Isolated bioactive fraction from Evanrat variety roses

Abstract

The present invention relates to a rose extract in the form of an isolated bioactive fraction, obtained from fresh roses of the Evanrat variety, its preparation process, a composition comprising it and its uses, in particular to promote and / or improve skin nutrition, promote and / or improve the skin barrier function, promote and / or improve the rhythmic process of skin cells, and / or prevent and / or slow down skin aging.

Description

Title of the invention: Isolated bioactive fraction from Evanrat variety roses FIELD OF INVENTION

[0001] The present invention relates to an extract of roses of the Evanrat variety, also known as 'Jardin de Granville®' roses, in the form of an isolated bioactive fraction, in particular an extract in the form of an isolated bioactive fraction of rose petals, as well as a method for its preparation. The invention further relates to a composition comprising an extract of roses of the Evanrat variety, or 'Jardin de Granville®' roses, in the form of an isolated bioactive fraction according to the invention, and its use for promoting and / or improving skin nutrition, the skin barrier function, the rhythmic process of skin cells, preventing and / or slowing down skin aging, and also preventing and / or slowing down the phenomena of chronic micro-inflammation of the skin. STATE OF THE ART

[0002] The skin is the body's first line of defense against the environment. It is subjected daily to the effects of factors of exogenous origin (e.g., UV radiation, temperature variations, air pollution, cigarette smoke...) or endogenous origin (e.g., hormones...). All these factors can disrupt its balance, particularly its micronutrient balance, and / or impair its barrier function, and / or induce micro-inflammatory stress and ultimately contribute to skin aging.

[0003] It is well known that nutritional quality is reflected in skin health and that certain skin disorders are associated with deficiencies, particularly in certain micronutrients (Park K. Role of micronutrients in skin health and function. Biomol. Ther. 2015; 23; 207-217). Micronutrients are essential for skin development and formation, the physiological renewal of the epidermis, and the skin's adaptation to its environment. In particular, the use of vitamins (A, E, C) and / or essential fatty acids is known in the formulation of cosmetic compositions intended for the skin, especially for their protective and / or nutritional effects.

[0004] The use of certain rose extracts is also known. LVMH Recherche's patent application FR3066388 describes, in particular, an aqueous rose extract in the form of a 'cryoextract' and its in vitro effects, in human keratinocyte cultures, on the stimulation of the expression of several clock genes such as CRY2, PERI and PER3, as well as on the protein expression of keratin 10 (KRT10), a marker of the maturation of the epidermis and desmoglein 1 (DSG1), a marker of epidermal cohesion.

[0005] However, there is a constant need to find new extracts to maintain and / or promote the nutritional balance of the skin and consequently to stimulate skin regeneration, the skin barrier, reduce micro-inflammation and act favorably on skin aging.

[0006] The Applicant has thus developed a new extract of roses of the Evanrat variety, or 'Jardin de Granville®' roses, in particular, a new extract of petals of said roses, in the form of an isolated bioactive fraction. Unexpectedly, the Applicant has demonstrated that this new extract in the form of a "bioactive serum fraction," also called the "isolated bioactive fraction of roses," exhibits even improved effects compared to those already known and described in the prior art. In particular, the extract in the form of an isolated bioactive fraction of roses of the Evanrat variety, or 'Jardin de Granville®' roses, according to the invention, improves skin nutrition by promoting cell survival under glucose deprivation conditions.Furthermore, the extract in the form of an isolated bioactive fraction of roses according to the invention acts on certain signaling pathways, and thus promotes and / or improves the skin barrier function, promotes and / or improves the rhythmic process of skin cells, prevents and / or slows down skin aging and prevents and / or slows down the phenomena of cutaneous micro-inflammation. Description of the invention

[0007] The present invention relates to a rose extract in the form of an isolated bioactive fraction, obtained from fresh roses of the Evanrat variety.

[0008] Preferably said rose extract in the form of an isolated bioactive fraction comprises at least 500 pg / pL of at least one monosaccharide sugar selected from fructose, glucose and sucrose, and / or at least 500 mg / Kg of at least one mineral selected from potassium, calcium and sodium.

[0009] Preferably, said extract in the form of a bioactive fraction isolated from roses according to the invention, is characterized in that said extract is obtained from fresh petals of roses of the Evanrat variety.

[0010] Preferably, said extract in the form of a bioactive fraction isolated from fresh roses of the Evanrat variety according to the invention is characterized in that it is obtained by a process comprising the steps:

[0011] a) cleaning of plant material, maceration, pressing then mechanical separation of plant material to obtain an intracellular colloidal dispersion (ICD) and a material enriched in fibers (fraction A);

[0012] b) destabilization of the DCI with electromagnetic waves followed by separation mechanics of intracellular colloidal dispersion (ICD) to obtain supernatant A and a Membrane Fraction (fraction B);

[0013] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0014] 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 D fraction (precipitate); and

[0015] e) Optionally, mixing the "bioactive serum fraction" with at least one preservative and / or stabilizer.

[0016] The present invention further relates to a method for preparing an extract in the form of an isolated bioactive fraction from fresh roses of the Evanrat variety or the 'Rose Jardin de Granville®' variety, as defined in the present invention, comprising the steps of:

[0017] a) cleaning of plant material, maceration, pressing then mechanical separation of plant material to obtain an intracellular colloidal dispersion (ICD) and a material enriched in fibers (fraction A);

[0018] 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);

[0019] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0020] 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 D fraction (precipitate); and

[0021] e) Optionally, mixing the "bioactive serum fraction" with at least one preservative and / or stabilizer.

[0022] According to another aspect, the present invention relates to a 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 an effective amount of at least one extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety as defined in the present invention.

[0023] Preferably, said composition is characterized in that the extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety is present in the composition in a content ranging from 0.001 to 50%, in particular from 0.01 to 20%, preferably from 0.01 to 10% and preferably again from 0.011 to 5% by weight of raw material in relation to the total weight of said composition.

[0024] The invention also relates to a cosmetic treatment method for the skin and / or lips, intended to promote and / or improve skin nutrition; promote and / or improve skin firmness; promote and / or improve the skin barrier function; promote and / or improve the rhythmic process of skin cells; and / or prevent and / or slow down skin aging, comprising the application to the skin and / or lips, in particular the skin of the face and / or neck, of a cosmetic composition as defined in the present invention.

[0025] The invention further relates to the use of at least an effective amount of at least one extract of fresh roses of the Evanrat variety as defined in the present invention in the form of an isolated bioactive fraction as an agent to promote and / or improve skin nutrition; promote and / or improve skin firmness, promote and / or improve skin barrier function, promote and / or improve the rhythmic process of skin cells, prevent and / or slow down the aging of the skin and / or lips, in particular the skin of the face and / or neck.

[0026] The invention further relates to an extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety according to the invention or composition comprising it for its use to prevent and / or slow down micro-inflammation phenomena induced by stress of the skin and / or lips, in particular the skin of the face and / or neck. DESCRIPTION OF THE FIGURES

[0027] [Fig. 1] extraction process used according to the invention

[0028] [Fig.2] Effect of different extracts on the protein expression of filaggrin in Human skin cultures.

[0029] [Fig. 3] Effect of different extracts on the gene expression of 11[3-HSD1 in human keratinocyte cell cultures.

[0030] [Fig.4] Effect of different extracts on the protein expression of the clock gene CLOCK in human skin cultures.

[0031] [Fig.5] Effect of different extracts on cell survival under deprivation conditions of glucose in human keratinocyte cell cultures.

[0032] [Fig.6] Effect of different extracts on the amount of collagen III in cultures of human skin.

[0033] [Fig.7] Effect of different extracts on the amount of collagen I in cultures of human skin.

[0034] [Fig.8] Effect of different extracts on the length of fibrilin-1 fibers in Human skin cultures.

[0035] [Fig.9A] Effect of different extracts on extracellular secretion of HMGB1 in human keratinocyte cell cultures under LPS stress conditions.

[0036] [Fig.9B] Immunostaining of the membrane after Westem-blot, quantification of results.

[0037] [Fig. 10] Effect of different extracts on COX-2 protein expression in human skin cultures under LPS stress conditions.

[0038] [Fig. 11] Effect of different extracts on the protein expression of transglutaminase-1 (TGM-1) in human skin cultures under LPS stress. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0039] The term “bioactive fraction,” “bioactive serum fraction,” “serum fraction,” or “isolated bioactive fraction of roses” refers to an extract of roses of the Evanrat variety, or the 'Jardin de Granville®' rose, comprising the enzymes, proteins, sugars, ions, and other active molecules present in the cytosol of the cells composing the various plant tissues of the roses. The extract according to the invention is distinct from a cryoextract of rose petals as described in application FR3066388.

[0040] By "keratinous material" we mean the skin and / or its appendages, and the lips, in particular the skin of the face and / or body and the lips.

[0041] According to the invention, "micro-inflammation" or "chronic micro-inflammation of the skin" refers to invisible (silent) inflammatory processes that occur locally in the skin and which, if they degenerate or are poorly resolved, become chronic. Over time, these repeated micro-inflammatory events contribute to skin aging.

[0042] The following definitions are related to the steps of the process of preparing the extract according to the invention.

[0043] By "cleaning" is meant the removal of debris from fresh Granville® roses, and preferably fresh petals, before further processing, in a manner that avoids damaging the plant or removing 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 significant amounts of plant pigments. Excess wash water is then removed from the washed plants.

[0044] “Maceration” means the process 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, a grinding wheel, or a grinder (e.g., a knife mill, a hammer mill, etc.). To prevent temperature-induced degradation of the plant material, the maceration step may include temperature monitoring and the selection of maceration parameters that ensure that there is no significant increase in the temperature of the plant material during this stage.

[0045] By "pressing" is meant the separation of the liquid matter from fresh Granville® roses, and preferably from the fresh petals, by the application of a mechanical force. This includes, but is not limited to, techniques such as ambient gravity drainage, pressing with a heavy object, the centrifugal force of a rotary expeller, the piston pressure of a hydraulic press, or rollers or a screw of a suitable type for a press.

[0046] By "fibre-enriched material" or "FEM" or "FEM" (Fibre Enriched Material), we mean a solid and / or semi-solid fraction enriched with fibers from fresh Granville® roses, preferably fresh petals, from which the liquid intracellular colloidal dispersion (ICD) has been removed by pressing.

[0047] By "intracellular colloidal dispersion" or "ICD" refers to 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 consist mainly of plant cell organelles, organelle fragments, and fiber-enriched residual material. The aqueous medium consists mainly of cytosols and vacuoles.

[0048] By "adjustment," we mean the modification of the activities of hydroxide and hydronium ions in the aqueous medium of the DCI or of aqueous fractions produced by subsequent treatment of the DCI, the hydronium ion activity remaining within the range found in viable plant cells (e.g., between pH 3 and pH 9). This alteration can be accomplished, for example, by a separation process such as S / L filtration, centrifugation, membrane filtration (with bipolar membranes, ultrafiltration and microfiltration, reverse osmosis), and / or by the addition of a weak acid or a weak alkali / base and precipitation. The adjustment parameters are selected to be sufficient for a particular change in physicochemical parameters, such as pH, or the surface potential at the electrolyte-air interface.Such adjustments facilitate subsequent destabilization and / or separation steps; or create the conditions for good preservation and stabilization.

[0049] By "destabilization" is meant the treatment of the adjusted DCI using electromagnetic waves to transiently modify physical properties (such as e'0, which is the real component of the low-frequency dielectric constant). It was unexpectedly found that particular modifications degrade the stability of the DCI by causing the agglomeration and / or aggregation of particles in assemblies that are sufficiently large and stable to permit and / or improve after separation into fractions, with certain desirable properties.

[0050] “Separation” means the separation of solid and / or semi-solid particles and non-aqueous liquid droplets from an aqueous liquid by exploiting particle density and / or size. This includes, but is not limited to, techniques such as draining, filtration (including pressure gradient filtration), skimming, ambient gravity sedimentation, decantation, centrifugation, or a combination thereof. Preferably, continuous flow mechanical separation is used, but this does not preclude batch processing. “Separation 1” and “Separation 2” refer to the respective process steps, carried out with respective parameters.

[0051] The term "supernatant" refers to an aqueous material from which particles have been separated. "Supernatant A" and "Supernatant B" denote the supernatants resulting from the respective separation steps of the process.

[0052] The term "precipitate" refers to the particles from which an aqueous material has been separated. "Fraction B" and "Fraction C" denote the precipitates resulting from the respective separation steps of the process.

[0053] The terms "serum fraction of fresh Granville® roses" and "serum fraction of fresh Granville® rose petals" refer to compositions produced by the process as shown above and in [Fig. 1] without preservatives and / or stabilizers added to protect the ingredient composition against environmental factors such as temperature, atmosphere (e.g., oxygen), light and microorganisms.

[0054] The term “preservatives and / or stabilizers” means substances which, when added to a “serum fraction of fresh Granville® roses,” preferably a “serum fraction of fresh Granville® rose petals,” protect it against environmental factors such as temperature, atmosphere (e.g., oxygen), light, and microorganisms. Specific suitable substances may include, but are not limited to, a preservative, a stabilizer, and / or a mixture thereof.

[0055] The term “Granville® rose serum” or “extract of fresh Granville® roses” as used herein means a combination of a fraction of serum from fresh Granville® roses and preservatives and / or stabilizers.

[0056] 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.

[0057] Rose extract: Bioactive fraction isolated from roses and preparation process

[0058] The present invention therefore relates to a new rose extract obtained by a process In particular, it employs a destabilization step using electromagnetic waves. The extract obtained from this process has superior activity compared to previously known aqueous rose extracts.

[0059] This extract is also called “Granville Rose Serum®” Plant material

[0060] In the context of the present invention, plant material will be used from roses of the Evanrat variety, also called 'Jardin de Granville®' roses, and preferably even more so from rose petals of the Evanrat variety, or 'Jardin de Granville®' roses.

[0061] The 'Jardin de Granville®' rose is a hybrid variety offered exclusively by "Roses anciennes André Eve SAS" and protected by Plant Variety Certificate No. 20110345, with the species name Rosa L. and the variety name EVANRAT. This bush rose belongs to the group of modern hybrids, which, from May to October, are continuously covered in roses, thus demonstrating excellent repeat flowering.

[0062] The extract of the invention is therefore an extract of roses, more particularly an extract of roses of the Evanrat variety, or 'Jardin de Granville®' roses.

[0063] The invention preferably uses selected roses, whose properties are preserved by an organic environment and farming method.

[0064] The rose extract according to the invention can be prepared from fresh, frozen, freeze-dried roses, or any mixture thereof. In the context of the invention, fresh roses are preferably used.

[0065] Fresh, i.e., living plants have maximum metabolic activity, thus representing an optimal source for capturing the full spectrum of natural complexes and compounds and preserving their properties. Therefore, the use of fresh roses is preferred. Plant viability can be verified, in particular, by measuring chlorophyll fluorescence.

[0066] The invention therefore relates primarily to an extract of roses of the Evanrat variety, or 'Jardin de Granville®' roses, in the form of an isolated bioactive fraction, obtained from fresh roses.

[0067] Two types of roses are distinguished according to the time of year they are harvested:

[0068] - Winter roses, generally harvested between November and April, - Summer roses, generally harvested between May and October.

[0069] In one particular embodiment, the invention uses summer roses, in particular summer rose petals. In another embodiment, winter roses, in particular winter rose petals, are preferably used.

[0070] The extract according to the invention can be prepared from the different parts of the plant, it can therefore be an extract of leaves, an extract of buds, an extract of flowers (petals), an extract of sepals, an extract of wood (stems), an extract of roots or mixtures thereof.

[0071] Advantageously, the extract according to the invention is a petal extract.

[0072] According to a preferred embodiment, the extract of the invention is an extract of fresh petals, and more particularly an extract of fresh petals of roses of the Evanrat variety, or 'Jardin de Granville®' roses.

[0073] 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 carotenes.

[0074] Rich in monosaccharide sugars means 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 selected from fructose, glucose and sucrose.

[0075] By mineral-rich, 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 selected from potassium, calcium and sodium.

[0076] Advantageously, calcium improves epidermal differentiation and strengthens skin structure, while potassium enhances skin hydration and boosts energy assimilation. Phyto-sugars (fructose, glucose, sucrose) help to saturate skin cells with energy.

[0077] The extract of Evanrat variety roses, or 'Jardin de Granville®' roses, preferably the extract of Evanrat variety rose petals or 'Jardin de Granville®' roses, according to the invention, is in the form of an isolated bioactive fraction, and more particularly in the form of a bioactive serum fraction. Compared to known aqueous extracts of the prior art (e.g., aqueous cryo-extract of rose flowers (petals) of application FR3066388), this extract is more concentrated in natural compounds, in particular, sugars such as fructose or glucose, but also polyphenols and minerals. Thus, by virtue of the particular process used to obtain this extract, the rose extract according to the invention is distinct from rose water. Extraction process

[0078] The extract of fresh roses of the Evanrat variety or 'Jardin de Granville®' roses according to the invention, also called "Granville Rose Serum", is advantageously obtained by implementing the extraction process described below as well as in [Fig.1].

[0079] Preferably, fresh roses of the variety are used as plant material Evanrat or 'Jardin de Granville®' roses. Depending on the preferred method, these are the fresh petals of roses of the Evanrat variety or 'Jardin de Granville®' roses.

[0080] Advantageously, said process does not require the addition of any solvent or exogenous liquid.

[0081] The process implemented to obtain the extract according to the invention comprises the main steps of:

[0082] a. cleaning of plant material, maceration, pressing then mechanical separation of plant material to obtain an intracellular colloidal dispersion (ICD) and a fiber-enriched material (fraction A); b. “Treatment A” then mechanical separation of the Intracellular Colloidal Dispersion (ICD) to obtain the 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” followed by mechanical separation of the Supernatant B to give a “Bioactive Serum Fraction” and a Fraction D (Precipitate); and e. Optionally, mixing of the "bioactive serum fraction" with at least one preservative and / or stabilizer.

[0083] The extract of fresh roses, preferably of 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" within the meaning of the invention.

[0084] In a preferred embodiment, the extract according to the invention is obtained by implementing the extraction process disclosed in patents EP2919757, JP 6130924, CN ZL201380057567.6, EP2491939B1, CN1929851B, U.S. patents No. 8,734,861 and No. 7,473,435; U.S. patent application No. 16 / 078925.

[0085] Advantageously, this extraction process does not involve organic solvents, such as hexane, which could lead to numerous safety problems for facilities and personnel, human health, and environmental protection. This technology thus reduces emissions of volatile organic compounds (VOCs). It is therefore a clean process that allows for the extraction, without the addition of solvents, of high-quality proteins, sugars, and other co-products in the form of an aqueous extract that is directly usable in cosmetics.

[0086] Preferably, the flowers of roses (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 in order to prevent disruption of the cellular structure of the flowers. The viability of the collected plants can be tested using an OS5p multimode chlorophyll fluorometer (Opti-Sciences Inc, Hudson, NH, USA).

[0087] According to a particular embodiment, fresh live flowers, including The petals, pistil, and stamen were removed from the stem, including the sepal and receptacle, and packed in storage bags. They were immediately placed at sub-zero temperatures between -20°C and -80°C, such as -20°C, -40°C, or -60°C. This allows the flowers to be stored for extended periods, such as several months or even years, before being used in the extraction process.

[0088] According to 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

[0089] Once harvesting is complete, the roses, preferably the rose petals, are immediately rinsed by spraying with water at 10°C to 15°C 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 can then undergo maceration, pressing, and separation by mechanical, roller, hydraulic, or juice pressing to extract the contents of the liquid intercellular colloidal dispersion (ICD) from the fiber-enriched material (“Fraction A”).

[0090] 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).

[0091] The INN typically comprises 2% to 20% dry matter, preferably 4% to 16% dry matter, and even more preferably 6% to 12% dry matter.

[0092] At this stage, the INN can be frozen for storage. Typically, it is frozen at -20°C. Step b

[0093] When the INN has been frozen for storage, it must first be gently thawed. Typically, it is placed to thaw at 4°C or in ice.

[0094] “Treatment A” is carried out by destabilizing 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 a decrease 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 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) in assemblies large and stable enough to allow and / or improve mechanical separation.

[0095] Indeed, the resulting intracellular colloidal suspension is considered 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 each other unless they have sufficient 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 the potential energy of the particles as a function of their parameters, their distance from each other, and the characteristics of the continuous phase.Modifying the values ​​of variables affecting the repulsive force impacts the stability of the dispersion. Under normal colloidal stability conditions, 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 modified conditions during treatment A allow the repulsive force of the double layer to decrease to the point where the potential energy barrier disappears, and the particles can approach and agglomerate freely.

[0096] Restoring the initial conditions does not restore stability, because the particles have irreversibly agglomerated. They are thus easily removed by mechanical means (Koganov et al., softw journal 2017).

[0097] Preferably, at the end of "treatment A", the mechanical separation step of the ICD is carried out by centrifugation in order to produce the "Supernatant A" and the "Fraction B".

[0098] Typically, the "supernatant A" has a turbidity of less than about 100 NTU.

[0099] The “fraction B” typically comprises 10% to 30% dry matter, preferably 13% to 27% dry matter, and even more preferably about 15.0% to 25.0% dry matter. Step c

[0100] The "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.

[0101] Following "treatment B", the separation step is preferably carried out mechanical centrifugation of “Supernatant A” to produce “Supernatant B” and “Fraction C”.

[0102] The “fraction C” typically comprises from 5% to 25% dry matter, preferably from 8% to 22% dry matter, and even more preferably about 10.0% to 20.0% dry matter. Step d

[0103] The "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 below 4.5 is obtained. Typically, a citric acid solution will be used as the preferred acid.

[0104] Following "treatment C", the mechanical separation step of "Supernatant B" is preferably carried out by centrifugation in order to produce the "serum fraction of fresh Granville® roses", preferably the "serum fraction of fresh Granville® rose petals" (Non-Preserved Extract) and the "Fraction D".

[0105] The “serum fraction of fresh Granville® rose petals” typically comprises 2% to 20% dry matter, preferably 4% to 16% dry matter, and even more preferably 6.0% to 10.0% dry matter. Step e

[0106] According to some 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 the fresh Granville® rose petal extract or "Granville® Rose Petal Serum".

[0107] 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.

[0108] Thus, according to a preferred embodiment, the process implemented to obtain the extract according to the invention comprises the steps of:

[0109] a) cleaning of plant material, maceration, pressing then mechanical separation of plant material to obtain an intracellular colloidal dispersion (ICD) and a fiber-enriched material (fraction A);

[0110] b) destabilization of the DCI with electromagnetic waves followed by separation mechanics of intracellular colloidal dispersion (ICD) to obtain supernatant A and a Membrane Fraction (fraction B);

[0111] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0112] 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 D fraction (precipitate); and

[0113] e) Optionally, mixing of the “bioactive serum fraction” with at least one preservative and / or stabilizer.

[0114] Thus, according to a preferred embodiment, the process implemented to obtain the extract in the form of a bioactive fraction isolated from fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses according to the invention, comprises the steps of:

[0115] a) cleaning of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, maceration, pressing then mechanical separation of the roses to obtain an intracellular colloidal dispersion (INN) and a material enriched in fibers (fraction A);

[0116] 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);

[0117] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0118] d) adjusting the pH level in supernatant B until a pH below 4.5 is obtained, then mechanically separating supernatant B to give a "serum fraction of fresh Granville® roses" and a fraction D (precipitate); and

[0119] e) Optionally, mixing of the “serum fraction of fresh Granville® roses” with at least one preservative and / or stabilizer.

[0120] According to another preferred embodiment, the process implemented to obtain the extract in the form of a bioactive fraction isolated from fresh rose petals of the Evanrat variety or 'Jardin de Granville®' rose according to the invention comprises the steps of:

[0121] a) cleaning of fresh rose petals of the Evanrat variety, or 'Jardin de Granville®' roses, maceration, pressing then mechanical separation of the roses to obtain an intracellular colloidal dispersion (INN) and a material enriched in fibers (fraction A);

[0122] 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);

[0123] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0124] d) adjusting the pH level in supernatant B until a pH below 4.5 is obtained, then mechanically separating supernatant B to give a "serum fraction of fresh Granville® rose petals" and a fraction D (precipitate); and

[0125] e) Optionally, mixing of the “fragment of fresh Granville® rose petal serum” with at least one preservative and / or stabilizer.

[0126] Preferably, the invention relates to an extract in the form of a bioactive fraction isolated from fresh roses of the Evanrat variety or 'Jardin de Granville®' roses, characterized in that it is obtained by a process comprising the steps:

[0127] a) cleaning of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, maceration, pressing then mechanical separation of the roses to obtain an intracellular colloidal dispersion (INN) and a material enriched in fibers (fraction A);

[0128] 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);

[0129] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0130] d) adjusting the pH level in supernatant B until a pH below 4.5 is obtained, then mechanically separating supernatant B to give a "serum fraction of fresh Granville® roses" and a fraction D (precipitate); and

[0131] e) Optionally, mixing of the “serum fraction of fresh Granville® roses” with at least one preservative and / or stabilizer.

[0132] According to a preferred embodiment, the invention relates to an extract in the form of a bioactive fraction isolated from fresh rose petals of the Evanrat variety or 'Jardin de Granville®' roses, characterized in that it is obtained by a process comprising the steps:

[0133] a) cleaning of fresh rose petals of the Evanrat variety, or 'Jardin de Granville®' roses, maceration, pressing then mechanical separation of the roses to obtain an intracellular colloidal dispersion (INN) and a material enriched in fibers (fraction A);

[0134] 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);

[0135] c) adjusting the pH level in the supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, then mechanically separating the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0136] d) adjusting the pH level in supernatant B until a pH below 4.5 is obtained, then mechanically separating supernatant B to give a "Granville® fresh rose petal serum fraction" and a D fraction (precipitate); and

[0137] e) Optionally, mixing of the “fragment of fresh Granville® rose petal serum” with at least one preservative and / or stabilizer.

[0138] According to a particular method, the rose extract, preferably of rose petals, obtained 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 rose extract, preferably of rose petals, in the form of a bioactive fraction isolated according to the invention, is designated by its INCI name Rosa Hybrid Flower Extract.

[0139] According to another particular mode, the rose extract, preferably of rose petals, obtained according to the invention comprises water in a content of 89 to 93%, a dry extract content of 6 to 10% and preservatives and / or stabilizers in a content of 0.3 to 1% by weight relative to the total weight of the extract.

[0140] The extract of roses of the Evanrat variety, or 'Jardin de Granville®' roses, in particular of rose petals of the Evanrat variety, or 'Jardin de Granville®' roses, according to the invention, includes in particular amino acids (aspartic acid, tyrosine, arginine), total sugars (glucose, fructose), minerals and other co-products, which, when applied to a keratinous material, in particular the skin, provides a benefit or an improvement in the appearance of the keratinous material, such as promoting and / or improving skin nutrition, promoting and / or improving the skin barrier function, promoting and / or improving the rhythmic process of skin cells, preventing and / or slowing down skin aging.

[0141] They also help to prevent and / or slow down micro-inflammation phenomena induced by stress. Composition and dosage

[0142] The present invention relates to a new composition comprising at least one extract in the form of a bioactive fraction isolated from fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses according to the invention, as defined above.

[0143] Preferably, the invention relates to a new composition comprising at least one extract in the form of an isolated bioactive fraction of fresh rose petals of the Evanrat variety, or 'Jardin de Granville®' roses according to the invention, as defined above.

[0144] The composition of the invention is preferably a cosmetic composition.

[0145] The term “cosmetic composition” means any composition intended for cosmetic purposes, that is to say, aesthetic, capable of coming into contact with the superficial parts of the human body and more particularly with keratinous materials, in particular the skin and / or lips, especially the skin of the face and / or neck.

[0146] The term "keratinous materials" according to the invention means skin and / or its appendages, and more particularly human skin and / or lips. In particular, this will refer to the skin of the face and / or neck and / or body, and the lips.

[0147] The keratinous materials according to the invention include, in particular, healthy keratinous materials (subjects "healthy"), that is to say, those not exhibiting any disorders or abnormalities that would indicate a pathological condition (subjects "unhealthy", suffering from a pathology). The terms "healthy skin" and / or "lips" or "skin" will be used interchangeably throughout the rest of this description.

[0148] The present invention therefore relates to a cosmetic composition comprising, in a physiologically acceptable medium, an effective quantity of at least one extract in the form of a bioactive fraction isolated from fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses as defined above.

[0149] By "physiologically acceptable medium" is meant any excipient suitable for topical use, in contact with keratinous materials, without risk of toxicity, incompatibility, instability and / or allergic response.

[0150] By "effective quantity" is meant the minimum quantity of "Jardin de Granville®" rose extract according to the invention which is necessary to obtain the beneficial effect according to the invention, namely a beneficial effect consisting of promoting and / or improving skin nutrition, promoting and / or improving the skin barrier function, promoting and / or improving the rhythmic process of skin cells, preventing and / or slowing down the aging of the skin and / or lips, in particular the skin of the face and / or neck.

[0151] The cosmetic composition according to the invention is a care composition for keratinous materials, in particular of the skin and / or lips and especially of the skin of the face and / or neck.

[0152] According to an advantageous variant, the extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, is present in the composition in a content ranging from 0.001 to 50%, in particular from 0.01 to 20%, preferably from 0.01 to 10% and preferably still from 0.011 to 5% by weight of raw material in relation to the total weight of said composition.

[0153] The physiologically acceptable medium generally represents from 1 to 99% by weight, relative to the total weight of said composition.

[0154] The cosmetic composition used according to the invention generally comprises, in addition to the "Jardin de Granville®" rose extract and the physiologically acceptable medium, one or more acceptable cosmetic excipients from among those known to the person skilled in the art in order to obtain a composition for topical application 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.

[0155] According to a particular mode, 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.

[0156] In a preferred embodiment, said composition used according to the invention is in the form of a cream or a serum.

[0157] The cosmetic composition according to the invention can be presented in any galenic form suitable for topical application on the skin and / or lips and in particular on the skin of the face and / or neck comprising the extract of roses “jardin de Granville®”, preferably the extract of rose petals “jardin de Granville®” and at least one cosmetic adjuvant chosen from antioxidants, perfumes, vitamins, thickening agents, emollients, moisturizing agents, anti-aging agents, lifting agents, tightening agents, plumping agents, soothing agents, anti-pollution agents, brightening or depigmenting agents, fillers, mother-of-pearls and mixtures thereof.

[0158] Also, according to a particular embodiment of the invention, the cosmetic composition according to the invention may further comprise at least one cosmetic adjuvant chosen from the group consisting of: antioxidant agents, emollient agents, moisturizing agents, anti-aging agents, perfumes, and mixtures thereof.

[0159] Depending on the nature of the composition, one or more cosmetically acceptable excipients will be selected from emulsifiers, polymers, surfactants, rheology agents, electrolytes, pH adjusters, antioxidants, preservatives, colorants, and mixtures thereof.

[0160] By way of particular example, the cosmetic composition according to the invention may include gelling agents, antioxidants, preservatives and mixtures thereof.

[0161] The cosmetic composition according to the invention may further comprise a fatty (solid fats) or oily phase.

[0162] The term "oil phase" refers to an oil or a mixture of oils, whether miscible or immiscible. For the purposes of this invention, "oil" refers to a fatty substance, insoluble in water, liquid at 25°C and atmospheric pressure. These oils may be volatile or non-volatile, vegetable, mineral, or synthetic.

[0163] An oily phase according to the invention may include natural, hydrocarbon, silicone oils, and mixtures thereof.

[0164] The oily or fatty phase content in the cosmetic composition of the invention will generally range from 0.2% to 45%, preferably from 0.5% to 30%, and even more preferably from 2% to 25% by weight relative to the total weight of said composition. Cosmetic and other uses

[0165] The invention also relates to a cosmetic treatment method for the skin and / or lips, intended to promote and / or improve skin nutrition, promote and / or to improve the skin barrier function, to promote and / or improve the rhythmic process of skin cells, to prevent and / or slow down skin aging, including the application to the skin and / or lips, in particular the skin of the face and / or neck, of a cosmetic composition as defined above.

[0166] The invention further relates to the use of at least an effective quantity of at least one extract of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, in the form of a bioactive fraction isolated according to the invention, as an agent to promote and / or improve skin nutrition, promote and / or improve the skin barrier function, promote and / or improve the rhythmic process of skin cells, prevent and / or slow down the aging of the skin and / or lips, in particular the skin of the face and / or neck.

[0167] The invention ultimately relates to an extract of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, in the form of an isolated bioactive fraction or a composition containing it, as defined in this application, for its use in preventing and / or slowing down micro-inflammation phenomena induced by stress of the skin and / or lips, in particular the skin of the face and / or neck.

[0168] The extracts of fresh roses of the Evanrat variety, or 'Jardin de Granville®' roses, in the form of an isolated bioactive fraction according to the invention, used for carrying out these processes and uses are those described in this application. In a particular and preferred embodiment, the rose extracts in the form of an isolated bioactive fraction are extracts in the form of an isolated bioactive fraction of rose petals, in particular roses of the Evanrat variety, and in particular roses from Jardin de Granville® rose bushes.

[0169] The invention will now be illustrated in the following non-limiting examples. Unless otherwise indicated, percentages are expressed by weight relative to the total weight of the composition. EXAMPLES Example 1: Plant extracts

[0170] 1.1 Bioactive fraction of 'Jardin de Granville®' rose petals according to the invention

[0171] Fresh petals of roses of the Evanrat variety or “Jardin de Granville®” rose are used as plant material.

[0172] The bioactive fraction of rose petals according to the invention is obtained according to the following protocol:

[0173] al) 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,

[0174] a2) maceration, pressing and then mechanical separation of the roses using a press mechanical screw (model CP-6 Vincent Corporation, FL) to extract the contents of the liquid intercellular colloidal dispersion (ICD) of the fiber-enriched material ("Fraction A");

[0175] bl) destabilization of the DCI with electromagnetic waves produced from magnetrons operating at a frequency between 2.45 and 5.8 GHz,

[0176] b2) centrifugation of the intracellular colloidal dispersion (ICD) to obtain the supernatant A and a Membrane Fraction (fraction B);

[0177] cl) adjustment of the pH level in the supernatant A by an alkali (i.e., potassium carbonate), in order to obtain a pH ranging from 6 to 7,

[0178] c2) mechanical separation of the supernatant A to obtain the supernatant B and the fraction C (cytoplasmic fraction);

[0179] dl) adjusting the pH level in the supernatant B by adding citric acid to obtain a pH below 4.5,

[0180] d2) mechanical separation of the supernatant B to give a "serum fraction of fresh petals of Granville® roses and a D fraction (precipitate); and

[0181] e) mixture of the “fragment of fresh rose petal serum from Granville®” with potassium sorbate and sodium benzoate.

[0182] Granville® Rose Petal Serum (also referred to as "Zeta Serum" or "Zf Serum" in the following examples) contains 8% dry matter (active ingredient), 91.5% water by weight, 0.15% potassium sorbate by weight, and 0.3% sodium benzoate by weight. These amounts are expressed as a percentage of the total weight of the extract. The INCI name for this extract is Rosa Hybrid Flower Extract, Potassium Sorbate, and Sodium Benzoate.

[0183] 1.2 Other rose extracts that can be used in combination

[0184] Other rose extracts, in particular from the Evanrat variety, preferably the Jardin de Granville® rose, can be used in combination with the rose extract, in the form of an isolated bioactive fraction according to the invention, to provide complementary effects. An example is the aqueous extract of rose flowers (petals) ('Cryoextract').

[0185] The aqueous extract of rose flowers (petals) (Cryoextract) is obtained by means of a cryo-extraction process such as that described in particular in patent application EP0425391. Such a process includes in particular the following steps:

[0186] - first crushing of rose flowers at a temperature between -10°C and -40°C, - second grinding of the rose fractions obtained in the previous step, at a temperature between -40°C and -100°C, in the presence of liquid nitrogen - sieving of the fractions obtained in the previous step, using a sieve with a particle size ranging from 2mm to 100pm and preferably less than 500pm, specifically ranging from 100p to approximately 500p, - pressing the fractions recovered in the previous step, and brought back to a temperature of 0°C ±5°C, then subjected to the following cycle of operations: freezing at a temperature between -10°C and -40°C, suspending these frozen fractions in a quantity of water approximately equal to the quantity of liquid obtained in the previous step, pressing the fractions suspended in water and brought back to a temperature of 0°C ±5°C, - Filtration of the quantities of liquid obtained and recovery of the filtrates, - concentration of filtrates by cold water removal, - advantageously freezing of the concentrated solutions obtained in the previous step.

[0187] A cryo-extract is obtained comprising 0.5% by weight of dry matter (active substance), 49-50% by weight of water, 49% by weight of glycerol, and preservatives. The INCI name of this aqueous rose extract is Water, Glycerin, Rose Extract or Rosa Hybrid Flower Extract, Water, Glycerin.

[0188] Example 2: Phytochemical characterization of rose extracts from Example 1

[0189] The results of the phytochemical analyses for the extract “rose petal serum” The results of the Granville® rose extract obtained according to Example 1.1 above (fresh petals) are shown in Tables 1 to 3 below. These data are compared, in particular, with the phytochemical analyses of the cryoextract of Granville® rose flowers (petals) obtained in parallel by the extraction process described in Example 1.2.

[0190] Table 1: Compounds detected by gas chromatography coupled with mass spectrometry in the serum of Granville® rose petals according to the invention and in the comparative extract, cryo-extract of Granville® rose petals. Compounds detected by gas chromatography coupled with mass spectrometry (in pg / pL) Granville® Rose Petal Serum Invention Flower Cryoextract (petals) Comparison 2-Oxoglutarate 233 - Alanine 43 0 Asparagine 1464 4 Aspartate 77 0 beta-Sitosterol 27 - Catechin 176 3 Citrate 138 0 Fructose 31236 200 Fructose-6-P 113 - Fumarate 48 0 GABA 52 1 Galactonate 45 0 Gluconate 59 0 Glucose 15396 100 Glucose-6-P 272 - Glutamate 527 0 Glutamine 268 0 Glycerate 14 0 Glycerol 284 - Isoleucine 33 0 Isoquercitrin 41 - Kaempferol 149 1 Kaempferol-3-O-glucoside 729 5 Malate 1319 11 Maltose 130 0 Mannose 32 0 myo-Inositol 1517 19 Phenylalanine 31 0 Phosphate 364 4 Proline 25 0 Quinate 2526 45 Ribulose-5-P 25 - Serine 82 1 Succinate 260 3 Sucrose (sucrose) 1541 - Threonine 19 0 U1747.6 / 392(Quercetin-3-O-glucuronide) 170 - Valine 28 0 Xylose 1323 7 Xylulose 24 -

[0192] It is therefore observed that, compared to the cryo-extract, the Granville® rose petal serum according to the invention is rich in sugars, in particular glucose, fructose, xylose, myo-Inositol and sucrose.

[0193] Other compounds of interest are also detected, such as phenols (catechin, Kaempferol-3-O-glucoside), citric acid, malic acid, and quinic acid. Overall, a total polyphenol concentration of 7474 ppm (rutin equivalent) is measured in the Granville® rose petal serum, compared to 3106 ppm in the cryoextract.

[0194] Table 2: Quantification of free amino acids in the serum of Granville® rose petals according to the invention and in the comparative extract, cryo-extract of Granville® rose petals. Free Amino Acids (in g / 100 mL) Granville® Rose Petal Serum Invention Cryoextract of Flowers (Petals) Comparison Alanine 0.027 0.002 Aspartic Acid 0.016 0.001 Asparagine 0.243 0.018 Glutamic Acid 0.021 0 Glutamine 0.015 0.001 Glycine 0.001 0 Histidine 0.003 0 Hydroxyproline 0.001 0 Isoleucine 0.008 0 Leucine 0.002 0 Phenylalanine 0.008 0 Proline 0.008 0.001 Serine 0.012 0 Threonine 0.005 0 Tyrosine 0.002 0 Valine 0.008 0

[0196] It is therefore observed that, compared to the cryo-extract, the Granville® rose petal serum according to the invention is richer in free amino acids.

[0197] Table 3: Quantification of minerals in the Granville® rose petal serum according to the invention and in the comparative extract, cryo-extract of Granville® rose petals. Minerals (in mg / kg) Granville® Rose Petal Serum Invention Cryoextract of flowers (petals) Comparative Ca 2094 25 Mg 215 17 P 180 19 K 2276 195 Na 509 29 Cu <1 - Fe <1 - Mn 3.5 - Zn 2 - Se <1 - Si - -

[0199] It is therefore observed that, compared to the cryo-extract, the Granville® rose petal serum according to the invention is richer in minerals. In particular, it is very rich in calcium and potassium.

[0200] Overall, these analyses show that the Granville® rose petal serum according to the invention is more concentrated in compounds of interest, compared to a cryo-extract of rose petals. Example 3: Materials and Method 3.1 Cell Cultures Keratinocyte cell cultures

[0201] The keratinocytes used in this study were derived from normal human epidermis of donors aged 26 to 61 years. They were cultured in serum-free keratinocyte medium (KSFM, Gibco) with 5 ng / mL of recombinant human EGF (Epidermal Growth Factor) (Gibco), 50 pg / ml of bovine pituitary extract (Gibco), and 100 pg / ml of Primocin (an antimicrobial agent, InvivoGen). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Fibroblast cell cultures

[0202] The fibroblasts used for this study are derived from normal human epidermis from a 48-year-old female donor. They were cultured in DMEM 1 g / L glucose (Lonza) supplemented with 10% FBS (Fetal Bovine Serum; Gibco), 2 mM LGlutamine (Lonza), and 100 pg / ml Primocine (InvivoGen). The cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. 3.2 Normal human skin or skin biopsy

[0203] Normal human skin is obtained from skin biopsies taken during plastic surgery procedures on the abdomen of donors aged 26 to 66 years. These biopsies were performed with a 6 mm diameter punch (medical pfm).

[0204] They were cultured on a culture medium containing 50% DMEM 1 g / L glucose (Lonza) and 50% Ham's-F12 culture medium (Lonza) supplemented with 10% FBS (Fetal Bovine Serum, Gibco), 2 mM L-glutamine (Lonza), and 100 qg / ml Primocine (InvivoGen). The skin biopsies were maintained in culture at 37°C in a humidified atmosphere containing 5% CO2. 3.3 Reagents

[0205] The reagents used for this study were:

[0206] - Lipopolysaccharide (LPS) (Sigma) diluted to 0.5 mg / mL for 6 hours, - Oxytocin (Sigma) diluted to 200pM for 24 hours, - 2-deoxy-D-glucose (2-DG) (Sigma) diluted to 2mM for 24 hours, - Neutral Red (Sigma) diluted to 0.05 mg / mL for 3 hours.

[0207] The primers and probes used for this study were of the "TaqMan Gene Expression Assays" type (Life Technologies):

[0208] - 11-HSD1 (Hs01547870_ml) - 18S endogenous control (Hs9999999901_sl) 3.4 Antibodies

[0209] The primary antibodies used for the study are:

[0210] - Anti-HMGBl (Abcam) rabbit monoclonal, diluted 1 / 500, overnight,

[0211] - Anti-COX-2 (Abcam) rabbit monoclonal, diluted 1 / 500, for 1h30,

[0212] - Anti-Filaggrin (Santa Cruz Biotechnology) mouse monoclonal, diluted 1 / 100, during hour 30 minutes,

[0213] - Anti-TGMl (Santa Cruz Biotechnology) mouse monoclonal, diluted 1 / 100, during at night,

[0214] - Anti-CLOCK (Abcam) rabbit polyclonal, diluted to 1 / 500, overnight.

[0215] - Anti-collagen III (Tebu) rabbit monoclonal, diluted 1 / 100, for 1h30,

[0216] - Anti-collagen I (Abcam) rabbit monoclonal, diluted 1 / 1000, overnight,

[0217] - Anti-fibrillin-1 (R&D Systems) rabbit monoclonal, diluted 1 / 500, for one hour and a half.

[0218] The secondary antibodies used for the study are:

[0219] - Rabbit-resistant goat peroxidase conjugated to goat peroxidase (Invitrogen), diluted 1 / 5000, for 1h,

[0220] - Alexa Fluor 488 donkey, anti-mouse (Invitrogen), diluted to 1 / 1000, for Ih,

[0221] - Alexa Fluor 488 donkey, anti-rabbit (Invitrogen), diluted to 1 / 1000, for Ih. 3.5 Western-Blot Protocol

[0222] In the cell lysate:

[0223] Cultured cells were homogenized in ice-cold RIPA (RadioImmunoPrecipitation Assay) buffer (Thermo Scientific) containing the protease inhibitor cocktail Hait and EDTA (Thermo Scientific). Cells were harvested mechanically and centrifuged at 10,000 rpm for 20 minutes at 4°C. Protein quantification was performed according to the kit recommendations (Thermo Scientific) on the cell supernatant, and Western blotting was performed on a standardized quantity of protein. The proteins were mixed with a loading buffer and a reducing agent, DTT (dithiothreitol, Sigma), and heated at 90°C for 5 minutes. The samples were then loaded onto a 4-12% Bis-Tris NuPAGE gel (Invitrogen), and electrophoresis was performed. The proteins were then transferred from the gel to a nitrocellulosic membrane using the iBlot Dry Blotting System (Invitrogen).Non-specific sites were saturated for 1 hour in a 5% milk solution diluted in TBS IX buffer (Tris buffered saline). The nitrocellulose membrane was incubated overnight at 4°C with the primary antibody, diluted in milk. After three 15-minute washes with a 0.05% Tween 20 solution in TBS IX, the secondary antibody, coupled to a peroxidase, was applied for 1 hour at room temperature. After a second series of three 15-minute washes with a 0.05% Tween 20 solution in TBS IX, protein visualization was performed with luminol (Thermo Scientific), and the photon emission was read by chemiluminescence using a Sensicam camera coupled with the Multilmage light cabinet (AIC).

[0224] In cellular supernatant:

[0225] The supernatants were collected. 20 µl of protein was mixed with loading buffer and DTT (Sigma) and heated at 90°C for 5 minutes. The samples were then loaded onto a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and electrophoresis was performed. The proteins were then transferred from the gel to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). Non-specific sites were saturated for 1 hour in a 5% milk solution diluted in TBS IX buffer. The nitrocellulose membrane was incubated overnight at 4°C in the primary antibody, diluted in milk. After three 15-minute washes with a 0.05% Tween 20 solution in TBS IX, the secondary antibody, coupled to a peroxidase, was applied for 1 hour at room temperature. After a second series of three 15-minute washes with a 0.05% solution of Tween 20 in TBS IX, protein detection was performed with luminol (Thermo Scientific), and the photon emission was read by chemiluminescence using a Sensicam camera associated with the Multilmage light cabinet (AIC).

[0226] The intensity of the bands was quantified with the Scion image analysis software. 3.6 Immunofluorescence Protocol

[0227] The sections were dewaxed and rehydrated with several successive baths of xylene, alcohol, and water. Then, an unmasking protocol was carried out as described for the different targets:

[0228] - for COX-2: microwave exposure at 600 W in EDTA buffer pH 9 (Sigma) until boiling, followed by digestion with 0.25% pepsin (Zymed, In-vitrogen) for 15 minutes at 37°C,

[0229] - for filaggrin: Digestion with 0.25% pepsin (Zymed, Invitrogen) for 15 minutes at 37°C,

[0230] - for TGM1: microwave exposure at 600 W in a pH 6 citrate buffer (Sigma) until boiling,

[0231] - for CLOCK: microwave exposure at 600 W in a pH 6 citrate buffer (Sigma) until boiling.

[0232] After washing with PB S and saturating nonspecific sites with a 5% BSA (Bovine Serum Albumin, Sigma) solution for 30 minutes, the primary antibody was applied and the slides were incubated with shaking at room temperature in a humid environment. After rinsing the slides with PBS (Phosphate Buffered Saline), the secondary antibody was applied in the dark, with shaking, at room temperature in a humid environment. Finally, the cell nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI, Molecular Probes) at 0.3 pM for 5 minutes and the sections were mounted in Fluoromount-G (Electron Microscopy Sciences). Detection was performed using a Zeiss Axiovert 200M microscope with a 20x or 40x objective. The photos were taken with a blue Qimaging EXI camera coupled with Volocity acquisition software (Improvision). 3.7 Quantitative PCR Protocol

[0233] Total RNA was first extracted using an isolation kit (Ambion) as follows:

[0234] After treatment, the culture medium was removed and the cells were rinsed with Cold PBS was collected and recovered with lysis buffer. Specific columns were used to purify the RNA, which was then eluted in 30 µL of RNase-free water. The total RNA was then reverse-transcribed using the cDNA (complementary DNA) reverse transcription kit containing the RNase inhibitor (Life Technologies). Reverse transcription was performed on 2 µg of total RNA using a thermocycler. (MJ Research). Finally, real-time PCR was performed on a thermocycler (Applied Biosystems) using a TaqMan Gene Expression Master Mix kit (Life Technologies) and TaqMan Gene Expression Assays (Life Technologies), which consisted of two primers and a sequence-specific probe. The TaqMan Gene Expression Master Mix contained DNA polymerase, dNTP, UDG (to prevent DNA contamination), ROX (a passive fluorescent reference), and a buffer.

[0235] TaqMan 18S primers were used as an endogenous control because the 18S gene encodes a ribosomal protein whose expression and corresponding mRNA are known to be invariant under all conditions. In addition, DNA was replaced with water as a negative control.

[0236] Each sample was analyzed in triplicate.

[0237] The comparative TC (Threshold cycle) method was used for the relative quantification of target expressions (Livak KJ and Schmittgen TD, 2001) and the StepOne software (Applied Biosystems) was used for data processing.

[0238] 3.8 Protocol for studying cell viability in “neutral red”

[0239] "Neutral Red" is a technique for detecting live cells by The absorption of a neutral red dye (eurhodine) is performed. Living cells absorb the neutral red dye via active transport and incorporate it into their lysosomes / endosomes. The cells are then lysed with an acidic solution, and the lysosomes / endosomes release the neutral red dye. Thus, the total number of living cells can be determined by measuring the amount of dye released. This measurement is performed by reading the optical density (OD) at 540 nm.

[0240] The cells were rinsed with Ca2+ / Mg2+ PBS (Lonza) and then incubated with a 0.05 mg / mL neutral red solution (Sigma) for 3 hours. After washing with Ca2+ / Mg2+ PBS, the cells were lysed with a lysis buffer (50% ethanol (VWR), 49% H2O, 1% acetic acid (Sigma)) and incubated for 10 minutes with shaking. Detection of the released dye at 540 nm was performed using a Sinergy 2 Biotek spectrophotometer.

[0241] In the following examples, the Applicant studied the impact of the bioactive fraction of rose petals as prepared above, on skin barrier function, skin cell aging, skin nutrition, the rhythmic process of skin cells, and skin regeneration, as well as in parallel on skin micro-inflammation.

[0242] For all these tests, the effects of the rose petal extract in the form of the bioactive fraction according to the invention "ZF serum" prepared according to the protocol described in Example 1.1 are compared to the effects of the "cryoextract" (comparative) prepared according to the protocol described in Example 1.2 and to the effects of a "placebo" control solution. consisting of a solution of preservatives (sodium benzoate, potassium sorbate, citric acid).

[0243] Percentages (%) are expressed as weight of raw material (RM) relative to the total weight of the composition unless otherwise stated.

[0244] Example 4: Effect of the isolated bioactive fraction of rose petals according to the invention on the skin barrier function

[0245] The effect of the isolated bioactive fraction of rose petals according to the invention, as prepared in Example 1.1, on the skin barrier function was tested.

[0246] HMGB1 is a central molecule in various physiological processes. It is located in chromatin, where it is notably involved in modulating the expression of different genes, particularly genes involved in the formation of the stratum comeum and epidermal growth. HMGB1 therefore plays a crucial role in the physiology of the epidermis and, more specifically, in the skin barrier function.

[0247] Capacity of the bioactive fraction isolated from rose petals according to the invention to modulate the expression of filaggrin

[0248] It has been shown in particular on skin cell models that stimulation of HMGBl negatively regulates the expression of filaggrin, a protein playing an essential role in the regulation of epithelial homeostasis and actively involved in the skin barrier function.

[0249] The ability of rose petal extract in the form of a bioactive fraction according to the invention to modulate filaggrin expression was evaluated on human skin biopsies.

[0250] Protocol:

[0251] - Human skin (biopsies according to Example 3.2) from two independent donors pendants.

[0252] - Conditions: the biopsies were processed twice daily for 48 hours by the various extracts or control solutions as indicated below:

[0253] (1) “Control”: diluted PBS,

[0254] (2) “ZF serum” at 0.3%,

[0255] (3) “Placebo” at 3%,

[0256] (4) “Cryo-extract” at 3%.

[0257] - Method of analysis: evaluation of filaggrin expression by immunofluorescence rescence, and quantification of results with Volocity according to the protocol detailed in Example 3.6.

[0258] The results of [Fig. 2] show that treatment with the isolated bioactive fraction of rose petals according to the invention significantly increases (+49%) the level of filaggrin protein expression in skin biopsies human. No significant effect was observed with the cryo-extract of rose petals (comparative) or with the placebo solution.

[0259] Example 5: Effect of the isolated bioactive fraction of rose petals according to the invention on cellular aging

[0260] The effect of the isolated bioactive fraction of rose petals on skin cellular aging was tested.

[0261] An increased amount of cortisol in the skin is associated with skin aging and its effects, such as decreased epidermal thickness, degradation of collagen fibers, and reduced cell proliferation. One of the major pathways involved in cortisol synthesis involves the activation of the enzyme 11[3-HSD1]. In skin cells, 11[3-HSD1 increases with age and, in conjunction with overexposure to UV radiation, contributes to the development of localized skin damage.

[0262] We therefore evaluated the ability of the rose petal fraction according to the invention to modulate the expression of 11[3-HSD1.

[0263] Protocol:

[0264] - Human keratinocyte cell cultures in culture medium for 6h.

[0265] - Conditions: keratinocytes were treated once during the 6 hours of culture using different extracts or control solutions as indicated below:

[0266] (1) “Control”: diluted PBS,

[0267] (2) “ZF serum” at 0.1%,

[0268] (2) “Placebo” at 1%,

[0269] (4) “Cryo-extract” at 1%.

[0270] - Analytical method: evaluation of 11[3-HSD1] expression by quantitative PCR in real time according to the protocol detailed in Example 3.7.

[0271] The results in [Fig. 3] show that treating keratinocytes with the isolated bioactive fraction of rose petals according to the invention significantly reduces (-28%) the expression level of 11[3-HSD1, known to regulate cortisol levels in skin cells. These effects are not observed with the cryoextract of rose petals (comparative) or with the placebo solution.

[0272] Thus, the bioactive fraction of rose petals according to the invention has a beneficial effect on the skin, making it possible to fight against skin cell aging.

[0273] Example 6: Effect of the isolated bioactive fraction of rose petals according to the invention on the circadian rhythm

[0274] The effect of the bioactive fraction of rose petals according to the invention on the rhythmic process of skin cells was tested. For this purpose, we studied, by immunofluorescence, the effect of the rose petal extract according to the invention on the expression of the CLOCK gene, a transcription factor playing a central role in the regeneration of skin cells. regulation of this pathway, notably by activating several elements of the signaling cascade enabling the generation of circadian rhythms.

[0275] Protocol:

[0276] - Human skin (biopsies according to Example 3.2) from two independent donors pendants.

[0277] - Conditions: the biopsies were treated twice during the 24h culture period by the various extracts or control solutions as indicated below.

[0278] (1) “Control”: diluted PBS,

[0279] (2) “ZF serum” at 0.3%,

[0280] (3) “Placebo” at 3%,

[0281] (4) “Cryo-extract” at 3%.

[0282] - Method of analysis: evaluation of CLOCK expression by immunofluorescence rescence, and quantification of results with Volocity according to the protocol detailed in Example 3.6.

[0283] The results in [Fig. 4] show that treatment with the bioactive fraction of rose petals according to the invention significantly increases (+16%) the expression of the CLOCK protein in human skin biopsies. Treatment with the cryo-extract of rose petals (comparative) or with the placebo solution does not result in a significant increase in CLOCK expression.

[0284] Thus, the bioactive fraction of rose petals according to the invention has a beneficial effect on the circadian rhythm of skin cells.

[0285] Example 7: Effect of the isolated bioactive fraction of rose petals according to the invention on skin nutrition

[0286] The effect of rose petal extract comprising an isolated bioactive fraction on skin nutrition was tested.

[0287] Nutrients and metabolites are essential for maintaining cellular homeostasis, particularly for mitochondrial respiration and ATP synthesis. Disruption of glucose metabolism can lead to cell death. 2-Deoxy-D-glucose (2-DG) is a well-known inhibitor of this metabolic pathway.

[0288] The potential protective effect of the rose petal extract according to the invention against glucose deprivation following 2-DG stress was therefore evaluated.

[0289] Protocol:

[0290] - Human keratinocyte cell cultures in culture medium for 48h.

[0291] - Conditions: the cell cultures were treated once during the first 24 hours of culture with the different extracts or control solutions as indicated below. Then, conditions (2) to (5) were treated with the 2-DG inhibitor at a concentration of 200pM for the last 24 hours of culture.

[0292] (1) “Control” diluted PBS, not stressed with 2-DG

[0293] (2) “Control”: diluted PBS,

[0294] (3) “ZF serum” at 0.1%,

[0295] (4) “Placebo” at 1%,

[0296] (5) “Cryo-extract” at 1%

[0297] - Method: Cell viability analysis by Neutral Red assessment according to the protocol described in Example 10.

[0298] The results in [Fig. 5] show that treatment with the 2-DG inhibitor leads to a significant decrease (-27%) in cell viability compared to the untreated PBS control. Advantageously, under 2-DG stress conditions, treatment with the isolated bioactive fraction of rose petals according to the invention significantly increases (+18%) cell viability compared to the PBS control condition. Under these same stress conditions, treatment with the cryoextract (comparative) or placebo does not result in a significant increase in cell viability compared to the PBS control condition.

[0299] Thus, the bioactive fraction of rose petals according to the invention has a protective effect against 2-DG stress and is shown to be beneficial for skin nutrition.

[0300] Example 8: Effect of the isolated bioactive fraction of rose petals according to the invention on skin firmness

[0301] The effect of rose petal extract comprising an isolated bioactive fraction on the firmness of skin tissue was tested.

[0302] Structural proteins, such as collagen and fibrillin, are essential components of the extracellular matrix, contributing to the firmness of skin tissue. Over time, and also with repeated stress, these proteins degrade and are less effectively renewed, contributing to tissue laxity and loss of skin firmness.

[0303] The potential protective effect of the isolated bioactive fraction of rose petals according to the invention on these extracellular matrix proteins was therefore evaluated.

[0304] 8.1. Materials and methods

[0305] The methods for obtaining biopsy tissues are described in Example 3.2.

[0306] The antibodies used are described in Example 3.4.

[0307] 8.1.1. Cryopreservation of biopsies

[0308] Following the treatments, to allow for the preservation and sectioning of the skin, the tissues were frozen in OCT medium (CellPath) using liquid nitrogen and stored at -20°C. The frozen skin biopsies were then cut with a cryotome (Leica) into 6 µm thick sections and placed on slides (Thermo Scientific).

[0309] 8.1.2. Immunofluorescence Protocol

[0310] The sections were dried for 30 minutes at 37°C, fixed in a bath The slides were soaked in cold acetone for 10 minutes and then rinsed in a PBS bath. After saturating the nonspecific sites with a 5% BSA solution (Sigma) for 30 minutes, the primary antibody (described in section 3.4) was applied, and the slides were incubated with shaking at room temperature in a humid environment. After rinsing the slides with PBS, the secondary antibody (described in section 3.4) was applied in the dark, with shaking, at room temperature in a humid environment. Finally, the cell nuclei were stained with 4',6'-diamidino-2-phenylindole (DAPI, Molecular Probes) at 0.3 pM for 5 minutes and the sections were mounted in Fluoromount-G (Electron Microscopy Sciences). Detection was performed using a Nikon Eclipse Ni-E microscope. Images were taken with a Nikon DS-Fi3 camera and processed using NiSAR acquisition software (Nikon).

[0311] 8.1.3. Imaging study (quantification)

[0312] The quantification of collagen I and collagen III immunostaining was performed using the Volocity image analysis software (Improvision). The area of ​​interest was selected based on fluorescence intensity. The results obtained are the sum of the intensities of the green pixels in the selected area. Finally, for each image, the sum obtained was adjusted to account for the area of ​​the dermis examined. Three to six images per condition were analyzed. The final quantification (Figures 6 and 7) represents the average of two independent experiments (n=6-12).

[0313] Fibrillin-1 quantification was performed using ImageJ software. Fibers from the upper dermis were selected at a constant depth for all conditions. This selection was used for quantitative analysis. The total length of the selected fibers was measured and normalized by the length of the dermo-epidermal junction. Five images per condition were analyzed. The final quantification ([Fig. 8]) represents the mean of two independent experiments (n=12).

[0314] 8.1.4. Statistical analysis

[0315] Statistical analyses were performed using the Student's t-test for independent samples with two-tailed rejection. p<0.05 was considered significant, p<0.01 highly significant, and p<0.005 highly significant.

[0316] 8.2, Study Protocol:

[0317] 8.2.1. For the study on collagen III:

[0318] Skin biopsies were treated, or not, in duplicate, with the extract according to the invention "ZF serum" diluted to 0.3% in PBS, twice daily for 72 hours. PBS alone was applied as the control condition (negative control).

[0319] For each experiment and each condition tested, 20 pL of solutions were applied to the biopsies.

[0320] 8.2.2. For the study on collagen I:

[0321] Skin biopsies were treated, in duplicate, with the rose flower cryo-extract, notably described in patent application EP0425391 and its examples (“benchmark” positive control), or with a 3% hydro-glycerin (water / glycerol) solution (“placebo” negative control), or with the extract according to the invention, “ZF serum” at 0.3% diluted in PBS, twice daily for 48 hours. PBS alone was applied as the control condition (second negative control). The total dry matter (active substance) contents are equivalent for the “ZF fraction” 0.3% condition (invention) and for the “cryo-extract” 3% condition (positive control).

[0322] Application of pro-senescent stress: A "senescent environment" type culture medium was prepared by mixing "fresh" skin biopsy culture medium with senescent fibroblast culture medium (in a 50 / 50 ratio). The "young environment" type culture medium, used as a control, was prepared by mixing "fresh" skin biopsy culture medium with non-senescent fibroblast culture medium (in a 50 / 50 ratio).

[0323] For each experiment and each condition tested, 20 pL of solutions were applied to the biopsies.

[0324] 8.2.3. For the study on fibrillin-1:

[0325] Skin biopsies were treated, in duplicate, with the rose flower cryoextract as described in the paragraph above) (benchmark, positive control) or placebo (hydroglycerin solution, negative control) at 3%, or with the extract according to the invention, "ZF serum" at 0.3% diluted in PBS, twice daily for 48 hours. PBS alone was applied as the control condition (second negative control).

[0326] Application of UV stress: the biopsies were subjected to stress with 5 J / cm2 of UVA (BLX-E365 type oven, Fisher Bioblock Scientific) followed by 200 mJ / cm2 of UVB (BLX-E312 type oven, Fisher Bioblock Scientific) after the first 24 hours of treatment.

[0327] For each experiment and each condition tested, 20 pL of solutions were applied to the biopsies.

[0328] 8.3. Results

[0329] 8.3.1. Results on collagen III expression

[0330] The results of [Fig.6] show that, in the condition treated with ZF serum In Rose de Granville® (according to the invention), biopsies show a significant increase in the amount of collagen III (+59%) compared to the control condition (PBS alone).

[0331] 8.3.2. Results on collagen I expression

[0332] The results of [Fig.7] show that skin biopsies cultured in a “Senescent environments” show a significant decrease (-27%) in the quantity of collagen I compared to biopsies cultured in a "young environment." Application of the ZF Rose de Granville® serum (according to the invention) for 48 hours is associated with a highly significant increase in the amount of collagen I (+21%, compared to the untreated control) in skin biopsies cultured in a senescent environment. This result is comparable to the positive control (cryoextract of rose flowers - "benchmark").

[0333] 8.3.3. Results on fibrillin-1 fiber length

[0334] The results in [Fig. 8] show that UV stress induces a decrease in fibrillin-1 fiber length (-17%). Under UV stress conditions, application of ZF Rose de Granville® serum (according to the invention) for 48 hours is associated with a highly significant improvement in fibrillin-1 structure. Under these conditions, application of the rose flower cryoextract (“benchmark”) showed no activity.

[0335] 8.4. Conclusions

[0336] Ex vivo tests were performed on biopsies of normal human skin treated with ZF Rose de Granville® serum (according to the invention). A comparison with the reference product and a placebo was carried out to evaluate the amount of collagen I and the length of fibrillin-1 fibers under UV stress conditions. First, an increase in the amount of collagen III was observed in association with the application of ZF Rose de Granville® serum (according to the invention).

[0337] Next, the Applicant was able to demonstrate, in a senescent model, a restoration of collagen I expression after application of the ZF Rose de Granville® serum (according to the invention). The same effect was observed after application of the cryo-extract of rose flowers (comparative).

[0338] Finally, the application of ZF Rose de Granville® serum (according to the invention) resulted in an improvement of fibrillin-1 fiber in skin exposed to UVA and UVB radiation. Under these conditions, no effect of the rose flower cryo-extract (comparative) was observed.

[0339] Taken together, these results show that the rose petal extract comprising a bioactive fraction isolated according to the invention is capable of promoting the expression of extracellular matrix proteins. It therefore has cosmetic potential for promoting and / or improving skin firmness and / or reducing loss of firmness, particularly related to aging.

[0340] Example 9: Effect of the isolated bioactive fraction of rose petals on microinflammation

[0341] The effect of the bioactive fraction of rose petals according to the invention on cutaneous micro-inflammation has been tested.

[0342] In addition to its role in the skin barrier function, HMGB1 plays an active role in the phenomena of cutaneous micro-inflammation. Under normal conditions, i.e. (without stress conditions), HMGB1 is localized in the cell nucleus, however in response to a stimulus, and particularly in response to pro-inflammatory signals, HMGB1 is actively relocalized to the cytoplasm and / or the extracellular space.

[0343] 9.1 Capacity of the bioactive fraction of rose petals according to the invention to produce an "oxytocin-like" effect

[0344] Oxytocin (OXT) is typically a stress response hormone. Under stressful conditions, oxytocin modulates the physiological and biological activities of cells, notably by reducing the release of HMGBl. Thus, by reducing cellular stress, oxytocin contributes to skin health.

[0345] The ability of the bioactive fraction of rose petals according to the invention to produce an "oxytocin-like" effect was thus evaluated by studying in particular its ability to limit the secretion of HMGB1 under conditions of cellular stress by LPS.

[0346] Protocol:

[0347] - Human keratinocyte cell cultures in culture medium for 30h.

[0348] Conditions: Keratinocytes were treated for 24 hours with the various extracts or control solutions as indicated below. Then, conditions (2) to (6) were treated with the stress factor LPS at a concentration of 0.5 mg / mL for 6 hours.

[0349] (1) “Control” diluted PBS, not stressed with LPS

[0350] (2) “Control”: diluted PBS,

[0351] (3) “Placebo” at 1%,

[0352] (4) “Cryo-extract” at 1%,

[0353] (5) “ZF serum” at 0.1%,

[0354] (6) “Oxytocin” (Sigma) at 200pM (positive control).

[0355] - Method: Western blot analysis of the quantities of HMGB1 protein in the supernatant and in the cell lysate according to the protocol detailed in Example 3.5.

[0356] The results in [Fig. 9A] and [Fig. 9B] show that under control conditions (PBS), treatment with the LPS stress factor induces the secretion of all (100%) of HMGB1 outside the cell. Under LPS stress conditions, treatment with the bioactive fraction of rose petals according to the invention induces a reduction (-31%) in HMGB1 secretion into the supernatant compared to the PBS control. This reduction is greater than that obtained with the positive oxytocin control (-22%) or with the cryoextract (-19%). The placebo has no effect on HMGB1 secretion.

[0357] The band observed on [Fig.9A] at 49KDa is not specific and was not taken into account for the analysis of the results.

[0358] The bioactive fraction of rose petals according to the invention therefore produces an effect "oxytocin-like".

[0359] 9.2 Capacity of the bioactive fraction of rose petals according to the invention to be emptied the expression of COX2

[0360] The initial phase of HMGB1 secretion requires a pro-inflammatory signal such as liposaccharide (LPS), IL-1 (interleukin 1), or TNF (tumor necrosis factor). HMGB1 can then interact with Toll-like receptors (TLRs) to activate certain signaling pathways leading to the production of cytokines and chemokines such as TNF-α. Under specific inflammatory conditions, HMGB1 induces the expression of the COX-2 protein, notably via the TNF-α signaling pathway.

[0361] The ability of the bioactive fraction of rose petals according to the invention to modulate the expression of COX-2 following pro-inflammatory stress (micro-inflammation) induced by LPS was therefore evaluated.

[0362] Protocol:

[0363] - Human skin (biopsies according to Example 3.2) from two independent donors pendants.

[0364] - Conditions: the biopsies were processed twice daily for 48 hours by the different extracts or control solutions as indicated below. Additionally, conditions (2) to (5) were treated with the stress factor LPS at a concentration of 0.5 mg / mL during the last 6 hours of treatment.

[0365] (1) “Control” diluted PBS, not stressed with LPS

[0366] (2) “Control”: diluted PBS,

[0367] (3) “ZF serum” at 0.3%,

[0368] (4) “Placebo” at 3%,

[0369] (5) “Cryo-extract” at 3%

[0370] - Method of analysis: evaluation of COX 2 expression by immunofluorescence, and quantification of results with Volocity according to the protocol detailed in Example 1.8.

[0371] The results in [Fig. 10] show that under LPS stress conditions, COX-2 expression in the PBS control condition is significantly increased (+55%) compared to the unstressed PBS control. Under LPS stress conditions, treatment with the bioactive fraction of rose petals according to the invention restores a level of COX-2 expression comparable to that observed in the absence of stress (unstressed PBS). This effect is not observed during treatment with the cryoextract or with the placebo solution.

[0372] The bioactive fraction of rose petals according to the invention therefore makes it possible to negatively regulate the expression of COX-2.

[0373] Thus, both by its effects on the retention of HMGB1 in cells, and by its effects on the expression of COX-2, the bioactive fraction of rose petals according to the invention exerts beneficial effects on cutaneous micro-inflammation.

[0374] 9.3. Capacity of the bioactive fraction of rose petals according to the invention to modulate expression of transglutaminase-i (TGM1}

[0375] Similarly, the ability of rose petal extract in the form of the isolated bioactive fraction according to the invention, as prepared in Example 1.1, to restore TGM1 expression after LPS (Lipopolysaccharide, Sigma)-induced inflammatory stress was evaluated. TGM-1 is a gene encoding transglutaminase-1, a protein essential for the keratinization of the stratum corneum of the skin.

[0376] Protocol:

[0377] - Human skin (biopsies according to Example 3.2) from two independent donors pendants.

[0378] - Conditions: the biopsies were processed twice daily for 48 hours by the different extracts or control solutions as indicated below. Additionally, conditions (2) to (5) were treated with the stress factor LPS at a concentration of 0.5 mg / mL during the last 6 hours of treatment.

[0379] (1) “Control” diluted PBS, not stressed with LPS

[0380] (2) “Control”: diluted PBS,

[0381] (3) “ZF serum” at 0.3%,

[0382] (4) “Placebo” at 3%,

[0383] (5) “Cryo-extract” at 3%

[0384] - Method of analysis: evaluation of TGM1 expression by immunofluorescence, and quantification of results with Volocity according to the protocol detailed in Example 3.6.

[0385] The results in [Fig. 11] show that under LPS stress conditions, TGM1 protein expression in the PBS control condition is significantly reduced (-49%) compared to the unstressed PBS control. Interestingly, under LPS stress conditions, treatment with the bioactive fraction of rose petals according to the invention results in a significant increase (+34%) in TGM1 expression levels compared to the PBS control. A greater increase in TGM-1 expression is observed with the placebo solution and the rose petal cryo-extract (comparative). It should be noted that the increase observed with the placebo was expected, as it contains citric acid, a compound commonly used in moisturizing formulations. Nevertheless, the effect observed with the extract according to the invention remains surprising given the lower concentration at which it is tested (lOx is less concentrated than the cryo-extract)..

[0386] Thus, by virtue of its effects on the expression of filaggrin and TGM-1, the rose petal extract in the form of a bioactive fraction according to the invention has, also under stress conditions, a beneficial effect on the skin barrier function. Example 10: Cosmetic formulations

[0387] 10.1 Composition in the form of an aqueous salt for the visase

[0388] [Tables4] Cosmetic Ingredients Content Isolated bioactive fraction of rose petals according to the invention * 50.00% Preservatives 0.50% Sugar 0.20% Carbomer 0.70% Purified water q.s. 100% Tetrasodium EDTA powder 0.20% Sodium hydroxide 0.17%

[0389] * as described in Example 1

[0390] The isolated bioactive fraction of rose petals according to the invention and the preservatives are mixed and homogenized at room temperature with stirring. The sugars are added with stirring, then the carbomer, then the water and EDTA with stirring. The aqueous gel is then neutralized by adding sodium hydroxide with stirring until a homogeneous gel is obtained.

[0391] Applied to the skin of the face, this aqueous gel gives a cooling effect, and brings suppleness and firmness to the skin.

[0392] 10.2 Composition in the form of a micronutrient gel-serum for the face

[0393] [Tables5] Cosmetic Ingredients Content 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% Nacres 1.0% Rose Cryoextract* 3.0% Centella Asiatica Extract 0.5% Horse Chestnut Extract 0.1% Isolated Bioactive Fraction of Rose Petals According to the Invention* 0.3% Tocopheryl Acetate 0.10%

[0394] * as described in Example 1.1 or 1.2

[0395] The aqueous phase ingredients (water, glycols, and carbomer) are mixed at 80°C with stirring. Preservatives are then added, followed by gelling agents at 80°C with stirring, and the temperature is then lowered to 75°C. Surfactants are then emulsified in the aqueous phase with stirring. The fillers and pearlescent pigments, previously mixed and homogenized, are added at 40°C. The cryo-extract, the isolated bioactive fraction of rose petals according to the invention, and the other extracts are then added at 40°C, with stirring until the mixture reaches 30°C.

[0396] After application to the face, the skin appears nourished, firmer, smoother (reduction in the appearance of wrinkles and fine lines), hydrated and more evenly coloured.

Claims

Demands

1. Rose extract in the form of an isolated bioactive fraction, obtained from fresh roses of the Evanrat variety, characterized in that it is obtained by a process comprising the steps: a) cleaning of the plant material, maceration, pressing and then mechanical separation of the plant material to obtain an intracellular colloidal dispersion (ICD) and a fiber-enriched material (fraction A); b) destabilization of the ICD with electromagnetic waves and then mechanical separation of the intracellular colloidal dispersion (ICD) to obtain supernatant A and a Membrane Fraction (fraction B); c) adjustment of the pH level in supernatant A until a pH greater than 6 is obtained, preferably a pH ranging from 6 to 7, and then mechanical separation of supernatant A to obtain supernatant B and fraction C (cytoplasmic fraction);d) Adjustment of the pH level in supernatant B until a pH below 4.5 is obtained, then mechanical separation of supernatant B to give a "Bioactive Serum Fraction" and a fraction D (precipitate); and e) Optionally, mixing of the "Bioactive Serum Fraction" with at least one preservative and / or stabilizer.

2. Rose extract in the form of an isolated bioactive fraction obtainable by the process according to claim 1, wherein said extract comprises at least 500 pg / pL of at least one monosaccharide sugar selected from fructose, glucose and sucrose, and / or at least 500 mg / Kg of at least one mineral selected from potassium, calcium and sodium.

3. Extract in the form of a bioactive fraction isolated from roses according to claim 1 or claim 2, characterized in that said extract is obtained from fresh rose petals of the Evanrat variety.

4. A process for preparing an extract in the form of an isolated bioactive fraction from fresh roses of the Evanrat variety or the 'Rose Jardin de Granville®' variety, as defined in any one of claims 1 to 3, comprising the steps of: a) cleaning the plant material, macerating, pressing, and then mechanically separating the plant material to obtain an intracellular colloidal dispersion (ICD) and a fiber-enriched material (fraction A); b) destabilizing the ICD with electromagnetic waves and then se- mechanical preparation of the intracellular colloidal dispersion (ICD) to obtain supernatant A and a Membrane Fraction (fraction B); c) adjustment of the pH level in supernatant A until a pH greater than 6 is obtained, 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 supernatant B until a pH less than 4.5 is obtained, then mechanical separation of supernatant B to give a "Bioactive serum fraction" and a fraction D (precipitate); and e) Optionally, mixing of the "Bioactive serum fraction" with at least one preservative and / or stabilizer.

5. 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 an effective amount of at least one extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety as defined in any one of claims 1 to 4.

6. Composition according to claim 5, characterized in that the extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety is present in the composition in a content ranging from 0.001 to 50%, in particular from 0.01 to 20%, preferably from 0.01 to 10% and preferably still from 0.011 to 5% by weight of raw material in relation to the total weight of said composition.

7. A cosmetic treatment method for healthy skin and / or healthy lips, intended to promote and / or improve skin nutrition; promote and / or improve skin firmness; promote and / or improve the skin barrier function; promote and / or improve the rhythmic process of skin cells; and / or prevent and / or slow down skin aging, comprising the application to healthy skin and / or healthy lips, in particular facial and / or neck skin, of a cosmetic composition as defined in any one of claims 5 or 6.

8. Use of at least an effective amount of at least one extract in the form of an isolated bioactive fraction of fresh roses of the Evanrat variety as defined in any one of claims 1 to 4 as an agent to promote and / or improve skin nutrition; promote and / or improve skin firmness; promote and / or improve the skin barrier function, promote and / or improve the rhythmic process of skin cells, prevent and / or slow down the aging of the skin and / or lips, especially the skin of the face and / or neck.

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