Method for obtaining plant extracts comprising an autofermentation step, compositions comprising such extracts and cosmetic uses thereof

Abstract

The invention relates to methods for preparing plant extracts comprising an autofermentation step and optional extraction steps for enriching the extracts with low-molecular-weight RNAs. The invention also relates to compositions comprising such extracts and to the cosmetic uses thereof for the in-vivo steps of combatting the signs of skin ageing and the loss of firmness or tone, improving barrier function and hydration, lightening the skin or improving the innate immune defences of the skin and wellbeing.

Description

Description Title of the invention: METHOD FOR OBTAINING PLANT EXTRACTS COMPRISING AN AUTOFERMENTATION STEP, COMPOSITIONS COMPRISING SUCH EXTRACTS AND THEIR COSMETIC USES technical field

[0001] The invention relates to the field of cosmetics and more particularly to the preparation of active extracts of plant origin used in cosmetic formulations to combat the signs of skin aging, loss of firmness, improve barrier function and hydration or lighten the skin. Technical background of the invention [2] The preparation of plant extracts usable in cosmetics uses all the purification techniques known in chemistry and phytochemistry, for example extraction methods using polar or non-polar organic solvents (EP3682865). [3] However, there has been a growing desire among consumers to turn to natural products containing as few synthetic ingredients as possible. To meet this new demand, the extracts described in this application are 100% natural. [4] One approach involves using microorganisms to orchestrate the biotransformation of plants. Numerous processes using selected or modified microorganisms are described in the literature and are used industrially. The most widespread are based on fermentation, known for millennia as a food preservation method. The main advantages of fermentation are preservation, improved taste, and enhanced nutritional quality of food, due to the presence of a higher quantity of easily assimilated bioactive molecules. [5] In the field of cosmetics, microorganisms are widely used for their direct beneficial effects on the skin or to produce fermented or biotransformed plant extracts. Examples include documents EP3744339A1 and CN1 10680774A, which describe plant fermentation processes, specifically for Citrus aurantium and jasmine flowers respectively, involving inoculation with yeasts of the species Saccharomyces cerevisiae. document CN101243897A which describes the use of lactic acid bacteria to produce cosmetic or food flower extracts. [6] In the same field, document FR3103826 describes the preparation of a consortium of microorganisms composed of at least one lactic acid bacterium, one yeast and one acetic acid bacterium, to prepare plant extracts rich in active compounds, suitable for use in the pharmaceutical, cosmetic and food fields. [7] Biotechnological processes employing microorganisms on synthetic nutrient media adapted to produce compounds of interest such as vitamins, citric acid, lactic acid or vanillin (US 20060269632) are also described. [8] However, the processes mentioned above involve the exogenous supply of microorganisms or the supply of specific nutrients to stimulate the growth of microorganisms which will ensure the biotransformation of plant matter. [9] Fermentation can also rely on the endogenous microflora present on the surface or in the internal structures of plants. This is a community of mutualistic or symbiotic bacterial and fungal microorganisms called phytobiota, hosted by virtually all plants or parts of plants (fruits, leaves, flowers, stems, roots, seeds, fungi, algae). This is then referred to as spontaneous fermentation or autofermentation.

[0010] Thus, document US20060269632A1 describes the preparation of an effective allergy drug obtained by mixing pine shoots with water and sugars and allowing spontaneous fermentation to develop for several months under anaerobic conditions.

[0011] One problem that the invention aims to solve is to provide a new process for preparing natural plant extracts, simple to implement, without the addition of any living microorganism, any enzyme, or any exogenous nutrient, and yet exhibiting remarkable biological effectiveness and an absence of toxicity.

[0012] To this end, the inventors have developed a process comprising a controlled self-fermentation step conducted essentially from the phytobiota of plant materials, including flowers, fruits, leaves, and roots, without any exogenous input.

[0013] Under these conditions, a significant amount of organic and phenolic acids are generated. The resulting plant extracts contain a wide range of phytomolecules, giving them proven biological efficacy.

[0014] The extracts thus obtained can be used in cosmetics for skin care and more particularly to combat the signs of skin aging, loss of firmness or tone, improve barrier function and hydration, brighten the skin or even improve the skin's innate immune defenses and in vivo well-being. Summary of the invention

[0015] The invention relates to a self-fermentation process for obtaining plant extracts enriched in organic acids and phenolic acids, comprising the following steps:

[0016] a) The plant material is mixed with water,

[0017] b) If necessary, adjust the pH to a value between 4 and 9.

[0018] c) The mixture is kept under gentle stirring for a time between 6 and 48 hours, at a temperature between 20 and 60°C, at a pH between 4 and 7, to allow the self-fermentation process, in an enclosure allowing gas exchange with the atmosphere.

[0019] d) the mixture obtained in c) is purified to remove residual solid plant matter and collect the liquid portion,

[0020] e) at least one filtration is carried out of the liquid portion obtained in the previous step,

[0021] f) The pH of the filtrate is checked and readjusted, if necessary, to a value between 4 and 8 and preferably between 4 and 7.

[0022] The invention also relates to a self-fermentation process coupled with a subsequent extraction allowing enrichment in small molecular weight RNA, comprising the following steps:

[0023] a) The plant material is mixed with water,

[0024] b) If necessary, adjust the pH to a value between 4 and 9.

[0025] c) The mixture is kept under gentle stirring for a time between 6 and 48 hours, at a temperature between 20 and 60°C, at a pH between 4 and 7, to allow the self-fermentation process, in an enclosure allowing gas exchange with the atmosphere.

[0026] d'J To the mixture obtained in step c), phytic acid is added at a concentration between 1 and 5 mM

[0027] e'J we adjust the pH if necessary to a value between 10 and 11,

[0028] f'J the mixture is kept under agitation for 1 to 3 hours, at a temperature between 50 °C and 80 °C.

[0029] g') the residual plant matter is separated

[0030] h') the liquid fraction obtained is purified by successive filtrations to clarify the extract,

[0031] i'J we adjust the pH of the resulting mixture to a value between 6 and 8,

[0032] Optionally, the extract is diluted with a physiologically acceptable solvent.

[0033] The invention further relates to diluted plant extracts, advantageously obtained from fresh jasmine flowers (Jasminum grandiflorum) by the process of auto-fermentation coupled with a subsequent extraction allowing enrichment in the above small RNAs and comprising at least 300 mg / kg of phenolic compounds of which at least 140 mg / kg of phenolic acids and at least 300 mg / kg of organic acids, of which at least 50 mg / kg of shikimic acid and 10 mg / kg of quinic acid.

[0034] The invention further relates to a composition comprising an effective quantity of extract obtained by one of the preceding processes and a physiological medium.

[0035] The invention also relates to the cosmetic use of the previous composition to combat the signs of skin aging, loss of firmness, improve barrier function and hydration, lighten the skin, mitigate the age-related decrease in the number or activity of mechanosensory touch receptors (Piezol) and oxytocin receptors (OXTR), or improve the skin's innate immune defenses and in vivo well-being.

[0036] The invention also relates to a composition comprising the plant extracts of the invention to improve the innate immune defenses of the skin. Brief description of the drawings

[0037] [Fig. 1] Protein profiles obtained by HPLC of jasmine extracts

[0038] [Fig. 2] HPLC analysis of phenolic compounds present in jasmine extracts

[0039] [Fig. 3] HPLC analysis of organic acids present in jasmine extracts

[0040] [Fig. 4] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on the expression of the Piezol receptor in reconstructed epidermis

[0041] [Fig. 5] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on the expression of the oxytocin receptor (OXTR) on human skin biopsies

[0042] [Fig. 6] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on the expression of oxytocin receptor (OXTR) messenger RNAs in cultured keratinocytes

[0043] [Fig. 7] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on viperin expression in cultured keratinocytes

[0044] [Fig. 8] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on the expression level of E-cadherin, on human skin biopsies pretreated with a blocker of Piezol activity (Dookul)

[0045] [Fig. 9] Evaluation of a self-fermented jasmine extract enriched with 2% small RNAs on the expression level of messenger RNAs of the enzyme 11p-HSDl in cultured keratinocytes

[0046] [Fig 10] Evaluation of the effect of a self-fermented jasmine extract enriched with 2% small RNAs on well-being using the WHO questionnaire, salivary oxytocin measurement, and emotional state assessment

[0047] [Fig 1 1 ] Evaluation of the effect of a self-fermented jasmine extract enriched with small RNAs at 2% of transepidermal water loss

[0048] [Fig 12] Evaluation of the effect of a self-fermented jasmine extract enriched with 2% small RNAs on the ITA (Individual Topology Angle)

[0049] [Fig 13] Evaluation of the effect of a self-fermented jasmine extract enriched with 2% small RNAs on skin roughness on the forehead

[0050] [Fig 14] Clinical expert evaluation of the effect of a 2% self-fermented jasmine extract enriched with small RNA on skin condition

[0051] [Fig 15] Diagram of the auto-fermentation process and the auto-fermentation process coupled with extraction allowing enrichment in small molecular weight RNA Detailed description of the invention Definitions

[0052] All terms used in this description have their most widely understood meanings unless otherwise stated. For the purposes of the invention, the following terms are defined as follows:

[0053] The term "extract" refers to the result of all aqueous extraction processes of plant matter.

[0054] The term “phytobiota” refers to all the microorganisms present on the surface or inside plant matter.

[0055] In this description, "plant matter" or "Plant" means a living organism belonging to the plant kingdom and possessing a phytobiota, including, among others, plants, mosses, lichens and algae.

[0056] The term "fresh plant material" refers to plant material used in the extraction process that has not undergone any chemical or mechanical treatment likely to alter its phytobiome. For example, the plant material was harvested shortly before being used in the process, or it was quickly frozen after harvesting, or it was dried under conditions that ensure good preservation of the phytobiome, such as at low temperatures. Fresh plant material may include plant residues obtained after processing.

[0057] "Autofermentation" refers to a fermentation process resulting from the plant's own phytobiota naturally present in or on the surface of the materials. plant-based products involved in the extraction process described in this application.

[0058] By "proteins" we mean large molecules composed of chains of amino acids, but also polypeptides and peptides smaller than 20 kDa (or 20 kg / mol).

[0059] By "small RNAs" or "small molecular weight RNAs" we mean a mixture of non-coding RNAs (ribonucleic acids), of small molecular weight, with a maximum length of 150 nucleotides, such as all types of small non-messenger RNAs, single and / or double stranded, for example micro-RNAs, interfering RNAs, introns, small nuclear RNAs or any RNA fragment.

[0060] The term "organic acids" refers to derivatives of the catabolism of amino acids, fatty acids, and sugars that include an acidic function. This group includes α-hydroxylated (AHA) or polyhydroxylated carboxylic acids such as glycolic, lactic, malic, citric, tartaric, shikimic, and quinic acids; carboxylic acids derived from fruit sugars or other plant parts such as uronic acids; and diacids such as succinic acid.

[0061] By "phenolic compounds" we mean all molecules possessing one or more aromatic rings themselves bearing one or more hydroxyl groups, such as phenolic acids, flavonoids or their derivatives, tannins or any other polyphenols.

[0062] By "phenolic acids" we mean phenolic compounds derived from benzoic acid and cinnamic acid, molecules possessing a single aromatic ring.

[0063] By "flavonoids" we mean phenolic compounds all sharing the same basic structure formed by two aromatic rings linked by three carbons.

[0064] By "glycosylated flavonoids" we mean flavonoids linked to one or more sugars.

[0065] By "sugars" we mean all carbohydrates such as mono- and disaccharides as well as oligo- and polysaccharides contained in the extract.

[0066] By "jasmintides" we mean peptides derived from jasmine characterized by a composition rich in cysteine ​​and non-classical disulfide bridges.

[0067] By "phytomolecules of interest", we mean all the molecules present in the extracts of the invention and in particular, proteins, sugars, phenolic compounds, organic acids, small RNAs with a length of up to 150 nucleotides.

[0068] When a range of values ​​is described, the bounds of that range should be understood as explicitly including all intermediate values ​​within the range. For example, a range of values ​​between 1% and 10% should be understood as including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all decimal values ​​between 1% and 10%.

[0069] Numerical percentage values ​​are percentages by weight, that is, the weight of a compound relative to the total weight of the mixture under consideration, unless otherwise specified.

[0070] The compositions described in this application may "include", "consist of" or "consist essentially of" the essential compounds or optional ingredients.

[0071] "Consist essentially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic characteristics or new characteristics of the composition or use described in this application.

[0072] A "physiologically acceptable medium" means a vehicle suitable for contact with the outer layers of the skin or mucous membranes, without causing toxicity, irritation, undue allergic response or similar intolerance reaction, and proportionate to a reasonable benefit / risk ratio.

[0073] “Skin” means the skin of the face and body including the scalp (including hair follicles and interfollicular skin spaces) and skin appendages (hair, body hair and nails).

[0074] The term "effective quantity" refers to the minimum quantity of extract according to the invention which is necessary to obtain at least one of the desired biological activities, without this quantity being toxic.

[0075] By "skin hydration" we mean the content and distribution of water in the upper layers of the epidermis.

[0076] By "signs of skin aging" we mean all changes in the external appearance of the skin due to aging such as, for example, wrinkles and fine lines, creases, bags under the eyes, dark circles, sagging, loss of elasticity, firmness and / or tone of the skin, pigmentary disorders such as senile lentigo or solar lentigo, but also all internal changes in the skin which do not systematically result in a changed external appearance such as, for example, the decrease in the number or activity of mechanosensory touch receptors (Piezo 1) and oxytocin receptors (OXTR), thinning of the skin, or all internal degradation of the skin resulting from environmental stresses such as pollution and solar radiation including UV.

[0077] By "in vivo well-being" we mean the emotional state of a person, the subjective feeling of well-being, the physiological production of oxytocin and the measurement of behavior or emotional expression.

[0078] It is understood that the invention relates to mammals and more particularly to human beings. Autofermentation and extraction processes and extracts obtained

[0079] The invention relates to a process for obtaining plant extracts enriched in organic acids and phenolic acids comprising the following steps:

[0080] a) The plant material is mixed with water,

[0081] b) If necessary, adjust the pH to a value between 4 and 9.

[0082] c) The mixture is kept under gentle stirring for a time between 6 and 48 hours, at a temperature between 20 and 60°C, at a pH between 4 and 7,

[0083] d) the mixture obtained in c) is purified to remove residual solid plant matter and collect the liquid part.

[0084] e) at least one filtration is carried out of the liquid portion obtained in the previous step.

[0085] f) The pH of the filtrate is checked and readjusted, if necessary, to a value between 4 and 8.

[0086] Fresh plant matter in the presence of water provides the nutrients necessary for the growth of the phytobiota. The nutrient supply is sufficient to allow the phytobiota to develop and survive for several days.

[0087] The process of the invention is implemented without the addition of exogenous nutrients and does not use detergents or solvents potentially toxic in cosmetics. It is therefore simple to implement and has a reduced environmental impact.

[0088] In step c), moderate agitation helps to keep the microorganisms of the phytobiota in suspension in the mixture.

[0089] The self-fermentation process in step c) is carried out in an open environment to allow gas exchange with the atmosphere, without any particular constraints to ensure sterility, but can alternatively be implemented in a fermenter in order to control gas exchange.

[0090] Auto-fermentation takes place when the microorganisms of the phytobiota, through their enzymes, metabolize plant-derived nutrients, such as sugars, proteins, and polyphenols, to produce an extract rich in various families of simpler and more easily assimilated compounds such as organic acids, peptides, and phenolic acids.

[0091] A study of microbial growth kinetics coupled with tests allowed us to observe a significant change in the environment during auto-fermentation, characterized by a decrease in flavonoid content, a decrease in sugar concentration, particularly glucose, and an increase in fermentation markers such as phenolic and organic acids, attesting to the fermentative activity of microorganisms.

[0092] In one particular embodiment, the plant materials used are plants from the Oleaceae, Poaceae or Fabaceae families.

[0093] In another particular embodiment, the plant materials used are plants from the Oleaceae family such as jasmine of the species Jasminum grandiflorum.

[0094] In one particular embodiment, the plant parts used are chosen from among the flowers, fruits, leaves and roots.

[0095] In a preferred embodiment, the plant parts used are the flowers.

[0096] In another embodiment, the plant materials used are algae.

[0097] In another particular embodiment, the plant material is a plant residue obtained after processing such as oilseed cakes or spent grains.

[0098] In step a), distilled, demineralized, or mineral-rich and / or trace-element water is used. Distilled water is preferred.

[0099] The plant material used in step a) can be whole or mechanically ground using a blade grinder, for example, to reduce the particle size from 0.5 mm to a few centimeters without affecting the viability of the microorganisms. Grinding can be carried out dry or in water.

[0100] When the plant material consists of flowers, it is preferably used whole, that is, uncrushed. Indeed, comparative extractions carried out with and without crushing have shown that the passive diffusion of compounds contained in flowers is high enough to make crushing unnecessary.

[0101] On the other hand, when the starting plant material is too thick or composed of large fragments, for example, a whole seaweed or a root, it is preferably ground beforehand before step a).

[0102] At step a) the ratio of plant matter to water is preferably between 3 and 30% by weight / weight, more preferably between 3 and 15%, even more preferably it is 3%, 5% or 15%.

[0103] At the beginning of step b), the pH is checked and adjusted to a value between 4 and 9, preferably between 6 and 8, and even more preferably to pH 7, by adding hydrochloric acid (HCl), citric acid, or sodium hydroxide (NaOH). The pH is then checked regularly throughout step b) and readjusted as needed.

[0104] When citric acid is used at this stage, this amount of citric acid is removed from the final calculations of organic acid concentration of the extract.

[0105] In step c), the mixture is advantageously stirred moderately, such as by stirring. The temperature is adjusted and maintained between 20 and 60°C, more preferably between 20 and 40°C, and even more preferably between 30 and 40°C. These temperature ranges are ideal for ensuring optimal microorganism development; indeed, below 20°C microbial growth is slowed, and above 60°C microorganism viability decreases.

[0106] Step c) must be carried out at an optimal pH and temperature to enrich the aqueous extraction solution with phytomolecules and create the growth conditions for the phytobiota present in the mixture. During this step, the pH must not fall below 5.

[0107] Step c) of auto-fermentation takes place over a period of between 1 and 48 h, preferably between 6 and 30 h, and more preferably between 15 and 30 h. A study of microbial growth kinetics has shown that growth is strong, under the process conditions, for a period of at least 30 hours, then reaches a plateau.

[0108] Optionally, at the end of step c) the residual solid material can be ground to release the cellular contents of the microorganisms and thus increase the yield of phytomolecule extraction.

[0109] To carry out step d), any method known to those skilled in the art may be used. The mixture obtained in c) may, for example, be filtered through filters with a porosity greater than 30 µm. Preferably, the mixture obtained in c) is centrifuged at low speed, for example for at least 10 min at 4000 g.

[0110] To carry out step e), preferably successive filtrations are performed, lowering the filtration threshold from 30 to 1 µm to clarify the mixture. Preferably, at least four filtrations are carried out with filters having porosity ranging from 30 µm, 4 µm, 2 µm, and 1 µm.

[0111] In step f), the pH of the filtrate is checked and readjusted, if necessary, to a value between 4 and 8, and preferably to a value between 4 and 7. The pH can be adjusted by adding a solution of either sodium hydroxide (NaOH) or acid. Hydrochloric acid (HCl) or any other equivalent acid compatible with cosmetic use, such as citric acid, is used. This step is essential to prevent the precipitation of phytomolecules of interest, such as sugars, phenolic compounds, organic acids, and proteins, and thus obtain a stable extract. Preferably, the pH is adjusted to a value between 4 and 7.

[0112] In step g) optionally the filtrate can be sterilized by any method known to a person skilled in the art, for example by steaming or preferably by sterilizing filtration on a 0.2 to 0.45 pm filter.

[0113] At the end of step f) or g) a concentrated self-fermented extract is obtained.

[0114] This concentrated autofermented extract is characterized by a dry weight of 4 to 30 g / kg, 0.5 to 6 g / kg of sugars, 300 to 900 mg / kg of organic acids (including 50-500 mg / kg of shikimic acid and 10-100 mg / kg of quinic acid), 300 to 2000 mg / kg of phenolic compounds (including 100-200 mg / kg of phenolic acids), and 1 to 10 g / kg of protein. However, the extracts obtained can exhibit significant variability depending on factors such as the location or year of harvest, the season, climatic conditions, biotic stress, etc.

[0115] The self-fermented extract does not contain ethanol since the process of the invention is not an alcoholic type fermentation.

[0116] h) The concentrated self-fermented extract thus obtained can be diluted in a physiologically acceptable solvent for cosmetic use.

[0117] The auto-fermentation process allows the extract to be enriched with phenolic acids from the transformation of polyphenols and with organic acids such as quinic, succinic and shikimic and lactic acids.

[0118] Thus, analysis by high-performance liquid chromatography unexpectedly showed that the concentrated self-fermented extracts obtained by the process described above contain at least 300 mg / kg of phenolic compounds of which at least 100 mg / kg of phenolic acids and at least 300 mg / kg of organic acids of which at least 50 mg / kg of shikimic acid and 10 mg / kg of quinic acid.

[0119] Indeed, in general, the analysis of organic acids contained in flowers and in particular in lavender flowers, does not contain shikimic and quinic acids.

[0120] In step h), among the physiologically acceptable solvents are water, glycerol, ethanol, propanediol and its natural version derived from corn, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, or any mixture of these solvents. One of the advantages of such dilution, besides obtaining precisely the desired phytomolecule concentrations, is to improve the stability and preservation of the self-fermented plant extract.

[0121] Preferably, the concentrated autofermented extract is diluted with butylene glycol, propanediol, or glycerin, and even more preferably diluted by the addition of 30% propanediol to ensure its stability and long-term preservation by preventing contamination. The concentrated extract can also be diluted by adding 30% glycerin combined with any type of water-soluble preservative, such as sodium benzoate or potassium sorbate, to a final concentration of 0.5%, or phenoxyethanol to a final concentration of 1.5%.

[0122] This so-called diluted extract comprises, by weight of the total weight of the extract, 2 to 20 g / kg of dry extract, 0.2 to 4 g / kg of sugars, 100 to 700 mg / kg of organic acids including 50 - 350 mg / kg of shikimic acid and 10 to 70 mg / kg of quinic acid, 100 to 1300 mg / kg of phenolic compounds including 70 to 140 mg / kg of phenolic acids and 0.1 to 5 g / kg of proteins.

[0123] The extract of the invention thus comprises a wide range of phytomolecules that may have beneficial effects on the skin, without presenting a risk of skin irritation or other harm to health.

[0124] In this particular embodiment, the plant material is advantageously a plant from the family of Oleaceae, Poaceae or Fabaceae.

[0125] In this particular embodiment, the plant material is even more advantageously the fresh jasmine flower, preferably of the species Jasminum grandiflorum.

[0126] Furthermore, after step c) additional extraction steps may be carried out according to any process known to a person skilled in the art.

[0127] Among these additional extraction steps, one can cite an extraction process allowing enrichment in small molecular weight RNA described in patents FR 1670672 and US 1 1,021,505.

[0128] To carry out this process, the following steps are performed:

[0129] Step d') To the mixture obtained in step c) phytic acid is added at a concentration between 1 and 5 mM.

[0130] Step e'J we adjust, if necessary, the pH to a value between 10 and 11.

[0131] Step f'J the mixture is kept under agitation for 1 to 3 hours, preferably for 2 hours, at a temperature between 50°C and 80°C, preferably at 80°C.

[0132] Step g') we separate the residual plant matter.

[0133] Step h') the liquid fraction obtained is purified by successive filtrations to clarify the extract.

[0134] Step i'J we adjust the pH of the mixture obtained to a value between 6 and 8.

[0135] One of the advantages of combining autofermentation with a complementary extraction process that enriches in small RNAs is that it increases the extraction yield by at least a factor of two compared to an extract obtained by autofermentation alone. The concentration of proteins, sugars, organic acids, and total phenolic compounds is also increased by approximately a factor of two to three.

[0136] Thus, the concentrated extracts enriched in small RNAs obtained by the processes described above have a dry weight at least 2 times greater than the dry weight of the extracts obtained by simple autofermentation and contain at least 500 mg / kg of phenolic compounds of which at least 600 mg / kg of organic acids.

[0137] Such concentrated extracts are characterized by a dry weight of 4 to 60 g / kg, and comprise 0.5 to 10 g / kg of sugars, 600 to 1200 g / kg of organic acids including 50 to 700 mg / kg of shikimic acid and 20 to 200 mg / kg of quinic acid, 500 to 3000 mg / kg of phenolic compounds including 200 to 300 mg / kg of phenolic acids, 2 to 20 g / kg of proteins, and 10 to 100 mg / kg of small molecular weight RNA.

[0138] In this particular embodiment, the plant material is advantageously a plant from the family of Oleaceae, Poaceae or Fabaceae.

[0139] In this particular embodiment, the plant material is even more advantageously the fresh jasmine flower, preferably of the species Jasminum grandiflorum.

[0140] When the plant material is fresh jasmine flower (Jasminum grandiflorum), the extract obtained has a dry weight at least 2 times greater than the dry weight of extracts obtained by simple autofermentation and comprises at least 500 mg / kg of phenolic compounds, at least 600 mg / kg of organic acids and at least one jasminide.

[0141] Jasmintides are part of the cyclotide family, which are thought to have activity similar to oxytocin in relation to well-being and anti-aging effects (Adriano Mollica et al. 2015, Cyclotides: a natural combinatorial peptide library or a bioactive sequence player?, Journal of Enzyme Inhibition and Medicinal Chemistry, 30:4, 575-580).

[0142] Step j') Optionally, the extract is diluted with a physiologically acceptable solvent.

[0143] To perform this dilution step, a physiologically acceptable solvent is chosen from among water, glycerol, ethanol, propanediol (including its natural corn derivative), butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, or any mixture of these solvents. One of the advantages of such dilution, besides obtaining precisely the desired phytomolecule concentrations, is to improve the stability and preservation of the self-fermented plant extract.

[0144] Preferably, the concentrated small RNA-enriched autofermented extract is diluted with butylene glycol, propanediol, or glycerin, and even more preferably diluted by the addition of 30% propanediol to ensure its stability and long-term preservation by preventing contamination. The concentrated extract can also be diluted by adding 30% glycerin combined with any type of water-soluble preservative, such as sodium benzoate or potassium sorbate, to a final concentration of 0.5%, or phenoxyethanol to a final concentration of 1.5%.

[0145] After dilution in a physiologically acceptable solvent, such extracts are characterized by a dry weight of 2 to 40 g / kg and comprise 0.2 to 5 g / kg of sugars; 300 to 900 mg / kg of organic acids including 50 to 500 mg / kg of shikimic acid and 10 to 200 mg / kg of quinic acid; 300 to 2000 mg / kg of phenolic compounds including 140 to 210 mg / kg of phenolic acids, 2 to 10 g / kg of proteins and at least 10 to 60 mg / kg of small molecular weight RNA.

[0146] When the plant material is the fresh jasmine flower (Jasminum grandiflorum), the extract further comprises at least one jasminetide. cosmetic compositions

[0147] Another object of the invention is a cosmetic composition comprising an effective amount of at least one plant extract obtained according to the process described in this application.

[0148] Advantageously, the plant extracts of the invention are used in diluted form and are added to a physiologically acceptable medium 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.

[0149] The composition usable according to the invention is formulated to be applied by any suitable route, in particular oral, or external topical, and the formulation of the compositions will be adapted by a person skilled in the art.

[0150] Preferably, the compositions according to the invention are presented in a form suitable for topical application. These compositions must therefore contain a physiologically acceptable medium, that is to say, one compatible with the skin and its appendages, without risk of discomfort during application, and cover all suitable cosmetic forms.

[0151] The compositions for implementing the invention may in particular be in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water emulsion, a water-in-oil emulsion or multiple emulsions; they may also be in the form of suspensions, or even powders, suitable for application on the skin, mucous membranes, lips and / or hair.

[0152] These compositions can be more or less fluid and may also have the appearance of a cream, lotion, milk, serum, ointment, gel, paste, or mousse. They can also be in solid form, such as a stick, or applied to the skin as an aerosol.

[0153] Examples of physiologically acceptable media commonly used in the intended field of application include formulation adjuvants such as solvents, thickeners, diluents, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.

[0154] In all cases, a person skilled in the art shall ensure that these additives and their proportions are chosen in such a way as not to impair the desired advantageous properties of the composition according to the invention. These additives may, for example, represent 0.01 to 20% of the total weight of the composition. When the composition according to the invention is an emulsion, the oil phase may 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 commonly used in the relevant field. For example, they may be used in a proportion ranging from 0.3 to 30% by weight relative to the total weight of the composition.

[0155] According to another advantageous embodiment of the invention, the plant extracts of the invention can be encapsulated or included in a cosmetic vector such as liposomes or any other nano capsule or microcapsule used in the field of cosmetics or adsorbed on powdered organic polymers, mineral supports such as talcs and bentonites.

[0156] Advantageously, the composition according to the invention may include, in addition to the plant extract according to the invention, at least one other active agent having cosmetic effects similar and / or complementary to those of the invention.

[0157] For example, the additional active agent(s) can be chosen from: anti-aging, firming, brightening, moisturizing, draining, microcirculation-enhancing, exfoliating, desquamating, and extracellular matrix-stimulating agents. activating energy metabolism, antibacterial, antifungal, soothing, anti-free radical, anti-UV, anti-acne, anti-inflammatory, anesthetic, providing a feeling of warmth, providing a feeling of freshness, slimming.

[0158] Such additional active agents may be chosen from groups including: - vitamins (vitamin A and its derivatives; vitamins B3, B5, B6 and B12; vitamin C; vitamins E, F, H, K, PP or even coenzyme Q10); - metalloproteinase inhibitors, or TIMP activators; - DHEA, its precursors and derivatives; - amino acids, natural or synthetic peptides, - Artemia salina extract, marketed under the name GP4G™ (FR2817748, ASHLAND®); - plant peptide extracts, yeast extracts, polyphenol extracts; - dehydroacetic acid (DHA); - phytosterols of synthetic or natural origin; - salicylic acid and its derivatives, alpha- and beta-hydroxy acids, silanols; - amino sugars and polysaccharides; - lipids such as ceramides or phospholipids; - cyclic AMP and its derivatives, the methylxanthines. cosmetic uses

[0159] The invention also relates to the cosmetic use of a composition comprising the plant extracts of the invention for skin care and more particularly to combat the signs of skin aging, loss of firmness or tone, improve barrier function and hydration, lighten the skin, mitigate the age-related decrease in the number or activity of mechanosensory touch receptors (Piezol) and oxytocin receptors (OXTR), or improve the skin's innate immune defenses and in vivo well-being.

[0160] Preferably, the cosmetic uses according to the present invention relate to cosmetic treatment methods by topical application on healthy skin.

[0161] In a particular embodiment, the invention relates to the cosmetic use of self-fermented extracts of fresh jasmine flowers (Jasminum grandiflorum).

[0162] Advantageously, the invention relates to the cosmetic use of a composition comprising an extract of fresh jasmine flowers (Jasminum grandiflorum) obtained by a self-fermentation process coupled with an extraction process allowing enrichment in small molecular weight RNA (process described in patents FR 1670672 and US 11,021,505).

[0163] The plant extracts of the invention have been tested on biological markers associated with aging and hydration, such as the expression of collagen and hyaluronic acid, and have demonstrated superior efficacy to conventional control plant extracts.

[0164] The plant extracts of the invention have been tested on biological markers associated with skin firmness or tone, such as e-cadherin, and have demonstrated superior efficacy to conventional control plant extracts.

[0165] The plant extracts of the invention have been tested on tyrosinase, an enzyme associated with melanin synthesis, and therefore with skin pigmentation, and have shown superior tyrosinase inhibitory activity compared to conventional control plant extracts.

[0166] A cosmetic composition comprising the plant extracts of the invention has made it possible to mitigate the age-related decline in the number or activity of mechanosensory touch receptors (Piezol).

[0167] A cosmetic composition comprising the plant extracts of the invention made it possible to stimulate the expression of the oxytocin receptor, a molecule involved in the prevention of skin aging.

[0168] In this particular embodiment, jasmine flower extracts have demonstrated their ability to stimulate the expression of viperin, a molecule associated with the skin's innate immune defenses.

[0169] In this particular embodiment, a composition comprising 2% of extracts of the invention, applied topically by healthy volunteers, produced a positive effect on well-being in vivo. In this test, emotional state was assessed through three distinct components: the subjective feeling of well-being, salivary oxytocin production, and the measurement of behavior or emotional state (Don Hockenbury and Sandra E. Hockenbury, Discovering Psychology, 5th edition, Page 344, Chapter 8, Worth Publishers).

[0170] In a particular embodiment, the invention also relates to a composition comprising the plant extracts of the invention to improve the immune defenses of the skin. Examples

[0171] For illustrative purposes, examples of embodiments of the process according to the invention are described below.

[0172] Example 1: Preparation of an extract from fresh, self-fermented jasmine flowers (Jasminum grandiflorum)

[0173] The species Jasminum grandiflorum belongs to the genus Jasminum and is widely used in industry as a fragrance or aroma.

[0174] In a first step a), 200 g of whole fresh jasmine flowers are mixed with 1800 g of distilled water, i.e. 10% of raw material involved in the process and 90% water for a total weight of 2 kg.

[0175] b) The pH is adjusted to 7.

[0176] c) The mixture is maintained at 30°C in an open beaker to maintain an aerobic atmosphere, favorable to the development of microorganisms, for 24 hours. During this step, the pH is regularly monitored, allowing for an acidification of the medium linked to the development of microorganisms, without it falling below 5.

[0177] d) The mixture obtained is centrifuged for 10 min at 4000 g, so as to sediment the residual plant matter in the pellet and collect the supernatant.

[0178] e) The resulting mixture is then subjected to successive filtrations by lowering the filtration threshold from 30 to 0.2 pm.

[0179] f) The pH of the filtrate obtained is adjusted to 6.3 using 10% concentrated citric acid.

[0180] g) The filtrate is sterilized by filtration.

[0181] The undiluted extract has a dry weight of 11.7 g / kg and contains a concentration of 5.2 g / kg of total sugars, 3.7 g / kg of protein, 411.1 mg / kg of total organic acids, 630 mg / kg of total phenolic compounds.

[0182] h) The extract is diluted with plant-derived glycerin to obtain a final concentration of 30% glycerin and 70% self-fermented jasmine flower extract.

[0183] The diluted extract has a dry weight of 8.1 g / kg, and exhibits a concentration of 3.6 g / kg of total sugars, 2.6 g / kg of protein, 287.7 mg / kg of total organic acids, 441 mg / kg of total phenolic compounds.

[0184] Example 2: Preparation of an extract of fresh jasmine flowers (Jasminum grandiflorum) by autofermentation coupled with a second extraction process allowing enrichment in small RNAs

[0185] Once the self-fermentation has been carried out according to steps a) to c) of example 1.

[0186] 2 g / L or 3 mM of phytic acid is added to the mixture.

[0187] The pH is adjusted to pH 1.1 to allow enrichment of the extract in small molecular weight RNA as well as various phytomolecules.

[0188] The mixture is heated for 2 hours at 80°C while stirring.

[0189] The mixture is then filtered using 30 pm porosity filters to separate the solid matter from the filtrate.

[0190] Sequential filtrations on filters of decreasing porosity are then carried out in order to clarify the plant extract down to a filtration at 1 pm porosity.

[0191] The pH is adjusted to 6.3 with a citric acid solution.

[0192] At this stage, the extract has a dry weight of 17.5 g / kg, and exhibits a concentration of 6.4 g / kg of total sugars, 6.6 g / kg of protein, 802.9 mg / kg of total organic acids, 1301 mg / kg of total phenolic compounds and 45 mg / kg of small molecular weight RNA and is devoid of DNA.

[0193] The absence of DNA was demonstrated by a DNase test, an enzyme that specifically degrades DNA and not RNA. The electrophoretic profile after The action of DNase is not modified, which demonstrates that the nucleic acid present in the extract is not sensitive to DNase and is therefore not DNA.

[0194] The extract is then diluted with plant-derived propanediol to achieve a final concentration of 30% propanediol and 70% self-fermented jasmine flower extract.

[0195] The diluted extract has a dry weight of 11.7 g / kg, and exhibits a concentration of 4.1 g / kg of total sugars, 4.6 g / kg of protein, 562.5 mg / kg of total organic acids, 874 mg / kg of total phenolic compounds and 31 mg / kg of small molecular weight RNA.

[0196] Example 3: Preparation of jasmine extracts as comparative elements 1 / Extraction of fresh jasmine flowers using a conventional process:

[0197] For comparative purposes, conventional extracts were made using the same quantity of whole, fresh jasmine flowers as in examples 1 or 2, i.e. 10% of plant raw material in distilled water.

[0198] The mixture is then heated for 1 hour at 25°C, and subsequently filtered through a first filtration using large-porosity filters (30 µm) to remove residual solid plant matter from the liquid. Sequential filtrations using filters of decreasing porosity are then performed to clarify the plant extract, culminating in a sterilizing filtration at 0.2 µm porosity.

[0199] The liquid portion obtained in the previous step constitutes a conventional control extract.

[0200] Comparative analytical data could thus be generated, as illustrated in [Fig. 1], [Fig. 2], [Fig. 5] and the examples. 2 / Extraction of fresh jasmine flowers using a process that allows enrichment in small RNAs:

[0201] This process is described in detail in patents FR 1670672 and US 1 1,021,505.

[0202] To obtain the fresh flower extract enriched with small RNAs, the same quantity of whole, fresh jasmine flowers is used as in examples 1 and 2, i.e. 10% of plant raw material in distilled water.

[0203] 2 g / L or 3 mM of phytic acid is added to the mixture.

[0204] The pH is adjusted to pH 1.1 to allow enrichment of the extract in small molecular weight RNA as well as various phytomolecules.

[0205] The mixture is heated for 2 hours at 80°C while stirring.

[0206] The mixture is then filtered using 30 pm porosity filters to separate the solid matter from the filtrate.

[0207] Sequential filtrations on filters of decreasing porosity are then carried out in order to clarify the plant extract up to a filtration at 1 m porosity.

[0208] The pH is adjusted to 6.3 with a citric acid solution.

[0209] The liquid portion obtained in the previous step constitutes an extract enriched in small control RNAs.

[0210] Comparative analytical data could thus be generated, as illustrated in [Fig. 1], [Fig. 2], [Fig. 5].

[0211] Example 4: Analysis of jasmine flower extracts from examples 1 and 2 versus a conventional extract and a small RNA-enriched extract from example 3

[0212] The analyses were performed on the extracts of examples 1 and 2 before dilution, on a conventional extract and on a small RNA enriched extract of example 3.

[0213] Methodologies

[0214] The total phenolic content of the extracts was measured spectrophotometrically at 760 nm after reduction of the Folin-Ciocalteu reagent by the phenols. Quantification was performed using a gallic acid standard curve, and the results are expressed as gallic acid equivalents.

[0215] The total protein content was measured by spectrophotometry at 550 nm after a calorimetric reaction of Biuret combined with Folin-Ciocalteu reagent. Quantification was performed using a standard BSA (Bovine Serum Albumin) curve.

[0216] The detailed composition of phenolic compounds was determined by liquid chromatography coupled with a UV detector fixed at 254 nm. The samples were separated on an UPTISPHERE CS EVOLUTION Cl 8-AQ column using an Agilent 1200 HPLC system (Agilent Technologies). The mobile phases consisted of a 0.1% formic acid solution and methanol.

[0217] The molecular weight distribution profile of the proteins was determined by size-exclusion chromatography coupled with a UV detector fixed at 254 nm. Samples were separated on a YMC Pack Diol 60 column (DL06503-2543WT) using an Agilent 1200 HPLC system (Agilent Technologies). The mobile phase consisted of an aqueous solution of 0.05% NaN3, 0.2 M NaCl, and 0.1 M phosphate buffer at pH 7.

[0218] The characterization and quantification of organic acids were performed by high-performance liquid chromatography coupled with mass spectrometry (Acquity Qda, Waters) equipped with a negative-mode electrospray ion source. Samples were separated on an EC 150 / 4.6 Nucleoshell RP 18plus-5|jm column (Macherey Nagel: 763236.46) using an Agilent 1260 HPLC system (Agilent Technologies). The flow rate was 0.3 mL / min. The mobile phases consisted of a 0.01% formic acid solution and acetonitrile.

[0219] The characterization and quantification of sugars were performed by high-performance liquid chromatography coupled with mass spectrometry (Acquity Qda, Waters) equipped with a negative-mode electrospray ion source. Samples were separated on a Luna Omega Suagr 100 A-3|jm column (Phenomenex: 00F-4775-E0) using an Agilent 1260 HPLC system (Agilent Technologies). The flow rate was 0.8 mL / min. The mobile phases consisted of a 10 mM aqueous solution of ammonium acetate and a 10 mM aqueous solution of ammonium acetate.

[0220] Jasmintide identification was performed by ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry. Samples were denatured and then separated on an Aurora column (15 cm x 75 m x 1.6 pm x 120 Å). The flow rate was 0.3 mL / min. The mobile phases consisted of a 0.1% aqueous solution of formic acid and a 0.1% acetonitrile solution. Jasmintide identification was performed by comparison with the Swissprot database. The overall screening of the extracts was carried out by Gas Chromatography (GC) coupled to Mass Spectrometry (MS). The samples were derived with (N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and then separated on an Optima 5HT column (Macherey Nagel 726104.15) by a gradient at 3°C / min from 50°C to 300°C.

[0221] Results

[0222] The protein profile of the extracts is illustrated in [Fig. 1]. The molecular weights of the conventional extract range from 7200 to 170 g / mol. In comparison, the autofermentation process in Example 1 broadens the molecular weight distribution of the extract's proteins, with weights ranging from 15800 to 170 g / mol. Comparisons of protein profiles highlight the increased protein content during the fermentation process compared to a conventional extract for weights above 1000 g / mol.

[0223] The process described in Example 2 (autofermentation coupled with extraction) increases the complexity of the extract, with the appearance of proteins with a molecular weight of approximately 19,000 g / mol and an increased signal for small molecules with a molecular weight of approximately 1,000 g / mol compared to the conventional extract. Quantification of total protein shows a 50% increase between the extract from Example 2 (autofermented with extraction) and the extract enriched in small RNAs.

[0224] The auto-fermentation process therefore brings about a modification of the protein profile of the extract and coupling it with an extraction step makes it possible to increase the total protein concentration.

[0225] Analysis of the total quantity of phenolic compounds indicates that the extract from Example 2 (autofermentation coupled with extraction) contains more phenolic compounds than a conventional extract (Example 3). Figure 2 shows targeted consumption of complex phenolic compounds (flavonoids or glycosylated flavonoids) and the appearance of simple phenolic compounds such as phenolic acids during fermentation. Figure 2 shows a 120% increase in the presence of phenolic acids between the conventional extract and the autofermented extract, and a 23% increase between the small RNA-enriched extract and the extract from Example 2 (autofermented coupled with extraction). Figure 2 shows a 23% increase in the presence of phenolic acids between the conventional extract and the autofermented extract. 2] also indicates an 87% decrease in the presence of flavonoids between the conventional extract and the self-fermented extract and a 93% decrease between the small RNA enriched extract and the extract from example 2 (self-fermented coupled with an extraction).

[0226] Analysis of the organic acids contained in the extracts, as illustrated by La [Fig. 3], shows the increase in the concentration of lactic, succinic, shikimic, quinic and glycolic acids during the fermentation process.

[0227] Table 1 details the presence of lactic and quinic acids in the self-fermented extract versus their absence in the conventional extract, and the 72% and 79% increases in succinic and shikimic acids in the self-fermented extract versus the conventional extract, respectively. It also details the 75%, 31%, 12%, 66%, and 27% increases in the concentration of lactic, succinic, shikimic, glycolic, and quinic acids in the self-fermented extract combined with an extraction versus a small RNA-enriched extract, respectively.

[0228] The concentration of malic acid undergoes a decrease during the fermentation process with a decrease of 38% in the self-fermented extract versus the conventional extract and a decrease of 19% in the self-fermented extract coupled with an extraction versus the extract enriched in small RNAs.

[0229] The overall concentration of organic acids is greater in the self-fermented extract coupled with an extraction than in conventional and small RNA enriched extracts that do not contain a fermentation step.

[0230] Sugar analysis shows a 54% decrease in glucose in the extract from example 2 (self-fermented coupled with extraction) versus the extract enriched in small RNAs due to sugar consumption by microorganisms in the self-fermented extract followed by extraction.

[0231] The overall concentration of phenolic acids is 57% higher in the self-fermented extract coupled with an extraction than in the self-fermented extract alone.

[0232] The analysis of jasmintides highlights the presence of at least one jasmintide in the extract of example 2 (self-fermented coupled with an extraction) as well as the absence of jasmintides in the conventional extract.

[0233] GCMS analysis of the extracts indicates that the autofermentation step increases the diversity of molecules present in the extract. Indeed, 109 molecules are detected in the profile of the autofermented extract compared to 95 in the conventional extract. 165 molecules are detected in the extract of Example 2 (self-fermentation coupled with extraction) versus 132 in the extract enriched in small RNAs. Coupling the self-fermentation process with extraction allows obtaining an extract with a more diverse composition than conventional extractions and an enrichment in small RNAs.

[0234] [Table 1]: Analysis of compounds in jasmine flower extracts

[0235] [Table 2]: Analysis of phenolic compounds in jasmine flower extracts (area under the curve)

[0236] Example 5: Demonstrating the role of phytobiota in the self-fermentation of jasmine flowers (Jasminum grandiflorum)

[0237] Fresh jasmine flowers were sterilized by autoclaving for 10 minutes at 121°C to kill microorganisms. This plant material, deprived of its live phytobiota, was then used in the process described in Example 1 to create a control extract.

[0238] Physicochemical analysis shows that the levels of phenolic compounds and proteins decrease sharply during the auto-fermentation process, while they are stable in the control condition.

[0239] These results demonstrate that the microorganisms of the phytobiota are directly responsible for the metabolic consumption of phenolic compounds and proteins during the self-fermentation process described in this application and in particular in example 1.

[0240] [Table 3]: Comparison of phenolic compound and protein consumption in self-fermentation or control conditions (phytobiota killed)

[0241] Example 6: In vitro evaluation of the hyaluronidase inhibitory capacity of fresh jasmine flower extracts obtained according to examples 1 and 3

[0242] Hyaluronidase catalyzes the degradation of hyaluronic acid (a glycosaminoglycan highly present in the dermis of the skin with hydrating and anti-aging properties) into mono or disaccharides as well as into smaller fragments of hyaluronic acid.

[0243] Hyaluronic acid has the ability to cause turbidity in the presence of an acidic albumin solution, which can be measured by spectrophotometer at 600 nm.

[0244] Protocol: The enzyme is incubated with the extracts obtained according to Examples 1 and 3 at the chosen concentration (vol / vol dilution) at a temperature of 37°C. The enzyme substrate, hyaluronic acid, is added to the mixture. The mixture is slowly homogenized and then incubated for exactly 45 minutes at 37°C. The hyaluronic acid remaining in the mixture is then contacted with an acid albumin solution for 10 minutes before the transmittance is read at 600 nM using a spectrophotometer.

[0245] The extract of self-fermented jasmine flowers prepared according to example 1 exhibits a significantly stronger enzyme inhibitory power than the extract prepared conventionally according to example 3. The results are presented in [Table 4].

[0246] [Table 4]: Comparison of the percentages of hyaluronidase inhibition obtained in vitro between conventional and self-fermented jasmine extracts

[0247] Example 7: Formula for a rich cream

[0248] [Table 5]

[0249] Preparation process

[0250] 1. Homogenize phase A in the main container and begin heating to 75-80°C;

[0251] 2. At 30°C, sprinkle into Phase B and homogenize while heating;

[0252] 3. In a separate beaker, prepare phase C, heat to 75-80°C until homogeneous;

[0253] 4. At 75°C, add phase C to the main container and homogenize for 10 minutes;

[0254] 5. Allow the temperature to cool and add phase D at 65°C. Mix well to homogenize for 10 minutes;

[0255] 6. Premix phase E before adding it to the main container;

[0256] 7. Add phase E at 60°C. Mix well to homogenize for 10 minutes;

[0257] 8. At 35°C, premix phase F before adding it and mix well;

[0258] 9. Premix phase G before adding it to the main container;

[0259] 10. Add phase G at 35°C. Mix well to homogenize;

[0260] 1 1. In a separate beaker, prepare phase H: sprinkle Natrosol™ into water at room temperature and homogenize while heating to 60°C;

[0261] 12. Add phase H at 30°C. Mix well to homogenize;

[0262] 13. Stop at 25°C.

[0263] The composition is thus presented in the form of a pink buttercream, with a pH between 4.90 and 5.40 and a viscosity (DO) of 160000 - 210000 cps (Brookfield RVT / Spindle D / 5 RPM / 1 minute / 25°C).

[0264] Example 8: Evaluation of the jasmine extract obtained according to example 2 on the expression of the Piezol receptor in reconstructed epidermis

[0265] The proteins Piezol and Piezo2 have been identified as ion channels mediating mechanosensory transduction in mammalian cells (Coste, Bertrand et al. “Piezol and Piezo2 are essential components of distinct mechanically activated cation channels.” Science (New York, NY) vol. 330, 6000 (2010): 55-60). In the skin, keratinocytes participate in mediating tactile sensation by detecting and encoding this information for sensory neurons. Piezol is the main mechanotransducer of keratinocytes (Holt, Jesse R et al. Spatiotemporal dynamics of PIEZO1 localization controls keratinocyte migration during wound healing. eLife vol. 10 e65415. Sep 27 2021 ).

[0266] Principle: The jasmine extract obtained according to example 2 was evaluated for its ability to modulate the expression of the mechanoreceptor Piezol in human skin ex vivo.

[0267] Protocol: Piezol receptor expression is assessed by indirect immunofluorescence on sections of reconstructed epidermis, previously treated by topical application of the jasmine extract from Example 2 diluted to 2% (v / v) for 48 hours (once daily). Reconstructed control epidermis incubated in parallel under the same conditions receives placebo (Phosphate Butter Saline, PBS). At the end of incubation, the reconstructed epidermis is fixed and paraffin-embedded for histological sectioning. Piezol receptor detection is performed by incubation with a primary anti-Piezol antibody (Proteintech). After 1.5 hours of incubation followed by rinsing, the sections are incubated with a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an epi-fluorescence microscope (Zeiss Axiovert 200M microscope). Piezol receptor expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0268] Results: As illustrated by [Fig. 4], when biopsies were treated with 2% jasmine extract, Piezol receptor expression increased by 39%.

[0269] Conclusion: Jasmine extract showed a positive effect on the expression of the Piezol receptor.

[0270] Example 9: Evaluation of the jasmine extract obtained according to example 2 on the expression of the oxytocin receptor (OXTR) on human skin biopsies

[0271] Principle: The aim of this experiment is to demonstrate an effect of jasmine extract on the synthesis of the oxytocin receptor in cultured human skin biopsies.

[0272] Protocol: Oxytocin receptor (OXTR) expression is assessed by indirect immunofluorescence on skin biopsies, previously treated by topical application of the jasmine extract from example 2 diluted to 2% (vol / vol) for 48 hours (twice daily). Control biopsies incubated in parallel under the same conditions receive the placebo (Phosphate Butter Saline, PBS). At the end of the incubation period, the biopsies are fixed and paraffin-embedded for histological sectioning. OXTR receptor detection is performed by incubation with anti-OXTR antibody (Proteintech). After 1.5 hours of incubation followed by rinsing, the sections are incubated with a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The sections are then examined under an epi-fluorescence microscope (Zeiss Axiovert 200M microscope). OXTR receptor expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0273] Results: As illustrated by [Fig. 5], when biopsies were treated with 2% jasmine extract, OXTR receptor expression increased by 63%.

[0274] Conclusion: Jasmine extract showed a positive effect on the expression of the olfactory receptor to oxytocin OXTR.

[0275] Example 10: Evaluation of the jasmine extract obtained according to example 2 on the expression of oxytocin receptor (OXTR) messenger RNA in cultured keratinocytes

[0276] Oxytocin is a natural peptide that controls a wide range of specific actions in its target tissues, from cell growth and differentiation to reproduction and social behavior (Carter, C. Sue et al. “Is Oxytocin?” Nature's Medicine Pharmacological Reviews vol. 72, 4 (2020): 829-861). A large body of scientific literature supports the beneficial role of oxytocin in skin physiology and aging (Hayre, Nicole. “Oxytocin Levels Inversely Correlate with Skin Age Score and Solar Damage.” Journal of Drugs in Dermatology: JDD vol. 19, 12 (2020): 1146-1148). Other research also highlights the importance of the oxytocin receptor in preventing skin aging (Cho, SY et al. “Oxytocin alleviates cellular senescence through oxytocin receptor-mediated extracellular signal-regulated kinase / Nrf2 signalling.” The British journal of dermatology vol. 181, 6 (2019): 1216-1225).

[0277] Principle: The aim of this experiment is to demonstrate an effect of jasmine extract on the synthesis of messenger RNA of the oxytocin receptor in human skin keratinocytes.

[0278] Protocol: The expression level of oxytocin receptor (OXTR) messenger RNA was assessed by qPCR (quantitative polymerase chain reaction) on cultured skin keratinocytes, pre-treated with jasmine extract obtained according to Example 2 for 48 hours at 1% (v / v dilution) in the culture medium (once daily). At the end of the culture period, the cells were lysed, the total RNA was extracted, and then converted to complementary DNA (cDNA) by reverse transcription. Quantification of OXTR receptor cDNA was performed by qPCR using a TaqMan probe for detection. The delta-ΔCt quantification method allowed for comparison of the difference in expression (ACt) between the gene of interest (OXTR) and the reference gene (GADPH).

[0279] Results: As illustrated by [Fig. 6], when cultured keratinocytes were treated with the jasmine extract obtained in Example 2 at 1%, the expression of OXTR receptor messenger RNAs was increased by 26%.

[0280] Conclusion: Jasmine extract showed a positive effect on the expression of OXTR receptor messenger RNAs.

[0281] Example 15: Evaluation of the jasmine extract obtained according to example 2 on the expression of viperin in cultured keratinocytes

[0282] Viperine is one of the factors of innate immune defense synthesized by keratinocytes (Garcia, Magali et al. “Innate Immune Response of Primary Human Keratinocytes to West Nile Virus Infection and Its Modulation by Mosquito Saliva.” Frontiers in Cellular and Infection Microbiology vol. 8387. 2 Nov. 2018). Viperine has been shown to catalyze the conversion of cytidine triphosphate (CTP) to 3'-deoxy-3',4'-didehydro-CTP (ddhCTP), a previously unknown ribonucleotide analog. The incorporation of ddhCTP causes the premature termination of RNA synthesis in certain viruses (Gizzi, Anthony S et al. “A naturally occurring antiviral ribonucleotide encoded by the human genome.” Nature vol. 558, 771 1 (2018): 610-614).

[0283] Principle: The aim of this experiment is to demonstrate an effect of jasmine extract on the level of viperin expression in cultured keratinocytes.

[0284] Protocol: Viper's bugloss expression was assessed by indirect immunofluorescence on keratinocytes. Cultured keratinocytes were treated with jasmine extract obtained according to Example 2 at 1% (vol / vol dilution) for 48 hours (once daily). At the end of the culture period, the cells were fixed. Viper's bugloss detection was performed by incubation with an anti-viper's bugloss antibody (Sigma). After 1.5 hours of incubation followed by rinsing, the cells were incubated with a secondary anti-mouse antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen). The cells were then examined under an epifluorescence microscope (Zeiss Axiovert 200M microscope). Viper's bugloss expression was then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0285] Results: As illustrated by [Fig. 7], the jasmine extract obtained according to example 2 showed a positive effect of +40% on viperin expression in cultured keratinocytes.

[0286] Conclusion: Jasmine extract showed a positive effect on viperine expression.

[0287] Example 16: Evaluation of the jasmine extract obtained according to example 2 on the level of E-cadherin expression, on human skin biopsies pretreated with a blocker of Piezol activity (Dookul)

[0288] Principle: The aim of this experiment is to demonstrate an effect of jasmine extract on the level of expression of E-cadherin, a molecule involved in tissue tone, in biopsies of cultured human skin.

[0289] Protocol: E-cadherin expression is assessed by indirect immunofluorescence on skin biopsies pretreated with a Piezol activity blocker (Dookul), then treated by topical application of jasmine extract obtained according to Example 2 for 48 hours (twice daily), or with a Piezol activator (Jedil) for 48 hours (once daily). Control biopsies incubated in parallel under the same conditions receive placebo (Phosphate Butter Saline, PBS). At the end of incubation, the biopsies are fixed and paraffin-embedded for sectioning. Histological analysis. E-cadherin detection is performed by incubation with anti-E-cadherin antibodies (Abeam). After 1.5 hours of incubation, followed by rinsing, the sections are incubated with a 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). OXTR receptor expression is then observed and quantified by image analysis (Volocity® image analysis software, Improvision).

[0290] Results: As illustrated in [Fig. 8], when biopsies were pretreated with a piezolytic blocker (Dookul), a 44% decrease in E-cadherin expression was observed. When biopsies were treated with a piezolytic activator (Jedil), E-cadherin expression increased by 23%. When biopsies were treated with jasmine extract, E-cadherin expression increased by 95%.

[0291] Conclusion: The jasmine extract obtained according to example 2 showed a positive effect on E-cadherin expression, mimicking the effect of the Piezol receptor activator (Jedil).

[0292] Example 17: Evaluation of the jasmine extract obtained according to example 2 on the expression level of 11-HSDl enzyme messenger RNA in cultured keratinocytes

[0293] Principle: The aim of this experiment is to demonstrate the effect of jasmine extract on the expression level of the messenger RNA of the enzyme 11p-HSDl, also known as cortisone reductase, in cultured human skin keratinocytes. An increase in enzyme expression 1 1 p-HSDl has been implicated in the degradation of epidermal barrier function during skin aging (Kim, BJ et al. Increased Expression of 1 1 -Hydroxysteroid Dehydrogenase Type 1 Contributes to Epidermal Permeability Barrier Dysfunction in Aged Skin. Int. J. Mol.Sci. 2021 , 22, 5750.).

[0294] Protocol: The expression level of messenger RNAs of the enzyme 11p-HSD1 is assessed by qPCR (quantitative polymerase chain reaction) on cultured human skin keratinocytes, pre-treated with jasmine extract obtained according to Example 2 for 48 hours at 1% in the culture medium (once a day). At the end of the culture period, the cells are lysed, the total RNAs are extracted, and then converted to complementary DNA (cDNA) by a reverse transcription reaction. Quantification of the cDNAs of the 11p-HSDl enzyme was performed by quantitative polymerase chain reaction using a TaqMan probe for detection. The delta-ΔCt quantification method allowed for comparison of the difference in expression (ACt) between the gene of interest (11p-HSDl) and the reference gene (GADPH).

[0295] Results: As illustrated by [Fig. 9], when cultured keratinocytes were treated with the jasmine extract obtained according to example 2 at 1%, the level of expression of the enzyme 1 1 p-HSDl mRNAs was decreased by -24%.

[0296] Conclusion: Jasmine extract showed an inhibitory effect on the expression level of the 11p-HSDl enzyme.

[0297] Example 18: Evaluation of the jasmine extract obtained according to example 2 to combat the signs of skin aging, improve barrier function, skin radiance and in vivo well-being.

[0298] Principle: To study the efficacy of the 2% Jasmine extract. Protocol: A double-blind, placebo-controlled comparative study was conducted on the face and forearms of 34 volunteers (aged 36 to 66 years) divided into two groups of 17 volunteers homogeneous in terms of age and sex. The treatment group received the Jasmine extract from Example 2, formulated at 2% in the skincare formula detailed in Table 6, and the placebo group received the same skincare formula but without the jasmine extract. Product allocation was performed according to a randomization list generated by a random number generator.

[0299] [Table 6: Skincare formula containing the Jasmine extract from Example 2 at 2% used during the clinical trial] phase

[0300] Study duration: 28 days including 3 follow-up visits at D0 (first day of the study), D14 (after 14 days of application) and D28 (end of the study after 28 days of application).

[0301] - Measurement of well-being using three different methods at 0.01 and 0.28: a. Anonymized questionnaire on the WHO Well-being Index. b. Quantification of oxytocin in saliva by ELISA (enzyme-linked immunosorbent assay). Oxytocin is a nonapeptide that plays a key role in several physiological functions (e.g., milk ejection) as well as in certain types of behavioral regulation such as attachment, social recognition, and anxiety. Several studies suggest that pleasant touch is associated with the release of oxytocin and induces relaxing behavioral effects. After rinsing the mouth thoroughly for 30 seconds, 2 ml of saliva from the volunteers was collected in a pre-inflated tube and immediately stored at -80 °C. Salivary oxytocin was quantified using an ELISA kit (Enzo Life Sciences). c. Measurement of emotional state using the Emotion Capture® system patented by ICONIK®. This biometric technology measures a person's stress level in real time, using 3 different sensors that measure: i. changes in the ionic charge of the sweat glands via a skin-sensitive galvanic sensor; ii. changes in heart rate via an optical sensor; iii. changes in skin temperature via a skin temperature sensor. All the data collected is then normalized and processed by an algorithm developed by ICONIK; the emotional state of the volunteers and their stress level is calculated.

[0302] - Transient water loss (TEWL) - on the forearm measured by a closed chamber method using the AquaFlux® AF200 (Biox®).

[0303] - Skin tone - measured on the face with the CM-700d spectrophotometer (Konica Minolta Sensing, Inc.). The ITA° (Individual Topology Angle) parameter was calculated using the following equation: ITA° = (Arc tan L*-50 / b*)lx 80 / n).

[0304] L*: skin luminance value

[0305] b*: values ​​between blue and yellow (negative values ​​indicate blue and positive values ​​indicate yellow)

[0306] - Forehead skin topography using silicone replicas, analyzed by combining fringe projection and stereometry technologies with the AEAVA-HE® system (Eotech®). Various skin microrelief parameters were calculated, such as surface and profile roughness. Furthermore, 2D and 3D representations of the skin topography were generated using the same system.

[0307] - Colour photos of the face. The photos were taken with the HeadScan* V03 bench (Orion Concept*).

[0308] - Evaluation on a scale of 0 to 100 by an expert of the condition of the facial skin by touch and according to the visual appearance of the following parameters:

[0309] - Skin tone: 0 corresponds to very dull skin and 100 to perfect luminosity and complexion.

[0310] - Skin homogeneity: 0 corresponds to non-homogeneous skin with redness and spots and 100 to very homogeneous skin without redness and spots.

[0311] - Skin hydration: 0 corresponds to very dry skin and 100 to very hydrated skin.

[0312] - Crow's feet: 0 corresponds to smooth skin without fine lines and 100 to skin with marked microrelief and visible fine lines.

[0313] -Overall skin appearance: 0 corresponds to poor skin hydration, very rough, dull and uneven, and 100 to very hydrated, luminous, even and smooth skin.

[0314] All measurements were taken on the subject after a rest period of 15 minutes in a room where the temperature (21°C + / -1) and relative humidity (50% + / - 5) were controlled.

[0315] Results of well-being measurements: As illustrated in [Fig. 10], the results show that after one month of application, volunteers in the treatment group experienced improved well-being. Analysis of the WHO Well-being Index questionnaire shows a 20% increase in perceived well-being compared to baseline (D0) for the treatment group, which was not the case for the placebo group. A significant increase in salivary oxytocin was also observed in the treatment group compared to the placebo group, suggesting that the application of jasmine extract may help enhance oxytocin release. Finally, the treatment group showed a significant improvement in their emotional state between D0 and D28, with volunteers feeling less stressed, whereas no significant improvement was recorded in the placebo group.

[0316] Conclusions from the well-being measurements: these results demonstrate that the 2% formulated Jasmine extract can improve in-vivo well-being.

[0317] Results of transepidermal water loss (TEWL) measurements: As shown in [Fig. 11], transepidermal water loss is significantly lower for the treated group than for the placebo group from 14 days of application until the end of the study.

[0318] Conclusion of the measurements of insensible water loss: this result demonstrates that the skin barrier was strengthened after just 14 days of application of the Jasmine extract formulated at 2%.

[0319] Results of skin tone measurements: [Fig. 12] shows that after one month of application of the cream containing 2% jasmine extract, the ITA° increases significantly compared to placebo.

[0320] Conclusion of skin tone measurements: this result demonstrates that the jasmine extract formulated at 2% improves facial tone.

[0321] Results of forehead skin topography measurements and color facial photographs: as illustrated in [Fig. 13], surface roughness and profile parameters on the forehead decreased in the treated group compared to the placebo group after just 14 days of application. Furthermore, smoother skin and less visible microrelief were observed in 3D photographs of volunteers in the treated group, unlike those in the placebo group. These results are confirmed by observation of color facial photographs.

[0322] Conclusion of the measurements of the skin topography of the forehead and color photos of the face: these results highlight an anti-aging effect of the jasmine extract formulated at 2%.

[0323] Expert evaluation results: According to [Fig. 14], after 14 days of application with 2% jasmine extract, the expert observed an improvement in skin tone, hydration, and overall appearance, and no improvement after 14 days of placebo application. Similarly, after one month of application, the expert noted an improvement in all parameters assessed in the volunteers in the treatment group but not in the volunteers in the placebo group.

[0324] Conclusion of expert evaluation: these results demonstrate that the Jasmine extract formulated at 2% visually reduces facial fine lines and improves facial hydration and complexion.

[0325] Test conclusion: Under current conditions, the above results demonstrate that the 2% Jasmine extract improves the well-being of volunteers and reduces the effects of skin aging.

Claims

AMENDED CLAIMS received by the International Bureau on June 8, 2023 (08.06.2023)

1. A process for obtaining plant extracts enriched in organic acids and phenolic acids, comprising the following steps: a) the plant material is mixed with water, b) the pH is adjusted, if necessary, to a value between 4 and 9, c) the mixture is kept under gentle stirring for a time between 6 and 48 hours, at a temperature between 20 and 60°C, at a pH between 4 and 7, to allow the auto-fermentation process, in a chamber allowing gas exchange with the atmosphere, d) the mixture obtained in c) is purified to remove residual solid plant material and the liquid portion is collected, e) at least one filtration is carried out of the liquid portion obtained in the previous step, f) the pH of the filtrate is checked and readjusted, if necessary, to a value between 4 and 8, and preferably to a value between 4 and 7, g) the filtrate is sterilized.(h) The extract is diluted with a physiologically acceptable solvent to obtain diluted plant extracts characterized in that they comprise, by weight of the total weight of the extract, 2 to 20 g / kg of dry extract, 0.2 to 4 g / kg of sugars, 100 to 700 mg / kg of organic acids of which 50 to 350 mg / kg of shikimic acid and 10 to 70 mg / kg of quinic acid, 100 to 1300 mg / kg of phenolic compounds of which 70 to 140 mg / kg of phenolic acids and 0.1 to 5 g / kg of proteins.

2. A process for preparing plant extracts according to claim 1, characterized in that step c) of self-fermentation takes place over a period of between 15 and 30 h, at a temperature between 30 and 40°C.

3. A process for preparing plant extracts characterized in that it comprises steps a) to c) according to any one of claims 1 or 2, and then the following subsequent steps: Step d') To the mixture obtained in step c), phytic acid is added at a concentration between 1 and 5 mM, Step e') the pH is adjusted if necessary to a value between 10 and H, Step f') the mixture is kept under stirring for 1 to 3 hours, at AMENDED SHEET (ARTICLE 19) at a temperature between 50°C and 80°C, Step g') the residual plant matter is separated, Step h') the liquid fraction obtained is purified by successive filtrations to clarify the extract, Step i') the pH of the resulting mixture is adjusted to a value between 6 and 8, Step j') Optionally, the extract is diluted with a physiologically acceptable solvent.

4. A process for preparing plant extracts according to any one of the preceding claims, characterized in that the plant materials used are plants of the Oleaceae, Poaceae, Fabaceae families.

5. A process for preparing plant extracts according to any one of the preceding claims, characterized in that the plant material used is the fresh jasmine flower, preferably of the species Jasminum grandiflorum.

6. Diluted plant extracts obtained by the process of claim 3 characterized in that they comprise at least 300 mg / kg of phenolic compounds of which at least 140 mg / kg of phenolic acids and at least 300 mg / kg of organic acids, of which at least 50 mg / kg of shikimic acid and 10 mg / kg of quinic acid.

7. Diluted plant extracts obtained by the process of claim 3, characterized in that they comprise, by weight of the total weight of the extract, 2 to 40 g / kg dry weight; 0.2 to 5 g / kg of sugars; 300 to 900 mg / kg of organic acids of which 50 to 500 mg / kg of shikimic acid and 10 to 200 mg / kg of quinic acid; 300 to 2000 mg / kg of phenolic compounds of which 140 to 210 mg / kg of phenolic acids and 2 to 10 g / kg of proteins, as well as 10 to 60 mg / kg of small molecular weight RNA.

8. Diluted plant extracts obtained by the process of claim 3, characterized in that the plant material used is fresh jasmine flower, preferably of the species Jasminum grandiflorum, and that it further comprises at least one jasminetide.

9. Composition comprising an effective amount of extract obtained according to one of the processes of claims 1 to 3, preferably comprising from 0.05 to 5% of said extract, and even more preferably from 0.1 to 2.5% of said extract by weight relative to the total weight of the composition, and a physiologically acceptable medium.

10. Cosmetic use of the composition of claim 9 to combat the signs of skin aging, loss of firmness or AMENDED SHEET (ARTICLE 19) toning, improving barrier function and hydration, brightening the skin or improving the skin's innate immune defenses and in vivo well-being.

11. Cosmetic use of the composition of claim 9 to mitigate the age-related decline of touch receptors (Piezo 1) and oxytocin receptors (OXTR), of the skin and in vivo well-being.

12. Composition according to claim 9 for its use in enhancing the skin's innate immune defenses. AMENDED SHEET (ARTICLE 19)

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