 (Law 94-954 of July 29, 1994).

Primary cultures of human fibroblasts, melanocytes, and keratinocytes were established from a healthy skin biopsy obtained from an adult patient. Normal human keratinocytes (NHK), melanocytes (MHN), and dermal fibroblasts (FHN) were isolated from human skin as previously described (Germain et al., 1993).

Cytotoxicity assessment

KHN, MHN, and FHN cells were trypsinized and seeded in 48- or 96-well plates at a density of 30,000 or 10,000 cells/well, respectively, and cultured with glyoxal at a concentration of 300 µM for FHN and 100 µM for KHN and MHN, until confluence.

The active ingredients and their combinations were dissolved in DMSO (DiMethyl SulfOxide) or in the cell culture medium. The cells were treated daily with the active ingredients for 4 days. At the end of the treatments, a solution of tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at a concentration of 0.5 mg/ml in PBS (Phosphate Buffered Saline) was added to the cells for 3 hours at 37°C. The formazan crystals were then dissolved with DMSO for 30 minutes under agitation and the absorbance was then measured by spectrometry at 550 nm. The percentage of viability for each condition was then calculated after normalization of the values with respect to the untreated control, representing a viability of 100%. A reproducibility equal to 8 was achieved for each condition.

At the following concentrations, no toxicity was observed:

ZETA fraction (0.2%),

ZETA + Bud (ZETA 0.025%, Bud 0.15%),

ROSAP P = complex according to the invention (ZETA 0.025% + Bud 0.15%, peptide 0.0625%).

These concentrations are those that were used in the examples below.

Cultivation of a 3D bioprinted full-thickness skin model

The 3D bioprinted full-thickness skin model was designed in two steps. A unique collagen-glycosaminoglycan-chitosan polymer matrix was used to seed FHN cells cultured in DMEM medium supplemented with bovine serum, L-ascorbic acid, and antibiotics. These dermal equivalents were cultured for 3 weeks at 37°C in a 5% CO2 atmosphere.

After this time, KHN and MHN were inkjet deposited using a bioprinter equipped with a piezoelectric nozzle onto the dermal construct at a density of 10,000 cells/cm2 in a precise and optimized arrangement and geometries without any support. After 72 hours of submerged culture, the 3D bioprinted constructs were lifted at the air-liquid interface to allow epidermal differentiation. The total duration of the protocol is 42 days.

Treatments with glyoxal at a concentration of 300 µM were carried out from D10 to D21 and with glyoxal at a concentration of 100 µM from D24 to the end of the culture (D42). Treatments with aminoguanidine (a molecule known to inhibit the deleterious effects of glyoxal) or with active ingredients were carried out from D10 to D21 and then from D24 to the end of the culture (D42).

The different treatments evaluated are as follows:

- Untreated condition (“CONTROL”)

- Glyoxal treatment (“GO”)

● Glyoxal at a concentration of 300 µM from D10 to D21 and 100 µM from D24 to D42.

- Glyoxal treatment + rose petal extract (“GO-ZETA”)

● Glyoxal at a concentration of 300 µM from D10 to D21 and 100 µM from D24 to D42, and

● Rose petal extract as described in example 1.2 (ZETA) at a concentration of 0.2%.

- Glyoxal treatment + rose petal extract + rose bud extract (“GO-ZETA+BOURGEON”)

● Glyoxal at a concentration of 300 µM from D10 to D21 and 100 µM from D24 to D42,

● Rose petal extract as described in example 1.2 (ZETA), at a concentration of 0.025%, and

● Extract of rose buds as described in example 1.1, at a concentration of 0.15%.

- Treatment with the complex according to the invention (“ROSAP P”)

● Extract of rose petals as described in example 1.2 (ZETA), at a concentration of 0.025%,

● Extract of rose buds as described in example 1.1, at a concentration of 0.15%, and

● Dipeptide-4 as described in Example 1.3, at a concentration of 0.0625%.

- Glyoxal treatment + complex according to the invention (“GO-ROSAP P”)

● Glyoxal at a concentration of 300 µM from D10 to D21 and 100 µM from D24 to D42,

● Extract of rose petals as described in example 1.2 (ZETA), at a concentration of 0.025%,

● Extract of rose buds as described in example 1.1, at a concentration of 0.15%, and

● Dipeptide-4 as described in Example 1.3, at a concentration of 0.0625%.

- Glyoxal + aminoguanidine treatment (“GO-REFERENCE”)

● Glyoxal at a concentration of 300 µM from D10 to D21 and 100 µM from D24 to D42, and

● Aminoguanidine at a concentration of 0.8mM.

Methodology and timetable of the study

Supernatants were collected after each sample collection. Samples collected at D35 were fixed in 4% neutral buffered formalin solution for 24 hours and embedded in paraffin.

Samples collected at D42 were fixed in 4% neutral buffered formalin for 24 h and embedded in paraffin or in OCT compound (“Optimal cutting temperature”; used to embed samples) and frozen at -80°C for histological and immunohistological analyses respectively.

For each cell culture condition and each analysis, 3D skin equivalents were produced in triplicate.

Histological analysis

Formalin-fixed, paraffin-embedded samples were then cut into 5 μm sections. After deparaffinization and rehydration, sections were stained with hematoxylin, phloxine, and saffron (HPS) and Masson's trichrome (light green version) for histological analysis.

Immunohistological analysis

Immunostaining was performed on 5 μm formalin-fixed, paraffin-embedded samples. After deparaffinization and antigen demasking, sections were incubated with PBS containing 5% BSA to block nonspecific antigen binding. Sections were then incubated with primary antibodies against filaggrin, aquaporin-3, transglutaminase, elastin, type I collagen, or carbonyl proteins. A secondary antibody conjugated to AlexaFluor-568 was incubated for 1 hour at room temperature. Nuclear counterstaining using Hoechst was performed routinely.

Image acquisition

HPS-stained specimens were observed using an Axioskop 2 Plus optical microscope (Zeiss), and images were captured using the DS-Ri1 CCD camera (Nikon) and NIS-Elements software (Nikon). Sixteen-bit images were saved in an uncompressed tagged image file format (tiff). At least nine representative images were captured for each condition in the same manner.

Image analysis

Image processing and analysis were performed using MBF_ImageJ software for microscopy (http://www.macbiophotonics.ca/imagej). The parameters that were analyzed were epidermal thickness for HPS-stained sections and positive immunolabeled surface for epidermal and dermal markers.

Thickness of the epidermis

Epidermal thickness was obtained using a Euclidean distance map. Pixels corresponding to the epidermis were selected from the other pixels. Images were converted to an 8-bit binary image. Images corresponding to the area of interest were converted to a 16-bit distance map. Each epidermis pixel (non-zero) in the binary image of the distance map was assigned a value equal to its distance from the nearest background pixel (zero). The basal line of the epidermis was selected and then applied to the distance map. The mean intensity of the basal line corresponds to the mean distance between the basal line and the stratum corneum (Dos Santos et al., 2015). Data are expressed in μm.

Surface measurement

Positively stained tissue areas were automatically detected and segmented from other pixels. Images were then converted to binary images, processed by mathematical morphology, and filtered to isolate regions of interest. The area of interest was measured automatically.

For dermal markers, tissue areas stained red or green were automatically detected and segmented relative to other pixels. Images were then converted to binary images, processed by mathematical morphology, and filtered to isolate regions of interest. The area of interest was measured automatically. Data were normalized by dermal area and expressed as percent density.

For epidermal markers, red-positive tissue areas were automatically detected and segmented from other pixels. Images were then converted to binary images, processed by mathematical morphology, and filtered to isolate regions of interest. The area of interest was automatically measured. Data were normalized by the length of the epidermal-dermal junction.

Assessment of transepidermal water loss in a 3D bioprinted skin model

Transepidermal water loss was measured using non-invasive techniques based on a closed chamber instrument (Vapometer).

Statistical analysis

For all data, statistical significance was assessed using the one-way Student's t test, and statistically significant differences are indicated as follows: *p < 0.05, **p < 0.01, and ***p < 0.001 and **** p < 0.0001.

3.2. Results

Morphological analysis on bioprinted skins

The Applicant evaluated the impact of Glyoxal stress on the morphology of skin tissue (3D bio-printed skin).

It has been observed that 3D bioprinted skins subjected to Glyoxal stress present alterations in morphology. In particular, there is a decrease in the thickness of the epidermis of -44%, and a decrease in the density of the dermis of -30%.

Unexpectedly, it was found that when bioprinted skins, stressed with Glyoxal, are placed in the presence of the complex according to the invention, they are protected from these alterations. Under these conditions, the thickness of the epidermis is increased by +73%, and the dermal density by +51% (compared to the glyoxal alone condition). The thickness and density are therefore similar to the untreated control condition. It is also observed that these results are comparable to those obtained with aminoguanidine, a molecule known to inhibit the deleterious effects of glyoxal ( (A) and (B)).

Immunohistological analysis of the epidermis on bioprinted skins

The Applicant evaluated the impact of Glyoxal stress on the protein expression of several epidermal markers (3D bio-printed skin).

It was observed that glyoxal treatment induced a decrease in the protein expression of two markers of epidermal differentiation: filaggrin (-75%) and transglutaminase 1 (-88%).

Unexpectedly, it was found that when bioprinted skins, stressed with Glyoxal, are placed in the presence of the complex according to the invention, they are protected from these effects and the expression of filaggrin and transglutaminase 1 is restored, or even increased (respectively + 300% and + 2082% compared to the glyoxal-only condition). These data show that the complex according to the invention allows the maintenance of epidermal differentiation and preserves the barrier function of the skin.

In addition, the complex according to the invention makes it possible to increase the expression of aquaporin 3, a major channel allowing the passage of water at the level of the epidermis. After treatment with glyoxal, the protein expression of aquaporin 3 is reduced by 41%. This expression is, however, increased by +156% with the complex according to the invention (compared to the glyoxal condition alone). These data show that the complex according to the invention promotes the hydration of the epidermis ( ).

Immunohistological analysis of the dermis on bioprinted skin

The Applicant evaluated the impact of Glyoxal stress on the protein expression of several dermal markers (3D bio-printed skin).

Collagen I fibers are a major component of the extracellular matrix that contribute to giving the dermis its density. Similarly, elastin fibers give the dermis its elastic properties.

It was observed that glyoxal treatment induced a decrease in the protein expression of these two dermal markers: collagen I (-47%) and elastin (-49%).

Unexpectedly, it was found that when bioprinted skins, stressed with Glyoxal, are placed in the presence of the complex according to the invention, they are protected from these effects and the expression of collagen I and elastin is increased, or even restored (respectively + 61% and + 127% compared to the glyoxal condition alone).

These data show that the complex according to the invention makes it possible to protect the dermis and the essential components of the extracellular matrix ( ).

Measurement of carbonylated proteins in the dermis and epidermis on bioprinted skins

The Applicant evaluated the impact of Glyoxal stress on protein carbonylation in the dermis and epidermis (3D bioprinted skins).

It was observed that glyoxal treatment induced a strong increase (+56%) in protein carbonylation in the dermis and epidermis. This carbonylation impacts the structure and function of proteins, decreasing light transmission at the epidermis (dull complexion), and stiffening collagen fibers in the dermis (loss of suppleness).

Unexpectedly, it was found that when bioprinted skins, stressed with Glyoxal, are placed in the presence of the complex according to the invention, they are protected from this phenomenon, with a decrease of -25% of carbonylated proteins (compared to the glyoxal condition alone) ( ).

3.3. Conclusions

Taken together, these results show that the complex according to the invention has numerous cosmetic effects, in particular making it possible to prevent and/or reduce the signs of skin aging, in particular to promote and/or improve the density and/or elasticity of the skin, promote and/or improve the skin barrier function, promote and/or improve the hydration of the epidermis, preserve the thickness of the epidermis, and/or prevent and/or avoid the appearance of a dull and uneven complexion.

Example 4: Measurement of gene expression of the senescence marker p21 from bioprinted skin

4.1. Materials and methods

The protocols relating to the obtaining and culturing of bio-printed skins and the different treatments applied to them, in particular the nature and concentration of the active ingredients, are identical to those in example 3.

RNA extraction

A sample of the bioprinted skins recovered at D35 were collected in tubes containing 1 ml of RNAlater (Thermofisher) and stored at -80 ° C. On the day of extraction, the skin samples were collected and blotted on sterile absorbent paper then placed in 350 µL of RA1 lysis buffer (Qiagen) containing 1% beta-mercaptoethanol. The tubes were placed in a Precellys tissue grinder homogenizer with the program 3x23 seconds / 6300 rpm (rotation per minute) / 2 minutes pause. Once the program is finished, 800 mL of TRIzol (Qiagen) are added to the ground material and the samples are shaken vigorously by hand for 15 seconds then left for 5 minutes at room temperature with shaking. 800 µL of Trizol are then added and the tubes are shaken vigorously by hand for 15 seconds and then left for 3 minutes at room temperature. The samples are then centrifuged for 15 minutes at 12,000 g at 4°C. The aqueous phase (~750 µL) is transferred to a 1.5 mL tube. The RNAs are then extracted with the Nucleospin RNA kit (Macherey Nagel) according to the supplier's recommendations.

TaqMan low density array (TLDA) PCR

Real-time PCR was performed on a thermal cycler (Applied Biosystems) with a TaqMan Gene Expression Master Mix kit (Life technologies) and TaqMan Gene Expression Assays (Life technologies) that consisted of two primers and a sequence-specific probe. The Taqman Gene Expression Master Mix contained DNA polymerase, dNTP, UDG (to avoid DNA contamination), ROX (a passive fluorescent reference), and buffer.

The data were normalized with the expression obtained for the housekeeping gene beta-2-microglobulin, whose expression is constitutive and allows to control for variations of the experiment.

Each sample was analyzed in triplicate.

Quantification was performed by the comparative ΔΔCt method. The relative quantification (RQ) values obtained correspond to the expression level compared to the control. The RQ is obtained by the following calculation, where the control is equal to 1:

RQ = 2–ΔΔCt = 2–(ΔCt treated – ΔCt untreated)

ΔCt treated = Ct target gene treated – Ct housekeeping gene treated

ΔCt untreated = Ct target gene untreated – Ct housekeeping gene untreated

Statistical analysis was performed using Student's t test. Results were considered significant when p<0.05(*) or p<0.01(**).

4.2. Results

The applicant evaluated the impact of treatment of bioprinted skins exposed to glyoxal on the gene expression of a senescence marker, p21.

It was observed that bioprinted skins subjected to Glyoxal stress exhibit a +73% increase in p21 gene expression, confirming the presence of senescent cells in the tissue.

Expression of the senescence marker p21 Glyoxal-treated skins versus control skins Relative quantification by qPCR 1,730 Variation after treatment +73%

Unexpectedly, it was found that when bioprinted skins, stressed with glyoxal, are placed in the presence of the complex according to the invention, they are protected from this expression. A decrease of -29% in the expression of p21 is observed in the presence of the ingredient ZETA, a decrease of -41% in the expression of p21 is observed in the presence of a complex with ZETA and Bud, and a decrease of -51% of p21 is observed in the presence of the complex comprising ZETA, Bud and Peptide (see Table 3). Thus, these results show that the complex according to the invention makes it possible to reduce the phenomena of senescence induced in the skin in response to stress.

Expression of the senescence marker p21 Skins treated with glyoxal + Zeta versus glyoxal Skins treated with glyoxal + Zeta/Bud versus glyoxal Skin treated with glyoxal + Rosapeptide versus glyoxal Relative quantification by qPCR 0.710 0.593 0.493 Variation after treatment -29% -41% -51%

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% Conservatives 0.6% Chelating 0.04% Carbomer (Carbopol® 981) 0.3% Sodium polyacrylate (Covacryl® MV60) 0.2% Sodium Hydroxide 0.15% Extract of rose petals according to the invention * 0.3% Extract of rose buds according to the invention * 1%

Table 4: Aqueous phase

Dipeptide-4* 2% 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

Table 5: Fatty phase

The composition is prepared according to the following procedure:

- the gelling agents are dispersed in the aqueous phase (excluding rose extracts and sodium hydroxide) which is heated to 70°C;

- the fatty phase (excluding perfume concentrate, antioxidant) is heated to 70°C;

- the emulsion is produced by introducing the fatty phase into the aqueous phase with vigorous stirring;

- the gelling agents are neutralized by adding sodium hydroxide and the emulsion is cooled under moderate stirring with the introduction of the perfume concentrate, the antioxidant and the rose extracts at low temperature.

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

5.2. Composition in the form of a gel-serum for the eye contour

Purified water Qsp 100.00% Glycols 13.0% Conservatives 0.60% Carbomer 0.80% Glyceryl stearate citrate 0.70% Lecithin and sodium acrylates copolymer (Lecigel PCR negative) 1.20% Isostearate isostearyl 9.0% Bis-diglyceryl polyacyladipate-2 (Softisan 649 MB) 1.0% Silica 2.0% Mother of pearls 1.0% Extract of rose petals according to the invention * 0.8% Extract of rose buds according to the invention * 0.5% Dipeptide-4* 0.5% Tocopheryl acetate 0.1% Scent of roses 0.2%

* as described in Example 1

The ingredients of the aqueous phase (water, glycols and carbomer) are mixed at 80°C with stirring. The preservatives are then added, followed by the gelling agents at 80°C with stirring and then the temperature is lowered to 75°C. Then the surfactants are emulsified in the aqueous phase with stirring. The fillers and pearlescent agents, previously pasted and homogenized, are added at 40°C. The rose extracts are then added at 40°C, with stirring until reaching 30°C.

After application to the eye contour, this gel-serum helps to smooth out wrinkles and fine lines, the eye area is firmed. The youthfulness of the eyes is restored over the course of applications.

Claims (11)

Cosmetic composition comprising, in a physiologically acceptable medium:
(i) an extract of rose buds of the Evanrat variety,
(ii) an extract of rose petals of the Evanrat variety, and
(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4. Cosmetic composition according to claim 1, characterized in that the rose bud extract is obtained by a cryoextraction process comprising the steps of:
a) crushing the buds
b) extraction in a polar solvent,
c) clarification,
d) addition of glycerin,
e) optional addition of preservatives,
f) freezing, and
g) filtration. Cosmetic composition according to claim 1 or claim 2, characterized in that said rose petal extract is in the form of an isolated bioactive fraction comprising at least 500 µg/µL 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. Cosmetic composition according to any one of claims 1 to 3, characterized in that:
(i) The extract of rose buds of the Evanrat variety is present in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition,
(ii) The extract of rose petals of the Evanrat variety is present in a content ranging from 0.01 to 5% by weight relative to the total weight of the composition, and
(iii) The synthetic dipeptide is present in a content ranging from 0.1 to 10% by weight relative to the total weight of the composition. Cosmetic composition according to any one of claims 1 to 4, characterized in that it is a composition for caring for and/or making up the skin and/or lips, in particular a skin care composition. Non-therapeutic cosmetic process for the care and/or makeup of 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 any one of claims 1 to 5. Cosmetic process according to claim 6, 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, in particular to promote and/or improve the density and/or elasticity of the skin, to promote and/or improve the skin barrier function, to promote and/or improve the hydration of the epidermis, to preserve the thickness of the epidermis, and/or to prevent and/or avoid the appearance of a dull and uneven complexion. Association of cosmetic active ingredients including:
(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,
(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and
(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4. Non-therapeutic cosmetic use of the combination of cosmetic active ingredients as defined in claim 8, as an active combination for preventing and/or reducing the signs of skin aging, in particular promoting and/or improving the density and/or elasticity of the skin, promoting and/or improving the skin barrier function, promoting and/or improving the hydration of the epidermis, preserving the thickness of the epidermis, and/or preventing and/or avoiding the appearance of a dull and uneven complexion. Cosmetic set including:
(i) an extract of rose buds of the Evanrat variety, preferably collected before the first flowering,
(ii) an extract of rose petals of the Evanrat variety, preferably from fresh roses from the first flowering, and
(iii) a synthetic dipeptide, in particular the dipeptide with INCI name: dipeptide-4,
characterized in that at least one of the active ingredients (i) to (iii) is packaged in a composition distinct from the other active ingredients, and in particular each active ingredient is packaged in a distinct composition. Cosmetic process comprising the successive application, to the skin and/or the lips, of the compositions of the cosmetic assembly as defined in claim 10.