EP4099975A1 - Method for obtaining an aqueous extract of lavender, compositions comprising such an extract and their cosmetic uses - Google Patents

Abstract

The invention relates to a method for obtaining an aqueous extract of lavender enriched in small RNAs being a maximum of 150 nucleotides in length, sugars, phenolic compounds and organic acids, and free of DNA, obtained by the following method: the aerial parts of lavender are placed in water; phytic acid is added at a pH of between 10 and 11; the pH of the obtained mixture is then adjusted to a value between 6 and 8, and the obtained mixture is purified. The invention also relates to cosmetic compositions comprising such an extract and their cosmetic uses for protecting the skin from external aggressions and oxidation, combating the signs of skin ageing, increasing photoprotection, lightening the skin, improving hydration, improving the barrier function, soothing the skin or improving the biological mechanisms associated with skin repair during the night.

Description

Description

Title of the invention: PROCESS FOR OBTAINING AN AQUEOUS LAVENDER EXTRACT, COMPOSITIONS INCLUDING SUCH EXTRACT AND THEIR USES

COSMETICS

Technical area

[1] The invention relates to the field of cosmetics and more particularly to active ingredients of natural origin used in the preparation of cosmetic formulation for improving the appearance of the skin or protecting it.

[2] The invention relates to a process for obtaining an aqueous extract of lavender as well as the extract enriched in small RNA, in sugars, in phenolic compounds, in organic acids obtained by the process, the cosmetic compositions comprising such extracts and their cosmetic uses for the care of the skin, scalp and integuments and more particularly to protect the skin from external aggressions and oxidation, fight against the signs of skin aging, increase photoprotection, lighten the skin, improve skin hydration, strengthen the barrier function, soothe the skin or to improve the biological mechanisms associated with skin repair at night.

Technical background of the invention

[3] Plants of the genus Lavandula, commonly known as lavender, form a group of 47 species. Among the best known and used species are Lavandula angustifolia, also called true lavender, a wild species native to Provence (south-eastern France), or lavandin, a hybrid resulting from the crossing of true lavender and lavender aspic.

[4] True lavender is widely used for the production of essential oils in Europe, North Africa, the Middle East and Asia, particularly for the perfume industry.

[5] Essential oil (ET) is generally obtained by hydrodistillation. The previously dried flowers are subjected to a stream of water vapor which entrains the volatile or soluble constituents are then recondensed to obtain a hydrosol (or floral water) and a supernatant comprising the lipid parts of the plant and constituting the essential oil.

[6] Lavender EOs are very fragrant and predominantly include volatile monoterpenes such as linalool and linalyl acetate. In true lavender IΉE, both are present at about 30%. Lavender EO is particularly recognized for its sedative, analgesic, analgesic, anti-inflammatory, antiseptic and antibacterial properties. It is also antispasmodic and decongestant and indicated to soothe skin conditions and promote healing. Lavender EO is also indicated to improve sleep disorders and various tensions (anxiety, stress, etc.).

[7] Lavender floral waters contain less than a few percent of essential oil-like volatile organic compounds, as well as the water-soluble compounds of the plant. Floral water has the same properties as lavender essential oil, but more attenuated due to the lower concentrations of terpene compounds.

[8] The very limited cosmetic use of lavender essential oils and floral waters is in part due to the majority presence of terpene molecules known to have irritant effects on the skin. Linalool, for example, has been recognized as a potentially allergenic and the use of lavender essential oil is not recommended for pregnant women and children under 12 years old.

[9] Most of the other lavender extraction methods described in the prior art use non-polar organic solvents which mainly extract volatile odorous compounds (CN10338583,

JP11199469) or a supercritical fluid (KR2015042999). In the latter case, the extract obtained is rich in polyphenols and flavonoids, but does not contain other compounds of interest such as amino acids, organic acids or proteins. In addition, phenolic acids are not extracted by this type of technique, in fact these molecules are mainly extracted in a polar solvent and ideally with water. [10] Consumers of cosmetic products want to use formulas that are as natural as possible, while exhibiting as great or even better efficacy than synthetic products.

[11] Despite the various anti-aging cosmetic products already on the market, the need for new and effective cosmetic ingredients of natural origin is growing.

[12] One problem which the invention proposes to solve is to provide a new aqueous extract of lavender meeting the requirements of the current cosmetic market concerning the criteria of naturalness and nevertheless exhibiting remarkable biological efficacy.

[13] Another problem which the invention proposes to solve is to provide a new aqueous extract of lavender which does not represent the defects of the extracts currently known, namely a strong odor, an instability of the EOs in the formulas for topical application, or else an irritant or allergenic nature due to the presence of terpene molecules such as linalool.

[14] A problem which the invention still proposes to solve is to provide a new aqueous extract of lavender enriched with compounds known for their effectiveness on the skin such as small RNAs, sugars, phenolic compounds and organic acids.

[15] The inventors have developed a new extract of lavender flower specifically enriched with small RNAs, sugars, phenolic compounds and organic acids, and which does not have the drawbacks of the methods of the prior art cited, such as, for example, use of potentially toxic detergents and solvents in cosmetics.

[16] The extract thus obtained can be used in cosmetics for the care of the skin, scalp and integuments and more particularly to protect the skin from external aggressions and oxidation, to fight against the signs of skin aging, increase photoprotection, lighten the skin, improve skin hydration, strengthen the barrier function or even soothe the skin.

Summary of the invention [17] The first subject of the invention is a process for obtaining an aqueous extract of aerial parts of lavender, comprising the following steps: a) the aerial parts of lavender are brought into contact with water; b) phytic acid is added at a concentration of between 1 and 10 mM in the mixture obtained in a) at a pH of between 10 and 11; c) the pH of the mixture obtained in b) is then adjusted to a value between 6 and 8; d) the mixture obtained in c) is purified so as to eliminate the residual solid plant material and to obtain a purified aqueous crude extract; and e) the pH is checked and it is readjusted if necessary to a value between 6 and 8, preferably between 6 and 6.5.

[18] In addition, a second subject of the invention is an aqueous extract of aerial parts of lavender enriched in small RNA with a length of at most 150 nucleotides, in sugars, in phenolic compounds and in organic acids, devoid of DNA, obtainable by the process of one of claims 1 to 5, characterized in that it comprises, by weight of the total weight of the extract, 10 to 30 g / kg of dry weight, containing 2 to 10 g / kg of sugars, 100 to 1500 mg / kg of organic acids, 500 to 2000 mg / kg of phenolic compounds and 40 to 200 mg / kg of low molecular weight RNA up to a length of 150 nucleotides.

[19] The third object of the invention is a cosmetic composition comprising, as an active ingredient, an effective amount of the extract of one of claims 6 or 7, and a physiologically acceptable medium.

[20] A fourth subject of the invention is the cosmetic use of the composition of the invention, for the care of the skin, the scalp and integuments, more particularly for protecting the skin from external attacks and from oxidation. , fight against the signs of skin aging, increase photoprotection, lighten the skin, improve skin hydration, strengthen the barrier function, soothe the skin, or even to improve the biological mechanisms associated with the repair of the skin during night.

Brief description of the drawings [21] The invention and the advantages which result therefrom will be better understood on reading the description and the non-limiting embodiments which follow, illustrated with reference to the appended figures in which:

[22] [Fig. 1] Characterization of organic acids by HPLC-MS analysis

[23] [Fig. 2] Analysis of RNAs of small molecular weight by the Bioanalyzer 2100. A: lavender extract obtained according to the process of the invention - B: conventional lavender extract.

Detailed description of the invention

Definitions

[24] All the terms used in the present description have the most widely known meaning unless otherwise specified. For the purposes of the invention, the following terms are defined as follows:

[25] By "lavender" is meant all species of the genus Lavandula, as well as their hybrids (such as lavandin).

[26] By "aerial parts" is meant the stems and flowers of lavender. The seeds are included in the "aerial parts" within the meaning of the invention.

[27] In the course of the present description, the terms “aerial parts” and “flowers” will be used interchangeably to designate the flowers and the thinnest stems bearing the flowers.

[28] By "small RNA" or "RNA of small molecular weight", or "small RNA of a maximum length of 150 nucleotides" is meant non-coding RNAs (ribonucleic acids), of low molecular weight, of a length of at most 150 nucleotides, such as all types of small non-messenger RNAs, single and / or double strands, for example microRNAs, interfering RNAs, introns, small nuclear RNAs or even any fragment of RNA. Electrophoresis analysis shows that the small RNAs present in the lavender extract of the invention have varied molecular weights of approximately between 30 and 150 nucleotides. [29] By "organic acids" is meant α-hydroxy acids (or AHAs), that is to say carboxylic acids derived from fruit or plant sugars, such as glycolic, malic, citric or tartaric acids. , succinic and uronic.

[30] By phenolic compounds (or polyphenols) is meant molecules of plant origin which have an aromatic ring carrying one or more hydroxyl groups, such as phenolic acids, flavonoids or their derivatives. Polyphenolic compounds are known to be powerful antioxidant molecules.

[31] The term “sugars” means monosaccharides and more particularly glucose and fructose as well as oligo and polysaccharides.

[32] The term "phytomolecules of interest" means all of the molecules present in the lavender extract of the invention and in particular, small RNAs with a length of at most 150 nucleotides, sugars, phenolic compounds and organic acids.

[33] When a range of values is described, the bounds of that range should be understood as explicitly including all intermediate values of the range. For example, a range of values between 1% and 10% should be understood as including 1%, 2%, 3%, 4%, 5%, 6%, 1%, 8%, 9%, and 10%, as well as all decimal values between 1% and 10%.

[34] The numerical values in percent are percentages by weight, that is to say the weight of a compound compared to the total weight of the mixture envisaged, unless specified otherwise.

[35] The compositions described in the present application can

"Include", "consist of" or "consist essentially of" the essential compounds or optional ingredients.

[36] "Consist essentially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not modify the basic characteristics or the new characteristics of the composition or the use described herein. request. [37] A "physiologically acceptable medium" means a vehicle suitable for contact with the outer layers of the skin or mucous membranes, without toxicity, irritation, undue allergic and similar response or intolerance reaction, and proportionate to a ratio. reasonable benefit / risk.

[38] By "topical application" is meant the fact of applying or spreading the aqueous extract enriched in small RNA with a length of at most 150 nucleotides, in sugars, in phenolic compounds and in organic acids according to invention, or a composition containing it, on the surface of the skin or of a mucous membrane.

[39] "Skin" refers to the skin of the face, including the area around the eyes and mouth, nose, forehead, neck, hands, but also the skin of the whole body.

[40] "Scalp" refers to the skin covering the skull, including hair follicles and inter-follicular skin spaces.

[41] “Integuments” in products designate hair follicles (hair and body hair) as well as the nails, which are rich in keratin.

[42] The term “reinforcing the barrier function” means that the protective properties of the skin against external aggressions (UV radiation, visible or infrared light, pollution, microorganisms, etc.) are improved.

[43] "Soothing the skin" is understood to reduce the irritant reactions that manifest as a feeling of discomfort, which may be accompanied by redness.

[44] "Lightening the skin" means reducing the intensity of the color of the skin linked to the melanin content of the epidermis, either homogeneously or locally by acting on pigment disorders, such as dark spots. senescence or senile lentigo.

[45] By "effective amount" is meant the minimum amount of extract according to the invention which is necessary to obtain at least one of the desired biological activities, in particular to exhibit an antioxidant activity vis-à-vis the reactive species of l. oxygen, increase melatonin or decrease melanin, or any other biological marker studied, without this quantity being toxic. [46] By “skin hydration” is meant the water content and distribution of the upper layers of the epidermis.

[47] “Improvement of skin hydration” means any improvement in changes in the external appearance of the skin due to dehydration such as, for example, dryness, tightness and discomfort, whether this condition is related to internal or external factors, such as adverse environmental conditions.

[48] By "signs of skin aging" is meant any changes in the external appearance of the skin due to aging such as, for example, fine lines and wrinkles, cracks, bags under the eyes, dark circles, wilting, loss of elasticity, firmness and / or tone of the skin, but also any internal skin changes that do not systematically result in a modified external appearance such as, for example, thinning of the skin, or all internal degradation of the skin resulting from environmental stresses such as pollution and solar radiation including UV rays.

[49] The term “signs of skin aging” also means pigmentary disorders such as senile lentigo or solar lentigo.

[50] By "external attacks" we mean solar radiation, including visible light, UV and infrared radiation, pollution, which may come from the ambient atmosphere outside or inside homes and including particles of different sizes (10 µm for PM 10, 2.5 µm for PM 2.5, or less than 100 nm for ultrafine particles) as well as several chemical elements (volatile organic compounds, polycyclic aromatic hydrocarbons, heavy metals. .).

[51] By "improving the appearance of the skin" we mean that the skin texture appears finer, the luminosity more intense and the complexion more homogeneous.

[52] It is understood that the invention relates to mammals and more particularly to humans.

Extraction process [53] The conventionally described ribonucleic acid (RNA) extraction protocols use solvents unsuitable for cosmetic use (Zumbo, P. 2014 "Phenol-chloroform Extraction", 2014). These methods aim to obtain nucleic acids (RNA or DNA or small RNA) completely purified, that is to say free of any other molecule of interest such as secondary metabolites, vitamins, sugars, peptides, etc. which may have beneficial effects for the skin and are therefore of cosmetic interest.

[54] Document FR2831168 is also known, which describes a process for obtaining a plant extract rich in nucleic acids (DNA and / or RNA).

The process uses cellulolytic enzymes.

[55] Patent documents EP1723958 and W003101376 are also known from the prior art which describe a composition for topical application comprising a synthetic double-stranded RNA oligonucleotide with a length of 12 to 40 nucleotides, of known sequence having a siRNA function ("short interfering").

[56] Document FR 1502361 (also published under the number WO2017084958) is also known, which describes a process for obtaining an aqueous extract of plants, enriched with low molecular weight ribonucleic acids (RNA), to prepare cosmetic compositions. . The process uses EDTA at a concentration of between 2 and 15 mM.

[57] Using phytic acid instead of EDTA has the following advantages: Unlike EDTA, phytic acid is a naturally occurring molecule found in the husk of seeds, such as grains and legumes. Therefore, the use of phytic acid makes it possible to obtain a lavender extract that is 100% of natural origin while maintaining good extraction efficiency of the phytomolecules contained in the plant. The efficiency of extracting small RNAs as well as those of other compounds present in lavender flowers such as sugars, phenolic compounds, or organic acids such as tartaric, malic or citric acid. [58] The first subject of the invention is thus an extraction process used to obtain an aqueous extract of the aerial, dried or fresh parts of lavender.

[59] The extraction process of the invention makes it possible to obtain an extract rich in phytomolecules of cosmetic interest such as small RNAs with a maximum length of 150 nucleotides, sugars, phenolic compounds and acids. organic, avoiding the use of solvents that are not considered cosmetic solvents.

[60] The process of the invention has a reduced impact on the environment.

[61] In a first step a) of the process according to the invention, the aerial parts of lavender are mixed with water. The water used is distilled, demineralized water or water rich in mineral salts and / or trace elements. The water preferably used is distilled water.

[62] Preferably the aerial parts of lavender are the lavender flowers and the small stems bearing the flowers.

[63] Preferably the species of lavender used is Lavandula angustifolia or true lavender.

[64] Preferably the aerial parts of lavender are in dry form.

[65] Preferably, the aerial parts of lavender are ground into powder form before being placed in the presence of water in step a). Grinding the aerial parts of lavender is a mechanical action that allows better extraction. Mechanical grinding to obtain the plant material in powder form, followed by alkaline lysis in the presence of phytic acid promotes the complete destructuring of cell membranes and in particular of the nuclear membrane. Preferably, the aerial parts of lavender previously ground in powder form are mixed with water, in step a) in a plant material / water ratio of 3 to 20% by weight / weight, more preferably in a ratio between 3 and 10%, for example in a ratio of 3%, 5% or 10% (weight / weight).

[66] Phytic acid is then added in step b) to the mixture obtained in a). The pH at this stage should be basic, at a value between 10 and 11 and must therefore be adjusted, if necessary, by adding sodium hydroxide (NaOH). During step b) it is essential that the pH is basic, between 10 and 11. Preferably, the pH is adjusted to a value between 10.5 and 11. Indeed, this pH level, associated with the action of phytic acid, causes the destructuring of the cell membrane, including the nuclear membrane, the lysis of cells and the denaturation of DNA (the 2 strands of the double helix are separated). Phytic acid weakens and destructures the pecto-cellulosic membranes of plant cells by sequestering by complexation the divalent ions such as calcium ions which form ionic bridges between the pectin molecules surrounding the cellulose microfibrils. The consequence of this is to promote the release of the cellular content during the extraction. The step of treatment with phytic acid is essential for enriching the extract in RNA of small molecular weights and more generally ensuring a better yield of extraction of the other phytomolecules of interest; namely sugars, phenolic compounds and organic acids.

[67] The pH control shows that it remains basic and stabilizes between 9 and

11 at the end of step b).

[68] The extraction process makes it possible to enrich the final extract with low molecular weight RNA through the use of an aqueous extraction solution containing in particular a natural chelating agent such as phytic acid. Phytic acid is a molecule found naturally in the husk of seeds, such as grains and legumes. Phytic acid is present in the form of calcium salts or sometimes magnesium, and it plays an important role for the plant, for example, it is the main source of phosphorus.

[69] Preferably, the phytic acid used is a phytic acid powder in the form of sodium salt at a concentration preferably between 1 and 10 mM, preferably between 1 and 5 mM and more preferably the concentration is 3. mM.

[70] The invention works particularly well for a phytic acid concentration between 2 and 3 as described in Table 1 below. It is thus observed that for only 2.25 mM of phytic acid the same results are obtained as with EDTA at 10 mM in terms of concentration of small RNA, as well as a similar overall extraction yield. A concentration of 3 mM allows an optimum extraction yield of RNAs of small molecular weights. The concentration of 3 mM is also optimal for having a better yield of other compounds of interest such as sugars, phenolic compounds and organic acids. With a phytic acid concentration of 4.5 mM the extraction yield of RNAs of small molecular weight is even higher, but the overall extraction yield decreases.

[71] [Table 1]

Table 1: Small molecular weight RNA yield and overall extraction yield as a function of phytic acid concentration

[72] Stage b) of treatment with phytic acid preferably lasts at least 1 hour, at a temperature between 20 and 80 ° C. During this step, the mixture obtained in a) is advantageously stirred.

[73] Advantageously, it is possible at the end of step b) to add diatomaceous earth in order to subsequently facilitate the separation between the solid residual plant material and the extract (soluble fraction) in step next.

[74] In a step c), the pH of the mixture obtained in b) is then adjusted to a value between 6 and 8.

[75] The pH can be adjusted by adding a solution of hydrochloric acid (HCl) or any other equivalent acid, compatible with cosmetic use. Acidification causes the sudden renaturation of DNA (re-pairing of the double helix strands). However, the chromosomal DNA, which is very long, does not not to re-pair completely and form insoluble tangles. On the contrary, the small RNAs remain in solution. DNA and small RNAs are thus separated into two distinct phases; a solid phase containing, among other things, chromosomal DNA, and a liquid phase containing, among other things, small RNAs. The pH adjustment step in step d) of the process according to the invention is an essential step for the optimum extraction of low molecular weight RNAs, as well as other phytomolecules of interest; namely sugars, phenolic compounds and organic acids.

[76] In step d), the mixture obtained in c) is purified so as to remove the aerial parts of residual solid lavender and to recover the soluble part which constitutes the crude aqueous extract according to the invention. Any method known to those skilled in the art can be used. For example, the mixture obtained in c) can be filtered through filters with a porosity greater than 30 μm so as to collect the filtrate. Preferably, the mixture obtained in c) is centrifuged at low speed, for example for at least 10 min at 4000 g, so as to sediment the residual plant material in the pellet and recover the crude aqueous extract in the supernatant.

[77] In a step e) the pH is checked and it is readjusted to a value between 6 and 8. Preferably the pH is readjusted to a value between 6 and 6.5, even more preferably to a value of 6, 5. The pH is readjusted by adding a solution of hydrochloric acid (HCl) or any equivalent acid compatible with cosmetic use.

[78] Indeed, a pH below 6 can lead to the precipitation of nucleic acids in general, and therefore that of small molecular weight RNAs of a maximum length of 150 nucleotides. The step of adjusting the pH in step e) of the process according to the invention is an essential step in order to have optimum stability of the low molecular weight RNAs in the extract.

[79] At the end of step e), a concentrated crude extract is obtained.

[80] Advantageously, the readjustment of the pH of step e) is preceded by at least one filtration of the crude aqueous extract obtained in d). Preferably Successive filtrations will be carried out by lowering the filtration threshold from 20 to 50 mih until a sterilizing filtration of 0.1 - 0.3 mih.

[81] The extract obtained in step e) can then be diluted in a physiologically acceptable solvent for cosmetic use, so that the dry weight is between 4 and 20 g / kg of dry extract relative to total weight of the diluted extract. This step improves the stability of the extract over time.

[82] The second subject of the invention is an aqueous extract of aerial parts of lavender enriched with small RNAs of a maximum length of 150 nucleotides, in sugars, in phenolic compounds, in organic acids and devoid of DNA, capable of to be obtained by the process described above. This extract does not contain DNA (deoxyribonucleic acid).

[83] A subject of the invention is also an aqueous extract of aerial parts of lavender enriched with small RNAs of a maximum length of 150 nucleotides, in sugars, in phenolic compounds and in organic acids, directly obtained by the method described. above. This extract does not contain DNA (deoxyribonucleic acid).

[84] By using the process of the invention according to steps a) to e), an aqueous concentrated crude extract of amber to dark amber lavender is obtained having a dry weight of 10 to 30 g / kg, containing 2 to 10 g / kg of sugars, 100 to 1500 mg / kg of organic acids, 500 to 2000 mg / kg of phenolic compounds and 40 to 200 mg / kg of low molecular weight RNA with a maximum length of 150 nucleotides . However, for aerial parts, in particular the lavender flower of the Lavandula angustifolia species, the extracts obtained can exhibit significant variability depending on factors such as the place or year of harvest, the season, the climatic conditions, biotic stress, etc.

[85] The extract thus obtained can then be diluted in a physiologically acceptable solvent for cosmetic use, so that the concentration of the extract is then adjusted to a dry weight of between 4 and 20 g / kg of extract. dry relative to the total weight of the diluted extract.

[86] By way of illustrative and nonlimiting examples of physiologically acceptable solvents, mention may be made of water, glycerol, ethanol, propanediol as well as its natural version called Zemea® from corn, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols or any mixture of these solvents.

Thus the extract obtained can be diluted to obtain a final concentration of 50% of butylene glycol derived from plants, or 50% of propanediol derived from plants or even 30% of glycerin derived from plants.

[87] Preferably, the extract obtained by the process according to the invention is diluted in butylene glycol so that the diluted extract comprises a final butylene glycol concentration of 50%.

[88] This so-called diluted extract comprises by weight of the total weight of the extract from 4 to 20 g / kg of dry extract, 0.5 to 10 g / kg of sugars, 50 to 700 mg / kg of organic acids , 50 to 1500 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA with a maximum length of 150 nucleotides.

[89] By way of nonlimiting example, mention may be made of a diluted extract of Lavandula angustifolia more particularly containing sugars at a concentration of 1.7 g / kg, organic acids at a content of 570 mg / kg, 620 mg. / kg of phenolic compounds and 45 mg / kg of low molecular weight RNA with a maximum length of 150 nucleotides.

[90] On the contrary, floral water and lavender essential oil mainly contain odorous molecules of terpene nature and do not contain RNA of small molecular weights of a maximum length of 150 nucleotides, nor of sugars. , nor of phenolic compounds or organic acids.

[91] The extract of the invention thus comprises a wide range of phytomolecules which may have beneficial effects on the skin, without presenting a risk of skin irritation or other damage to health.

[92] For example, sugars actively participate in the hydration of the epidermal layers, and in doing so in resistance to external aggressions, without exhibiting any undesirable effects. The lavender extract of the invention more particularly contains mono- and polysaccharides, which are present neither in floral water nor in essential oil of lavender. [93] True lavender is part of the Lamiaceae family which has a particular metabolism known as CAM for Crassulacean Acid Metabolism. The plant stores organic acids and more particularly malic acid, citric acid and tartaric acid inside its cells. The process described in the invention makes it possible to extract these organic acids or AHAs. Applied to the skin, these AHAs reduce cell cohesion between the corneocytes, cause the desquamation of the horny layers and thus stimulate cell renewal.

[94] The lavender extract according to the invention is also enriched in phenolic compounds, such as phenolic acids. These water-soluble molecules known for their antioxidant activity contribute to the antioxidant and protective potential of the lavender extract of the invention.

[95] A third aspect of the invention is a cosmetic composition comprising an effective amount of an aqueous extract enriched in small RNA with a length of at most 150 nucleotides, in sugars, in phenolic compounds and in organic acids obtained according to the invention, as an active ingredient, and a physiologically acceptable medium.

The aqueous extract enriched in small RNA with a length of at most 150 nucleotides, in sugars, in phenolic compounds and in organic acids, obtained according to the invention is advantageously used for the preparation of cosmetic compositions, as an ingredient. active.

[96] Advantageously, the extract of aerial parts of lavender according to the invention is added to the composition at a concentration of 0.05 to 5% by weight relative to the total weight of the composition, preferably at a concentration of 0, 1 to 2.5% by weight relative to the total weight of the composition and even more preferably at a concentration of 0.1 to 1.0% by weight relative to the total weight of the composition.

The composition which can be used according to the invention may be applied by any suitable route, in particular oral, or external topical, and the formulation of the compositions will be adapted by those skilled in the art.

[97] Preferably, the compositions according to the invention are provided in a form suitable for topical application. These compositions must therefore contain a physiologically acceptable medium, that is to say compatible with the skin and integuments, without risk of discomfort during their application and cover all suitable cosmetic forms.

[98] The compositions for implementing the invention may in particular be in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water, water-in-oil or multiple emulsions; they can also be in the form of suspensions, or even powders, suitable for application to the skin, mucous membranes, lips and / or hair.

[99] These compositions can be more or less fluid and also have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or mousse. They can also be in solid form, such as a stick or be applied to the skin in the form of an aerosol.

As physiologically acceptable medium commonly used in the field of application envisaged, there may be mentioned, for example, adjuvants necessary for the formulation, such as solvents, thickeners, diluents, antioxidants, dyes, sunscreens, agents. self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[100] In all cases, a person skilled in the art will ensure that these adjuvants as well as their proportions are chosen so as not to harm the desired advantageous properties of the composition according to the invention. These adjuvants can, for example, correspond to 0.01 to 20% of the total weight of the composition. When the composition according to the invention is an emulsion, the fatty phase can represent from 5 to 80% by weight and preferably from 5 to 50% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition are chosen from those conventionally used in the field under consideration. For example, they can be used in a proportion ranging from 0.3 to 30% by weight relative to the total weight of the composition. [101] According to another advantageous embodiment of the invention, the aqueous lavender extract of the invention can be encapsulated or included in a cosmetic vector such as liposomes or any other nanocapsule or microcapsule used in the field of cosmetics or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites.

[102] Advantageously, the composition according to the invention can comprise, in addition to the active ingredient according to the invention, at least one other active agent exhibiting cosmetic effects similar and / or complementary to those of the invention.

[103] For example, the additional active agent (s) can be chosen from: anti-aging, firming, brightening, moisturizing, draining agents, promoting microcirculation, exfoliating, desquamating, stimulating the extracellular matrix, activating energy metabolism, antibacterial agents , antifungal, soothing, anti-radical, anti-UV, anti-acne, anti-inflammatory, anesthetics, providing a feeling of heat, providing a feeling of freshness, slimming.

[104] Such additional active agents can be chosen from the groups comprising:

- vitamin A and in particular retinoic acid, retinol, retinol propionate, retinol palmitate;

- vitamin B3 and more particularly niacinamide, tocopherol nicotinate;

- vitamin B5, vitamin B6, vitamin B12, panthenol;

- vitamin C, in particular ascorbic acid, ascorbyl glucoside, ascorbyl tetrapalmitate, magnesium and sodium ascorbyl phosphate;

- vitamins E, F, H, K, PP, coenzyme Q10;

- metalloproteinase inhibitors, or an activator of Tl MP;

- DHEA, its precursors and derivatives;

- Amino acids such as arginine, ornithine, hydroxyproline, hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine, methionine and its derivatives, N-acylated amino acid compounds;

- natural or synthetic peptides, including di-, tri-, tetra-, penta- and hexapeptides and their lipophilic derivatives, isomers and complexed with others species such as a metal ion (eg copper, zinc, manganese, magnesium, and the like). As examples, mention may be made of the peptides commercially known under the names of MATRIXYL®, ARGIRELINE®, CHRONOGEN ™, LAMINIXYL IS ™, PEPTIDE Q10 ™, COLLAXYL ™ (patent FR2827170, ASHLAND®), PEPTIDE VINCI 01 ™ (patent FR2837098, ASHLAND®), PEPTIDE VINCI 02 ™ (patent FR2841781, ASHLAND®), ATPeptide ™ (patent FR2846883, ASHLAND®) or the synthetic peptide of sequence Arg-Gly-Ser-NH2, marketed under the name ATPeptide ™ by ASHLAND®;

- the extract of Artemia satina, marketed under the name GP4G ™ (FR2817748, ASHLAND®);

- plant peptide extracts such as extracts of flax (Lipigénine ™, patent FR2956818, ASHLAND®), extracts of soybean, spelled, vine, rapeseed, flax, rice, corn, peas;

- yeast extracts, for example Dynagen ™ (patent FR2951946, ASHLAND®) or Actopontine ™ (patent FR2944526, ASHLAND®);

- dehydroacetic acid (DHA);

- phystosterols of synthetic or natural origin;

- salicylic acid and its derivatives, alpha- and beta-hydroxy acids, silanols;

- amino sugars, glucosamine, D-glucosamine, N-acetyl glucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl mannosamine, galactosamine, N-acetyl galactosamine;

- extracts of polyphenols, isoflavones, flavonoids, such as grape extracts, pine extracts, olive extracts;

- lipids such as ceramides or phospholipids, oils of animal origin, such as squalene or squalane; vegetable oils, such as sweet almond, copra, castor, jojoba, olive, rapeseed, peanut, sunflower, wheat germ, corn germ, soybean oil, cotton, alfalfa, poppy, pumpkin, evening primrose, millet, barley, rye, safflower, passionflower, hazelnut, palm, apricot kernel, avocado, calendula ; ethoxylated vegetable oils, shea butter;

- all UV screens and sun filters;

- cyclic AMP and its derivatives, activators of the adenylate enzyme cyclase and inhibitors of the enzyme phosphodiesterase, extract of Centella asiatica, asiaticoside and asiatic acid, methyl xanthines, theine, caffeine and its derivatives, theophylline, theobromine, forskolin, l esculin and esculoside, ACE inhibitors, Val-Trp peptide, neuropeptide Y inhibitor, enkephalin, Ginkgo biloba extract, dioscorea extract, rutin, extract of yerba mate, guarana extract, oligosaccharides, polysaccharides, carnitine, ivy extract, wrack extract, hydrolyzed extract of Prunella vulgaris, hydrolyzed extract of Celosia cristata, extract 6 'Anogeissus leiocarpus, extract of leaves of Manihot utilissima, palmitoylcarnitine, carnosine, taurine, elderberry extract, seaweed extracts such as extract of Palmaria Palmata.

[105] A fourth subject of the invention is the cosmetic use of a composition comprising the lavender extract of the invention for caring for the skin, scalp and integuments, more particularly for protecting the skin from attacks. externalities and oxidation, fight against the signs of skin aging, increase photoprotection, lighten the skin, improve skin hydration, strengthen the barrier function, soothe the skin, or to improve the biological mechanisms associated with overnight skin repair.

[106] The skin is an organ composed of several layers (dermis, epidermis and stratum corneum), which covers the entire surface of the body and provides protective functions against external, sensitive, immune, metabolic, thermoregulatory or still barrier limiting dehydration.

[107] In particular, the stratum corneum acts as a protective physical barrier, commonly known as the “skin barrier function”. This function is of major importance in tissue homeostasis and in protection from the external environment.

[108] The appearance of the skin can be modified by internal alterations (intrinsic aging, diseases and hormonal changes such as pregnancy) or external (environmental factors, such as pollution, sunlight, pathogens, variations temperature, etc.). All of these alterations affect not only the skin, but also the keratinous appendages such as body hair, eyelashes, eyebrows, nails and hair.

[109] The extract of the aerial parts of lavender of the invention has been tested on the main biomarkers associated with the mechanisms of repair of the skin during the night. Mechanisms that take place in the skin overnight include increased DNA repair, rate of cell proliferation, skin temperature, skin blood flow, incidence of itching, as well as permeability. of the skin barrier, leading to a loss of hydration (Matsui MS et al, Biological rhythms in the skin, Int. J. Mol. Soi. 2016, 17,801). The extract of aerial parts of lavender of the invention was in particular evaluated on the repair rate of pyrimidine dimers (CPD for Cyclobutane Pyrimidine Dimers). The day / night rhythm perceived at the level of the central nervous system, includes the production of melatonin by the pineal gland (Slominski A.T. et al, Melatonin: A cutaneous perspective on its production, metabolism, and functions. J Invest Dermatol, 2018 March;

138 (3): 490-499). Melatonin is also produced locally in the skin from tryptophan. These functions help to reduce the level of reactive oxygen and nitrogen species (ROS and RNS, for reactive oxygen species and reactive nitrogen species, respectively). In fact, in addition to its direct role as a free radical scavenger, melatonin stimulates the production of antioxidant enzymes such as catalase and superoxide dismutase and is involved in DNA repair. By optimizing the functioning of the mitochondria, melatonin also helps to increase the level of ATP produced by the cell. The effect of the extract of aerial parts of lavender of the invention was evaluated on the level of melatonin in the skin and on the expression of the enzyme AANAT (Aralkylamine N-Acetyltransferase or serotonin N-acetyl transferase or timezyme ) which catalyzes the N-acetylation of serotonin to N-acetylserotonin, the final step in the synthesis of melatonin. In addition, topical application of exogenous melatonin has shown an effect on hair loss associated with androgenetic alopecia (Fisher TW, Topical melatonin fortreatment of androgenetic alopecia, Int J Trichology, 2012 Oct-Dec, 4 (4): 236-245). [110] The extract of aerial parts of lavender of the invention has been shown to be effective in increasing the production of melatonin in cultured skin biopsies.

[111] Alterations in the barrier function result from external aggressions such as UV rays. The consequences are major in terms of the loss of cell cohesion and mechanical integrity. Certain enzymes involved in the terminal phases of keratinocyte differentiation play a key role in protection against UV rays. Changes in the expression and / or activity of these enzymes have important consequences for the integrity of the barrier function and homeostasis of the skin.

[112] The extract of aerial parts of lavender of the invention has demonstrated its effectiveness in reinforcing the barrier function and thus improving the protection of the skin against external aggressions (UV radiation, pollution, microorganisms, etc.). ).

[113] The extract of the aerial parts of lavender of the invention has been shown to be effective in reducing the level of melanin in skin biopsies, and thus inducing a lightening effect.

Examples

[114] By way of illustration, exemplary embodiments of the method according to the invention are described below.

[115] Example 1: Preparation of an extract of lavender (Lavandula angustifolia) of the Lamiaceae family, enriched in small RNA

Extraction process

[116] The lavender flowers of the Lavandula angustifolia species are first dried in a ventilated place, protected from light. In a first step,

3% of dried lavender flowers and stems bearing the flowers are ground in the form of powder with a particle size ranging from 500 μm to 1 mm, preferably at 800 μm, i.e. the equivalent of 30 g of powder of dried lavender flowers in 968 g of distilled water. Then 2 g / L or 3 mM of phytic acid are added. The pH is adjusted to 10.8 for optimal enrichment of the extract in RNA of small molecular weights. [117] The mixture is then heated for 1 hour at 80 ° C. with stirring.

After this hour of extraction, diatomaceous earth is added to this mixture to facilitate subsequent separation between the residual solid plant material and the extract (soluble fraction).

[118] The mixture is then filtered using 30 µm porosity filters to remove solid matter. The pH is then adjusted to pH 7.5 using an HCl solution. Sequential filtrations on filters of decreasing porosity are then carried out in order to clarify the plant extract to sterilizing filtration at 0.2 μm.

[119] At the end of this step, the pH is checked, then it is adjusted to 6.3 with an HCl solution. 6 and 6.5 and preserve the small RNAs in the extract.

An aqueous extract is obtained having a dry weight of 12.6 g / kg.

Characterization of lavender extract

[120] The extract obtained at a dry weight of 12.6 g / kg.

Physico-chemical analysis shows that the extract obtained has a concentration of 3.7 g / kg of total sugars, 1160 g / kg of total organic acids, 1270 mg / kg of total phenolic compounds and 118 mg / kg of RNA of small molecular weights of up to 150 nucleotides in length.

The extract is then diluted with a physiologically acceptable cosmetic solvent to guarantee better stability and better conservation of the extract over time.

[121] The dilution is carried out with butylene glycol derived from plants so as to obtain a final concentration of 50% butylene glycol and 50% lavender extract. The extract thus diluted then has a dry weight of 6 g / kg, and has a concentration of 1.7 g / kg of total sugars, 570 mg / kg of total organic acids, 620 mg / kg of total phenolic compounds and 45 mg / kg of low molecular weight RNA with a maximum length of 150 nucleotides.

Assay methods used to determine the amount of the different compounds contained in the final lavender extract:

[122] The total sugar content of the extract was determined by spectrophotometric assay resulting from an adaptation of the assay described by Dubois et al. (1956) (Dubois et al., "Colorimetric method for the determination of sugars and related substances", Anal. Chem., 1956, 28 (3), 350-356). This analysis consists of dissolving the raw material in concentrated sulfuric acid and then reacting with phenol to form a colored complex. The absorbance of the complex is read on the spectrophotometer at 490 nm. The sugar content is determined using a standard glucose curve. A TLC analysis made it possible to demonstrate that the major sugars present in the extract of the invention are glucose and fructose molecules as well as sugars of high molecular weight (oligo and polysaccharides).

[123] The total polyphenol content of the lavender extract was determined by the Folin-Ciocalteu spectrophotometric assay method (Singleton et al., Analysis of total phenols and other oxidation substrates and antioxidants using the Folin-Ciocalteu reagent, 1999, 299: 152). The polyphenol-like compounds present in the sample react with the Folin-Ciocalteu reagent, oxidation of the reagent gives a blue color. The absorbance of the sample is read on the spectrophotometer at 760 nm. The content is expressed in gallic acid equivalents using a gallic acid standard curve.

[124] The characterization of organic acids was carried out on the Lavender extract of Example 1, a floral water and a reference essential oil.

High performance liquid chromatographic analysis coupled with a mass detector was used. All samples were separated on an EC 150 / 4.6 Nucleoshell RP 18plus-5pm (150x4.6mm) column (Macherey Nagel: 763236.46) by an Agilent 1260 HPLC system (Agilent Technologies). The flow rate was 0.3 ml / min. The mobile phase consisted of a solution of formic acid (HCOOH) at 0.01% (A) and acetonitrile (B). The gradient program facilitated the elution as described in Table 2.

[125] [Table 2]

[126] The temperature of the column was maintained at 25 ° C and the injection volume was 5 µL. Detection was performed by an ACQUITY Qda (WATERS) mass spectrometer detector with an electrospray ion source in negative mode. The source was set to a capillary voltage of 0.8 kV and a probe temperature of 600 ° C.

M / z and cone tension were targeted for each compound as described in Table 3.

[127] [Table 3]

[128] The identification of organic acids was performed by comparing retention times and mass spectral peaks of the sample with a standard. The quantitative estimate of organic acids was carried out on the basis of the maximum area of the sample concentrations compared to the maximum area of the standards.

[129] The HPLC-MS analysis, which makes it possible to quantify and identify the organic acids contained in the extract, shows that only the lavender extract contains different types of organic acids, mainly citric, malic and tartaric acid, as presented in FIG. 1. HPLC-MS analysis shows that these organic acids are present neither in the floral water of true lavender nor in the essential oil of true lavender.

[130] The quantification of low molecular weight RNAs was performed by a miniaturized electrophoresis technique on microfluidic chips specific for nucleic acid analysis such as that of low molecular weight RNAs (Bioanalyzer 2100®, Agilent). This method makes it possible to determine the size and concentration of nucleic acids contained in an extract from a few microliters. The result is presented as a graph with an arbitrary fluorescence unit (FU) on the ordinate and the number of nucleotides (nt) on the abscissa. An internal marker is added to each analysis (peak at 25 nt in Figure 2), and serves as an internal control to validate the proper conduct of the analysis.

[131] Figure 2 shows the analysis of small molecular weight RNAs by the 2100 Bioanalyzer. A: lavender extract according to Example 1. B: conventional lavender extract according to Example 2.

[132] Bioanalyzer analysis shows that the method described in the present invention extracts low molecular weight RNAs from lavender, as shown in Figure 2A. Figure 2A shows that the RNAs present in the lavender extract of the invention have molecular weights ranging from greater than 25 to about 150 nucleotides.

[133] A conventional extraction process such as the maceration described below in Example 2 does not make it possible to extract the RNAs of small molecular weights (FIG. 2B). The analysis also made it possible to demonstrate that there are no these molecules either in the floral water or in the essential oil. In addition, this analysis makes it possible to demonstrate the absence of DNA in the extract. [134] The analysis of volatile odorous compounds (VOC) was carried out on the extract of Lavender according to the invention, floral water and essential oil by GC-FID. All samples were separated on a 30 mx 250 µm x 0.25 µm OPTIMA 5HT GC column (Macherey-Nagel 726106.30) by Agilent GC / FID 7890A gas chromatography (Agilent Technologies). 3 µL of sample products were injected onto the column at a flow rate of 1.3 ml / min, using helium as carrier gas on a programmed oven which rises from 75 ° C to 320 ° C in 40 min.

[135] The molecules were detected on a flame ionization detector at 230 ° C, using hydrogen (30 ml / min) and air (400 ml / min) for the flame. The identification of VOCs is done by comparing the retention times of the compounds.

[136] For aqueous samples, liquid / liquid extraction in hexane (1: 1) is performed prior to injection. Magnesium sulfate is added to the organic phase to remove all traces of water.

[137] [Table 4]

Table A: Determination of volatile odorous compounds in lavender extract

[138] The results of the GC-FID analysis of Table 4 make it possible to demonstrate that the lavender extract of Example 1 does not contain any odorous molecule, of terpene nature, unlike floral water and essential oil of lavender known to mainly contain these molecules.

[139] Example 2 preparation of a lavender macerate [140] In order to compare a so-called classic extraction with the extract of the invention, a lavender macerate was carried out, using the same quantity of dried lavender flowers of the species Lavandula angustifolia as in Example 1, either 3% crushed dried lavender flowers put in distilled water. The mixture is then heated for 1 h at 80 ° C, then the mixture is filtered by a first filtration with a large porosity of 30 μm in order to remove the solid residual plant material from the liquid part, then by sequential filtration of decreasing porosity until 0.2 µm. The purpose of this process is to prepare a control extract in order to obtain comparative analytical data with respect to the lavender extract obtained by the process of the invention. The results obtained are illustrated in FIG. 2B and in the text of the application.

[141] Example 3: Evaluation of the Lavandula angustifolia extract of Example 1 on reactive oxygen species after application of visible light stress on normal human keratinocytes:

[142] Principle:

The aim of this study is to show the effect of the lavender extract prepared according to Example 1 on the decrease in reactive oxygen species generated by visible light stress. This type of light between 400 and 700 nm is intended to mimic daylight. Reactive oxygen species are involved in different mechanisms of protein and DNA damage, linked to skin aging.

[143] Protocol:

Normal human keratinocytes are treated with the extract of Example 1 overnight. The cells are then exposed to visible light generated by a spot of 24 Watt and 5000 ° Kelvin, making it possible to mimic daylight. This exposure is repeated 4 times for 10 minutes, during the day. The treatment is applied again overnight, then the cells are again subjected to the same visible light stress. After this stress, reactive oxygen species are detected by the mitochondrial probe MitoSOX ™ Red (ThermoFisher scientific).

[144] Results:

The application of daylight stress resulted in an increase in reactive oxygen species (ROS) by + 43% compared to unexposed cells. When the cells were treated with the extract of Example 1 at 0.1% (volume / volume dilution), the ROS are highly significantly reduced by -12% compared to the untreated cells. A lavender macerate obtained according to Example 2 is tested under the same conditions, allowing a smaller decrease of -5%.

[145] Conclusion:

Lavender extract has shown antioxidant activity, directed against reactive oxygen species at the mitochondrial level. This activity has been shown to be superior to that obtained with a lavender extract obtained from conventional maceration.

[146] Example 4: Evaluation of the extract from Example 1 on DNA damage in normal human melanocytes exposed to UVB stress:

[147] Principle:

Genomic instability can be defined as all the chemical changes in DNA that occur in different processes and can accumulate over time. DNA damage is a major component of photoaging. In the present study, we are interested in the damage created by UVB in melanocytes.

Pyrimidine dimers result from a direct effect of UVB on pyrimidine bases in DNA. There is formation of cyclobutane pyrimidine dimers (or CPD for Cyclobutane Pyrimidine Dimer) which is the most common DNA damage induced by UVB. In melanocytes, CPDs continue to be generated more than 3 hours after exposure to UVB. They are called "dark CPDs" and are caused by the chemexcitation of melanin, leading to energy transfer to DNA.

In this study, the ability of the extract from Example 1 to reduce the formation of "dark CPDs" in melanocytes was assessed.

[148] Protocol:

Melanocytes extracted from the human epidermis are treated with the extract of Example 1 at 0.1% (volume / volume dilution) overnight, then irradiated with UVB at 60 mJ / cm2 and again treated overnight. by lavender extract. the the next morning, the pyrimidine dimers are detected by immunostaining with an antibody directed against the CPDs (Cyclobutane Pyrimidine Dimers Mouse Monoclonal, Euromedex). After an hour and a half of incubation followed by rinsing, the cells are incubated in the presence of the secondary anti-mouse antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The cells are then examined under an Epi-fluorescence microscope (Zeiss Axiovert 200M microscope). The presence of the CPDs is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[149] Results:

Under non-irradiated conditions, the melanocytes do not exhibit CPDs. Their formation is induced as a result of UVB stress. The application of the extract of Example 1 on the melanocytes made it possible to reduce the induction of "dark CPDs" by UVB by -37% (highly significant according to Student's t test compared to irradiated cells. untreated). Under the same conditions and at an equal concentration of 0.1%, the lavender macerate obtained according to Example 2 did not have a significant effect.

[150] Conclusion:

The extract from Example 1 decreased "dark CPDs" produced in melanocytes by UVB stress. Through this activity, lavender extract has been shown to benefit in reducing DNA damage, contributing to the skin's repair mechanisms overnight.

[151] Example 5: Evaluation of the extract of Example 1 on the melatonin synthesis pathway in ex vivo skin biopsies and human hair follicles:

[152] Principle:

The aim of this experiment is to demonstrate an effect of the extract of Example 1 on the synthesis of melatonin in biopsies of human skin in culture. This evaluation includes two markers: 1 - the enzyme AANAT (Aralkylamine N-Acetyltransferase or serotonin N-acetyl transferase or timezyme) which catalyzes the N-acetylation of serotonin to N-acetylserotonin, the final step in the synthesis of melatonin, 2 - the evaluation of melatonin itself. The AANAT enzyme controls the day / night rate of melatonin production at the level of the pineal gland. As melatonin is also synthesized locally in the skin, we wanted to follow its synthesis in response to the application of lavender extract.

[153] Protocol:

The AANAT enzyme and melatonin are evaluated by indirect immunofluorescence on skin biopsies, previously treated with topical application of lavender extract for 48 hours (twice a day). In addition, the extract of Example 1 diluted to 0.5% (volume / volume dilution) in the culture medium was contacted with human hair follicles, isolated from scalp biopsies. Control biopsies incubated in parallel under the same conditions, as well as control hair follicles without addition of lavender extract, receive the placebo (Phosphate Buffer Saline, PBS). At the end of the incubation, the biopsies and the hair follicles are fixed and embedded in paraffin for the realization of histological sections. The detection of the enzyme AANAT and of melatonin is carried out by incubation with the respective antibodies: anti-AANAT (Invitrogen) and anti-melatonin (Abnova, Cliniscience). After an hour and a half of incubation followed by rinses, the sections are incubated in the presence of the secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an Epifluorescence microscope (Zeiss Axiovert 200M microscope). The expression of collagen I is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[154] Result:

Evaluation of the AANAT enzyme and melatonin showed an increase of + 20% and + 51%, respectively in skin biopsies treated with the extract of Example 1 at 0.5% (highly significant according to Student's t test, compared to biopsies receiving placebo). The lavender macerate obtained according to Example 2 gave an equivalent result with regard to AANAT, but led to a smaller increase for melatonin (+ 34%, highly significant according to Student's t test, compared to biopsies receiving placebo). In the hair follicles in culture, the extract of Example 1 at 0.5% has resulted in an increase in melatonin production of the order of + 23%, observed in the outer epithelial sheath of the follicles.

[155] Conclusion:

The extract from Example 1 showed activity on melatonin production in ex vivo skin biopsies and in hair follicles. This increase is related to an increase in the enzyme AANAT, which is increased in expression in the pineal gland during the transition from day to night. The properties of melatonin are linked to cell damage repair activities, especially at the DNA level and due to its antioxidant activity. The increase in melatonin in the skin by lavender extract therefore appears to be beneficial for the damage repair processes of the skin overnight. The increase in melatonin in the hair follicle indicates a beneficial effect on the physiology of the hair follicle, since melatonin is associated with the growth phase of the hair.

[156] Example 6: Evaluation of the lightening potential of the extract of Example 1 on ex vivo skin biopsies:

[157] Principle:

The aim of this study is to evaluate the lightening potential of the extract of Example 1 on ex vivo skin biopsies, using the histological staining of Fontana-Masson melanin, based on the reduction of a solution of ammoniacal silver nitrate in metallic silver. The color obtained reveals the melanin content and is quantified by image analysis.

[158] Protocol:

Ex vivo human skin biopsies are cultured and treated with the extract of Example 1 at 0.5% (volume / volume dilution) and 1% (volume / volume dilution) for 48 hours. After treatment, the biopsies are fixed for histological analysis and embedded in paraffin. After dewaxing, the sections are incubated with the ammoniacal silver nitrate solution at 60 ° C. for 10 minutes. After rinsing, they are treated with 5% sodium thiosulphate for 2 minutes, then rinsed again and mounted for examination under an Eclipse E600 microscope (Nikon). Photos are taken with the camera Qlmaging Retiga 2000R Fast1394 and analyzed by Q-Capture Pro 7 software (Qlmaging).

[159] Results and conclusion:

On ex vivo skin biopsies, a decrease in melanin content of minus 55% and minus 72% was observed after application of the extract of Example 1 at 0.5% (volume / volume dilution) and 1% (volume / volume dilution) respectively (highly significant by Student's t test compared to placebos biopsies), while the lavender macerate obtained according to Example 2 did not show a decrease to 0.5%, and a smaller decrease (from -47%) to 1%.

[160] This test therefore made it possible to conclude that there is a potential lightening effect of the extract of Example 1 of Lavandula angustifolia on ex vivo skin biopsies.

[161] Example 7: Formula of a rich cream

[162] [Table 5]

Ingredients (Brand name) INCI% w / w

[163] Preparation process:

1. Homogenize phase A in the main vessel and start heating to 75-80 ° C;

2. At 30 ° C, sprinkle in Phase B and homogenize while heating;

3. In a separate beaker, prepare phase C, heat to 75-80 ° C until homogeneous;

4. At 75 ° C, add phase C to the main vessel and homogenize for 10 minutes;

5. Allow the temperature to cool and add phase D to 65 ° C. Mix well to homogenize for 10 minutes;

6. Premix phase E before adding it to the main container;

7. Add phase E at 60 ° C. Mix well to homogenize for 10 minutes;

8. At 35 ° C, premix phase F before adding it and mixing well;

9. Premix phase G before adding it to the main container;

10. Add phase G at 35 ° C. Mix well to homogenize;

11. In a separate beaker, prepare phase H: sprinkle Natrosol ™ in water at room temperature and homogenize while heating to 60 ° C;

12. Add phase H at 30 ° C. Mix well to homogenize;

13. Stop at 25 ° C.

[164] The composition is thus in the form of a pink buttercream, with a pH between 4.90 and 5.40 and a viscosity (OD) of 160,000 - 210,000 cps (Brookfield RVT / Spindle D / 5 RPM / 1 minute / 25 ° C).

[165] Example 8: Formula of an anti-aging mask

[166] [Table 6]

Ingredients (Brand name) INCI% w / w [167] Preparation process:

1. At 25 ° C, homogenize phase A in the main vessel;

2. At 25 ° C, sprinkle in phase B and mix well until homogeneous;

3. At 25 ° C, add phase C and mix well until homogeneous;

4. Premix phase D in a separate beaker and add to the main vessel at 25 ° C;

5. At 25 ° C, add phase E to the main container and mix well;

6. Premix phase F and add slowly. Mix well until blended;

7. Premix phase G in a separate beaker and add to the main container until blended;

8. Stop at 25 ° C.

[168] The composition is thus in the form of a cream gel with scintillating green effects, with a pH between 5.30 and 5.80 and a viscosity (OD) of 70,000 - 100,000 cps (Brookfield RVT / Spindle C / 5 RPM / 1 minute / 25 ° C).

[169] Example 9: Formula of a Serum

[170] [Table 7]

Ingredients (Brand name) INCI% w / w

[171] Preparation process:

1. Add water to the main container and start mixing with a hi-lo propeller blade;

2. Add the remaining ingredients, one at a time, stirring between each addition.

[172] The composition is thus in the form of a smooth, semi-opaque serum, with a pH between 5.75 and 6.25 and a viscosity (OD) of 1.100 - 1.400 cps (Brookfield RVT / spindle 3/20 rpm / 25 ° C / 1 minute).

Claims

Claims

[Claim 1] A process for obtaining an aqueous extract of aerial parts of lavender, comprising the following steps: a) the aerial parts of lavender are brought into contact with water; b) phytic acid is added at a concentration of between 1 and 10 mM in the mixture obtained in a) at a pH of between 10 and 11; c) the pH of the mixture obtained in b) is then adjusted to a value between 6 and 8; d) the mixture obtained in c) is purified so as to eliminate the residual solid plant material and to obtain a purified aqueous crude extract; and e) the pH is checked and it is readjusted if necessary to a value between 6 and 8, preferably between 6 and 6.5.

[Claim 2] The method of claim 1 characterized in that in step a) the aerial parts of lavender, previously dried and then ground, are brought together with water in a plant material / water ratio of between 3 and 20% (w / w).

[Claim 3] Method according to one of claims 1 or 2 characterized in that step b) of treatment with phytic acid at a concentration of 3 mM and is carried out with stirring for a time of at least 1 hour and at a temperature between 20 and 80 ° C.

[Claim 4] Method according to one of claims 1 to 3, characterized in that step e) is preceded by at least one filtration of the crude aqueous extract obtained in d) and preferably by successive filtrations of the extract. crude aqueous by lowering the filtration threshold from 20-50 µm to 0.10-0.30 µm.

[Claim 5] A method according to one of claims 1 to 4 characterized in that the aerial parts of lavender are of the species Lavandula angustifolia.

[Claim 6] Aqueous extract of aerial parts of lavender enriched with small RNAs of up to 150 nucleotides in length, sugars, phenolics and organic acids, devoid of DNA, susceptible to be obtained by the process of one of claims 1 to 5, characterized in that it comprises, by weight of the total weight of the extract, 10 to 30 g / kg of dry weight, containing 2 to 10 g / kg of sugars, 100 to 1500 mg / kg of organic acids, 500 to 2000 mg / kg of phenolic compounds and 40 to 200 mg / kg of RNA of small molecular weights of up to 150 nucleotides in length.

[Claim 7] Extract according to claim 6, characterized in that it is subsequently diluted in a solvent and comprises by weight of the total weight of the extract, 4 to 20 g / kg of dry extract, 0.5 to 10 g / kg of sugars, 50 to 700 mg / kg of organic acids, 50 to 1500 mg / kg of phenolic compounds and 10 to 100 mg / kg of low molecular weight RNA with a maximum length of 150 nucleotides .

[Claim 8] A composition comprising, as an active ingredient, an effective amount of the extract of one of claims 6 or 7, and a physiologically acceptable medium.

[Claim 9] Composition according to Claim 8, characterized in that the extract is present at a concentration of between 0.05 and 5% by weight of the total weight of the composition and preferably between 0.1 to 1.0% by weight. weight of the total weight of the composition.

[Claim 10] Composition according to one of claims 8 or 9, characterized in that it is formulated to be applied topically to the skin, integuments and the scalp.

[Claim 11] Cosmetic use of the composition of one of claims 8 to 10 for caring for the skin, scalp and integuments and more particularly for protecting the skin from external attacks and oxidation, to fight against signs of skin aging, increase photoprotection, lighten the skin, improve skin hydration, strengthen the barrier function or even soothe the skin.

[Claim 12] Cosmetic use of the composition of any of claims 8 to 10 for improving biological mechanisms associated with overnight skin repair.