Method for obtaining extract of plant origin, composition containing same and cosmetic use thereof

Abstract

The invention provides a method for obtaining an extract of plant origin from an undifferentiated or dedifferentiated plant cell line by in vitro culture, the method comprising the addition of at least one aglycone flavonoid to the culture medium. The obtained extract contains a group of target secondary metabolites. It can be advantageously used for the non-therapeutic cosmetic treatment of the skin and appendages.

Description

[0001] The present invention relates to a new process for obtaining extracts of plant origin, compositions containing the same and their cosmetic use.

[0002] The present invention relates in particular to ingredients and compositions for use in the cosmetics, cosmeceuticals, dermatological pharmacy and hygiene and personal care products industries, for treating extracts of plant origin and their appendages, whether mammalian, human or animal.

[0003] Plant-derived extracts can be obtained directly from plants in a conventional manner, or by culturing plant cells, tissues or plant organs in vitro.

[0004] More particularly, the present invention relates to a preparation method by in vitro culture.

[0005] Obtaining plant-derived extracts by in vitro culture has many advantages over the agro-industrial route (planting in open fields and subsequently performing industrial extraction). Due to the complete control of culture conditions, the extracts obtained by in vitro culture do not contain toxic substances (herbicides, pesticides, fertilizers, heavy metals and other pollutants, such as pollutants that may come from plant parasites). In addition, strict control of in vitro culture conditions reduces the risk of spontaneous variation of strains and ensures a reproducible spectrum of secondary metabolites corresponding to the target molecule being sought, which is different from cultivating in open fields, where there will be variability issues related to climate, meteorological and geographical conditions and their hazards. In addition, this technology overcomes obstacles such as the natural biological cycle of plants and the seasonality of secondary metabolite production, allowing for safer and faster supply. In addition, since it significantly reduces water consumption, avoids the development of arable soil, and prevents soil contamination, the environmental impact is minimal. In addition, since plants or even seeds are sufficient to start new in vitro cultures, biodiversity is preserved. Finally, this technology offers the possibility of implementing controlled and relatively rapid protocols for increasing the production of certain molecules, especially those produced in plants in small quantities (for example, by using induction in in vitro cultures).

[0006] In the context of existing in vitro plant culture techniques, the present invention is more specifically directed to the cultivation of undifferentiated or dedifferentiated cells: the method involves first establishing a highly proliferative cell line in an agar medium from meristematic cells, which are undifferentiated cells, or from dedifferentiated cells, which grow as a healing mass (called callus) after removing and cutting plant fragments (also known as explants) from plants, leaves, stems, roots, or other parts. Thereafter, the cell line is cultivated first on an agar medium and then in a liquid medium to significantly increase the biomass. During the growth period, and under defined and optimized culture medium conditions, the plant cells of the biomass will synthesize secondary metabolites that will constitute the molecules of cosmetic interest. The cultivation is then stopped, and extraction is performed at the optimal time to obtain the maximum amount of the target molecule. Existing commercially available cell lines can also be used as starting material.

[0007] Therefore, the method of the present invention schematically comprises:

[0008] - optionally, in a first instance, a step of establishing and selecting a cell line capable of producing cell biomass on a large scale, according to pre-established criteria (constant phenotype and optimal and constant production of selected metabolites, proliferation capacity);

[0009] - then, at a second time, a pre-cultivation step performed with the selected cell line to increase the number of cells in the biomass;

[0010] - then, at a third time, a cultivation step in a bioreactor to multiply the cell biomass, optionally with a simultaneous induction step;

[0011] - Then, at a fourth time, a treatment step of the cellular biomass obtained in order to recover said cellular extract of plant origin according to the invention, which will comprise at least the expected secondary metabolites of cosmetic interest.

[0012] Said in vitro technology allows the preparation of extracts that are original compared to classical extracts to date, as they contain caffeic acid derivatives as secondary metabolites in attractive concentrations to provide cosmetic activity. The following patents and applications can be cited.

[0013] EP 1 736 167 describes extracts of plant origin obtained from plant cells of Syringa vulgaris , said extracts comprising advantageous amounts of isocarboside, which belongs to the family of phenylpropanoid glycosides.

[0014] EP 2 319 914 describes a theoretical list of plant species from which extracts containing phenylpropanoid glycosides or caffeoylquinic acid, which are derivatives of caffeic acid, can be obtained in vitro.

[0015] WO 2016 / 113659 describes plant-derived extracts obtained from undifferentiated or dedifferentiated plant cells of Leontopodium alpinium, said extracts comprising advantageous amounts of phenylpropanoid glycosides, including leontopodic acids A and B.

[0016] WO 2017 / 163174 describes an extract of plant origin obtained from undifferentiated or dedifferentiated plant cells of Leontopodium alpinum, said extract comprising a favorable amount of phenylpropanoid glycosides, including Leontopodium acid A and B.

[0017] WO2020 / 165365 describes a plant-derived extract obtained from undifferentiated or dedifferentiated plant cells of Buddleja davidii Franch., which contains a favorable amount of phenylpropanoid glycosides, including verbascoside.

[0018] In order to improve the biological activity and originality of extracts, it is often proposed to stimulate plant cells to produce secondary metabolites that are only present in very small amounts in biomass by induction (e.g. chemical or physical induction).

[0019] The present invention aims to propose a novel preparation method by in vitro plant cell culture, which can produce extracts containing secondary metabolites other than those described in the prior art, said extracts exhibiting biological activity, in particular cosmetic activity. According to the first object, the present invention proposes a method for producing plant-derived extracts by in vitro plant culture, comprising the following steps, carried out in sequence from an undifferentiated or dedifferentiated plant cell line:

[0020] - a pre-cultivation step aimed at amplifying the biomass of said plant cells;

[0021] - a cultivation step of the biomass in a bioreactor, comprising at least one multiplication phase; and

[0022] - a processing step of the harvested biomass to prepare said extract comprising the secondary metabolites of interest;

[0023] In the culturing step in the bioreactor, at least one aglycone flavonoid is added to the culture medium.

[0024] Advantageously, the method according to the present invention yields a novel extract enriched in a novel class of secondary metabolites. At the end of the method, a plant extract is recovered that includes a group of glycosylated flavonoids among its secondary metabolites. In contrast, plant extracts obtained according to prior art methods do not contain this type of secondary metabolite.

[0025] Surprisingly, plant cells are able to incorporate aglycone flavonoids added to the culture medium and metabolize them. Aglycone flavonoids serve as precursors.

[0026] "Aglycone" generally refers to a compound without any osidic radical.

[0027] Several mechanisms may explain the surprising effects obtained according to the present invention.

[0028] Aglycone flavonoids can reveal specific metabolic pathways that do exist but have not yet been used due to a lack of precursors. By revealing and stimulating these metabolic pathways, aglycone flavonoids are converted into glycosylated flavonoids.

[0029] Flavonoid aglycones can also act as epigenetic probes, sending signals to the cell's DNA to produce and / or activate specific enzymes. These enzymes, responsible for cell differentiation, then trigger the activation of specific metabolic pathways.

[0030] Finally, aglycone flavonoids may act as epigenetic probes capable of unlocking certain regions of DNA reading.

[0031] These mechanisms can occur individually or in combination, advantageously providing for cumulative or synergistic effects to produce particularly attractive compositions of target molecules.

[0032] Glycosylated flavonoids are of interest in cosmetics because they can improve or beautify the overall condition of the skin and its appendages. They are known for their exceptional antioxidant properties. The detailed description given below provides examples of extracts obtained according to the method of the present invention, which have very attractive cosmetic properties supported by in vitro and in vivo tests.

[0033] Preferably, according to the present invention, the aglycone flavonoid added to the biomass is selected from at least one aglycone flavonoid and / or one aglycone flavanone.

[0034] Preferably, the at least one flavanone aglycone is selected from naringenin, eriodictyol, and buteflavone, or a mixture thereof, and the at least one flavonoid aglycone comprises luteolin and apigenin, or a mixture thereof. More preferably, according to the present invention, the aglycone flavonoid added to the biomass is naringenin and / or luteolin. These two flavonoids, each belonging to a different class, are exemplified in the detailed description.

[0035] Naringenin is also known as naraginin, naringin, naringinogen or 5,7-dihydroxy-2-(4-hydroxyphenyl)chroman-4-one (C 15 H 12 O5), and has the following structural formula:

[0036]

[0037] Luteolin is also known as luteolol or 5,7-dihydroxy-2-(3,4-dihydroxyphenyl)-chroman-4-one (C 15 H 10 O6), and has the following structural formula:

[0038]

[0039] The selection of one or more aglycone flavonoids may be made according to the type of glycosylated molecules that are sought to be derived from these flavonoids.

[0040] The amount of the aglycone flavonoid(s) to be added is determined based on the amount that can be taken up and utilized by the plant cells, ensuring firstly rapid growth of the biomass and secondly maximum yield of secondary metabolite synthesis, all without any toxic effects on the cells.

[0041] The addition of aglycone flavonoids is performed during the bioreactor cultivation step, preferably during the exponential phase of cell growth, more preferably during the exponential phase of the last proliferation cycle.

[0042] "Cycle" refers to the time period between the addition of fresh culture medium to the bioreactor, or the time period between the addition of fresh culture medium and the recovery of cells (the last cycle). The cycle can be repeated many times. It is used to proliferate the cells. Each cycle occurs in several stages, which are determined by their proliferation rate. During the exponential phase, the cells are most active and therefore most able to metabolize the aglycone flavonoids added to the culture medium into a group of glycosylated derivatives of these flavonoids.

[0043] According to another feature of the invention, the optional induction step may be performed during the bioreactor cultivation step, preferably also during the exponential phase of cell growth, more preferably during the exponential phase of the last proliferation cycle, more preferably simultaneously with the addition of aglycone flavonoids.

[0044] The amount of inducer to be added is chosen based on the amount that the cells can accept and utilize, just as with the aglycone flavonoids.

[0045] In general, the induction of the target compound can be carried out by adding microbial parts (especially Saccharomyces yeast) to the culture; adding molecules of biological origin, such as, for example, chitosan, methyl jasmonate, jasmonic acid and salicylic acid, to the culture; adding molecules of non-biological origin, such as, for example, paclobutrazol, to the culture; subjecting the culture to changes in temperature, pH or osmotic stress induced by non-metabolizable sugars (such as, for example, mannitol); using even more drastic depletion of the culture medium with macroelements and sugars; adding adsorption resins to the culture, which, in addition to inducing the production of the target compounds, can also capture them.

[0046] Preferably, according to the present invention, the added inducer is methyl jasmonate.

[0047] Surprisingly, the Applicant has noticed that the addition of methyl jasmonate in combination with the addition of at least one aglycone flavonoid not only makes it possible to increase the concentration of secondary metabolites naturally present in the cell only in very small amounts, such as rosmarinic acid, but also allows to increase the concentration of glycosylated flavonoids (see analytical results detailed below).

[0048] According to another feature of the method of the present invention, the biomass processing step for recovering the extract according to the present invention after the cultivation step includes a conventional separation step to eliminate the culture medium (supernatant) and recover the cell biomass. This step can be performed, for example, by filtration or centrifugation. The recovered biomass consists of partially lysed or intact cells in the form of clumps or single cells. The target secondary metabolites (including all glycosylated flavonoids) are present in the intracellular contents of the plant cells of the recovered biomass.

[0049] Thus, a cell extract according to the invention is obtained, containing intact cells or partially lysed cells, more precisely, an extract containing both intracellular contents (including the secondary metabolites of interest) and cell walls and / or fragments. This extract can be used to prepare a cosmetic composition, optionally after undergoing additional treatment to disrupt cell aggregates, in particular by homogenization under high pressure or any other appropriate technique. The composition according to the invention can be more or less concentrated in the cell extract to form a cosmetic ingredient, or a final cosmetic preparation for the end consumer that can be prepared from this cosmetic ingredient.

[0050] Optionally, the treatment step may include a subsequent step of releasing the cell contents outside the plant cells, which step may be performed according to various known methods, which may be combined: heating, by maceration, grinding, decoction, extraction, pressurization, leaching, diffusion, distillation, liquid / liquid separation, cell lysis using ultrasound, microwaves, or by any suitable chemical or physical method. Biomass may also be extracted using supercritical or subcritical fluids.

[0051] According to the present invention, the release of the intracellular contents containing the secondary metabolites of interest occurs preferentially by osmotic diffusion or by cell lysis.

[0052] Osmotic diffusion is performed by adding a water-poor solvent to the biomass. Preferably, according to the present invention, butanediol, propanediol, propylene glycol, pentanediol, glycerol or a mixture thereof is added. More preferably, glycerol is added.

[0053] An extract containing intracellular contents and cell debris is thus obtained. This extract can be used as such to prepare a cosmetic composition.

[0054] Optionally, the treatment step may include a subsequent step to eliminate cell debris from the obtained extract, for example by any type of filtration or centrifugation. This step advantageously results in a purified and clarified extract that can be used as an active ingredient in a cosmetic composition. The extract is more transparent and has advantageous formulation properties, such as being useful in the preparation of gel or serum-type products.

[0055] According to another particular feature of the method of the invention, the plant cell line used in the pre-cultivation step is preferably prepared extemporaneously.

[0056] The establishment of cell lines involves the following stages: 1) induction of callus (masses of undifferentiated or dedifferentiated cells), 2) selection of the best calli and 3) optimization of the selected calli in culture medium.

[0057] The culture medium (liquid or agar) contains macroelements, trace elements, hormones, vitamins and sugars, which are selected to promote the growth and production of the target molecule (i.e. the glycosylated derivative of the aglycone flavonoid added during the cultivation phase).

[0058] More specifically:

[0059] 1) Callus induction is carried out in agar medium and can be carried out with all parts of the plant, including leaves, fruits, roots, buds, seeds, stems, branches, meristems and cambium. Preferably, according to the present invention, the culture of plant cells is carried out from the leaves of the plant.

[0060] 2) According to the invention, before transfer to the liquid basal medium, a selection of the best cell lines is performed, in particular according to the following criteria: strong proliferation capacity, tender and crisp texture, uniform color, good dispersion in the liquid medium and stability of these parameters over time.

[0061] 3) Optimization of the growth and production characteristics of secondary metabolites of the selected cell line in liquid culture involves the selection of the most appropriate culture medium, firstly to ensure rapid growth of the biomass and secondly to allow maximum synthetic yield of primary and secondary metabolites by the cells at the end of the exponential phase (proliferation arrest) to facilitate their entry into the bioreactor.

[0062] The cell lines thus prepared can be used directly or stored for later use.

[0063] According to the method of the invention, the culture medium (liquid or agar) used in the different stages generally contains macroelements, trace elements, hormones, vitamins and sugars. They are selected to promote the growth and production of the target molecule (i.e., the glycosylated flavonoid according to the invention). The culture medium can also be selected to allow the growth of other secondary metabolites produced by the plant cells, in particular those that are naturally present in the cell line but in very small quantities, such as, for example, the classic caffeic acid and, in particular, rosmarinic acid, as shown in the examples given below. The extract obtained will advantageously include a whole group of secondary metabolites that can act synergistically and give the extract original and new properties in cosmetics.

[0064] The method of the present invention can be carried out starting from any plant cell line.

[0065] The plants may be chosen from plant families known to produce extracts of interest in cosmetics, in particular from Lamiaceae, Boraginaceae, Nyctaginaceae, Asteraceae or Apiaceae, preferably Lamiaceae and / or Nyctaginaceae.

[0066] More preferably, the plant can be selected from the following genera: Abronia, Acinos, Acleisanthes, Ajuga, Allionia, Andradea, Anulocaulis, Ballota, Belemia, Boerhavia, Bougainvillea, Calamintha, C aribea), Clinopodium, Colignonia, Commicarpus, Cyphomeris, Galeopsis, Glechoma, Guapira, Hyssopus, Lamium, Lavandula, Leucaster, Marrubium, Melissa ), Melittis, Mentha, Mirabilis, Monarda, Neea, Nepeta, Nyctaginia, Ocimum, Okenia, Origanum, Orthosiphon, Perilla, Phaeoptilum, Pisonia, Pisonia, and Pisonia spp. ella), Phlomis, Plectranthus, Prunella, Ramisia, Reichenbachia, Rosmarinus, Salvia, Satureja, Stachys, Teucrium, Thymus, Tripterocalyx, and Salpianthus.

[0067] More preferably, according to the present invention, the plant is selected from the following genera: Verbena, Powdered Windmill, Annular Grass, Bougainvillea, Lavender, European Summer Solstice, Melissa, Mentha, Mirabilis, Monarda, Basil, Origanum, Panax, Perilla, Frostflower, Microcarpa, Prunella, Rosmarinus, Salvia, Herba Lysimachiae and Thyme.

[0068] More preferably, the plant belongs to the genus Spermum, Monarda and / or Lavandula.

[0069] The genus Monarda belongs to the Lamiaceae family and includes several species, including those known as:

[0070] Monarda austroappalachiana, Monarda bartlettii, Monarda bradburiana, Monarda brevis, Monarda citriodora, Monarda aclinopodia, Monarda clinopodioides, Monarda didyma, Monarda eplingiana, Monardafistulosa, Monarda fruticulosa, Monarda humilis, Monarda lindheimeri, Monarda luteola, Monarda maritima, Monarda media, Monarda pectinata, Monarda pringlei, Monarda punctata, Monarda russeliana, Monarda stanfieldii, Monarda viridissima.

[0071] Applicants are particularly interested in American mint species.

[0072] Mentha piperita is a honey, nectar-producing, aromatic, and edible plant native to eastern North America. Mentha piperita was first described by Nicolas Monardes and is also known as Oswego tea, "Mélisse d'Or," or Bee Balm. Native Americans used a maceration of the leaves in oil for hair care or for its antiseptic properties on pimples or to combat colds.

[0073] The genus Lavandula belongs to the Lamiaceae family and includes several species, including those known as: Lavandula xalportelensis, Lavandula angustifolia, Lavandula antineae, Lavandula aristibracteata, Lavandula atriplicifolia, Lavandula austroapennina, Lavandula bipinnata, Lavandula bramwellii, Lavandula buchii, Lavandula x cadevallii, Lavandula canariensis, Lavandula xcavanillesii, Lavandula citriodora, Lavandula coronopifolia, Lavandula dentata, Lavandula dhofarensis, Lavandula erythraeae, Lavandulagalgalloensis, Lavandula gibsonii, Lavandula x ginginsii, Lavandula hasikensis, Sweet lavender (Lavandula x heterophylla), Eye-catching lavender (Lavandula x intermedia), Cotton lavender (Lavandula lanata), Broadleaf lavender (Lavandula latifolia), Lavandula xlimae, Lavandula x losae, Lavandula macra, Lavandula mairei, Lavandula maroccana, Lavandula minutolii, Fernleaf lavender (Lavandula multifida), Socotra lavender (Lavandulanimmoi Benth), Lavandula nooruddinii, Butterfly lavender (Lavandula pedunculata), Featherleaf lavender (Lavandula pinnata), Short-haired lavender (Lavandula pubescens), Lavandula qishnensis, Lavandula rejdalii, Roundleaf lavender (Lavandula rotundifolia), Lavandulasaharica, Lavandula samhanensis, Lavandula setifera, Lavandula somaliensis, Spanish lavender (Lavandula stoechas), Lavandula sublepidota, Lavandula subnuda, Lavandulatenuisecta, lemon lavender (Lavandula viridis).

[0074] The applicant is particularly interested in lavender species.

[0075] Lavender is a plant prized for its scent. It is also known as lavender, true lavender, or lavender. The flower is commonly used to obtain its essential oil, which has various benefits, such as anti-inflammatory, antiseptic, healing, and antibacterial properties.

[0076] The genus Abronia belongs to the family Mirabilisaceae and includes several species, including those known as Abronia alba, Abronia alpina, Abronia ameliae, Abronia ammophila, Abronia angustifolia, Abronia argillosa, Abronia bigelovii, Abronia carletonii, Abronia elliptica, Abronia fragrans, Abronia gracilis, Abronia insularis, Abronia latifolia, Abronia macrocarpa, Abronia maritima, Abronia mellifera, Abronia minor, Abronianana, Abronia neurophylla, Abronia platyphylla, Abronia pogonantha, Abroniaturbinata, Abronia umbellate, and Abronia villosa.

[0077] The applicant is particularly interested in the desert sand verbena species.

[0078] Desert sand verbena is a species of sand verbena known by the common names: desert sand-verbena and chaparral sand-verbena. The Paiute Native Americans used the plant as a diuretic. The Shoshone used the crushed root as a poultice for burns.

[0079] Other more specific features of the method according to the invention are:

[0080] Regarding the pre-cultivation step, the selected cell line is propagated to obtain a sufficient amount of undifferentiated or dedifferentiated cell biomass to proceed to the large-scale production step, ie the cultivation step in a bioreactor.

[0081] Perform the following sub-steps:

[0082] a) The selected cell line is inoculated into a liquid culture medium and cultured for a sufficient time to obtain a biomass amount that is doubled or even tripled compared to the original biomass. This first step is carried out in a container with a capacity of 0.5L to 2L.

[0083] b) Optionally, all or part of the suspension obtained in a) is transferred to a fresh liquid culture medium and cultivated again for a sufficient time to obtain a biomass amount doubled or even tripled compared to the amount of biomass at the beginning of the cycle.

[0084] This step b) constitutes a cycle, which can be repeated multiple times.

[0085] c) Optionally, repeat step b).

[0086] Regarding the bioreactor cultivation step, the best cell line stabilized in the pre-culture step is transferred to a bioreactor containing liquid culture medium for cell proliferation. Steps b) and c) performed for the pre-culture are repeated in a container having a volume suitable for the biomass.

[0087] The addition of one or more aglycone flavonoids is preferably performed during the exponential growth phase of the final cycle, ie, between approximately 7 and 12 days after the last initiation of culture in the bioreactor.

[0088] When the desired level of secondary metabolites is reached, preferably between about 2 and 7 days after the addition of the one or more aglycone flavonoids, the cultivation is stopped. To achieve this level, culture medium is used, if necessary, as seen above.

[0089] According to the method of the invention, the culture medium (liquid or agar) used in the pre-culture or culture step generally contains macroelements, trace elements, hormones, vitamins and sugars. It is selected to promote the growth and production of the target molecule (i.e. at least the glycosylated flavonoid according to the invention). As seen above, the culture medium can also be selected to allow the growth of other secondary metabolites produced by the plant cells, in particular those that are naturally present in the cell line but in very small quantities, such as, for example, the classic caffeic acid and, in particular, rosmarinic acid, as shown in the examples given below. The extract obtained will advantageously include a whole group of secondary metabolites that can act synergistically and confer original and new properties in cosmetics.

[0090] As regards the biomass treatment step, the recovery of the biomass produced in the bioreactor is carried out after a cultivation time of 7 to 21 days, preferably 10 to 14 days, to advantageously allow the production of the highest amount of biomass and with high viability.

[0091] The cell biomass recovered and released from the supernatant can be subjected to an additional drying step, in particular freeze drying, with the advantage that the biomass can thus be preserved in a more stable form for a long period of time. The dried biomass can be temporarily rehydrated or rediluted for its subsequent use.

[0092] The final step of extensive purification of the cell extract to eliminate its debris can be carried out by all industrially available methods, by liquid-liquid partitioning or chromatography, in particular using adsorption resins, in order to concentrate the target molecules, such as all glycosylated flavonoid derivatives produced by the cells.

[0093] According to a second object, the present invention provides a plant extract obtainable by a process according to the invention and as described above.

[0094] According to other features, the extract according to the invention is characterized in that it is obtained from a plant of the Lamiaceae family and in that it contains glycosylated flavonoids as secondary metabolites.

[0095] The extract obtained from peppermint contains in particular as secondary metabolites a group of glycosylated flavonoids originating from the added aglycone flavonoid. Preferably, the added aglycone flavonoid is part of the aglycone flavanone family, more preferably naringenin.

[0096] The extract obtained from lavender contains as secondary metabolites a group of glycosylated flavonoids derived from the added aglycone flavonoid. Preferably, the added aglycone flavonoid is part of the aglycone flavonoid family, more preferably luteolin.

[0097] The extract obtained from Verbena deserticola contains as secondary metabolites a group of glycosylated flavonoids derived from the added aglycone flavonoid. Preferably, the added aglycone flavonoid is part of the aglycone flavonoid family, more preferably luteolin.

[0098] Thus, the undifferentiated or dedifferentiated plant cells obtained and / or the extracts obtained can be used for the preparation of active ingredients for cosmetic or dermatological compositions aimed at improving the general condition of the skin and its appendages.

[0099] According to a third object, the present invention provides a composition, in particular a cosmetic composition, comprising as active ingredient an extract according to the second object and a physiologically acceptable medium.

[0100] According to the invention, the expression "physiologically acceptable medium" refers to, but is not limited to, aqueous or hydroalcoholic solutions, water-in-oil emulsions, oil-in-water emulsions, microemulsions, hydrogels, anhydrous gels, serums, vesicular dispersions or powders.

[0101] "Physiologically acceptable" means that the composition is suitable for topical use in contact with the mucous membranes, nails, scalp, hair and skin of mammals, especially humans, without risk of toxicity, incompatibility, instability, allergic reaction, etc.

[0102] This "physiologically acceptable medium" forms what is generally referred to as the excipient of the composition.

[0103] According to the present invention, the physiologically acceptable medium can be aqueous, hydroglycolic or hydroalcoholic medium, or be formed by water-in-oil emulsion, oil-in-water emulsion or microemulsion. More preferably, it is a hydroglycolic solution. More preferably, the physiologically acceptable medium is a mixture of water and glycerol.

[0104] Thus, according to the present invention, the appearance and general condition of the skin and / or appendages can be beautified or improved and imperfections treated by topically applying to the skin of a subject in need thereof an effective amount of at least one extract according to the present invention and / or a composition comprising such extract in a physiologically acceptable excipient.

[0105] According to a fourth object, the present invention provides the use of an extract according to the second object and / or a composition according to the third object for the non-therapeutic cosmetic treatment of the skin and its appendages. Preferably, according to the invention, the treatment is a topical treatment.

[0106] Several phenomena affect the quality of the skin, and various parameters make it appear less uniform, such as wrinkles and fine lines, dryness, loss of elasticity and uneven pigmentation. These skin imperfections can be made to disappear or masked by aesthetic treatments that beautify the skin by making it more uniform.

[0107] Hereinafter in the description are given the results of in vitro and in vivo tests demonstrating the beneficial cosmetic activity on the skin and its appendages of two examples of extracts prepared according to the method of the invention from cell lines of peppermint and lavender.

[0108] According to the invention, these cosmetic effects can be envisaged individually or in combination, which advantageously provide, for example, a combined aesthetic and sensory effect.

[0109] According to the invention, "topical treatment" or "topical application" means an application intended to act at the site of its application: skin, mucous membranes and / or appendages.

[0110] The present invention preferably provides the use of an extract according to the invention derived from plant cells of the genus Mentha, and more particularly Mentha spp., for at least one treatment selected from:

[0111] - anti-aging treatments; and / or

[0112] - anti-seborrheic treatment; and / or

[0113] - moisturizing treatment; and / or

[0114] - treatments to strengthen the skin barrier; and / or

[0115] - weight loss treatment; and / or

[0116] - Soothing treatment.

[0117] More particularly, the proposed anti-aging treatment is suitable for acting on pigmentation imperfections of the skin and, more particularly, according to the invention, it is advantageously suitable for treating white and / or brown age spots of the skin.

[0118] As skin ages, two opposing phenomena lead to the appearance of brown and white spots, which are often considered unsightly. Brown spots are due to hyperpigmentation, while white spots are due to hypopigmentation.

[0119] According to the present invention, "hyperpigmentation" refers to spots rich in (multiple) pigments due to the production of melanin in excessive amounts in certain areas of the skin.

[0120] In contrast, according to the present invention, "hypopigmentation" refers to spots that lack pigment. These spots result in an unattractive, uneven appearance of skin color.

[0121] Skin pigmentation originates from very specialized cells called melanocytes. These cells are responsible for the production of the skin pigment melanin.

[0122] With aging and / or overexposure to the sun, melanocytes age and become less efficient. They become fewer in number, lower in quality, and less evenly distributed. This uneven distribution leads to the brown and white spots that are signs of aging skin.

[0123] Below in the description are given the results of in vitro tests on this specific activity which improves the evenness of the skin colour, more particularly:

[0124] - preventing and / or treating skin loss of pigmentation, in particular to prevent and / or treat areas of white spots; and / or

[0125] -Preventing and / or treating skin hyperpigmentation, in particular for preventing and / or treating areas where brown spots are present.

[0126] In vitro tests have shown that the peppermint extract according to the present invention is suitable for acting in various aspects:

[0127] - Maintains melanocyte homeostasis and youthfulness by inhibiting oxidative and free radical effects known to accelerate cellular aging;

[0128] - Maintaining the environment near melanocytes in a favorable state by reducing the production of proteases that lead to the destruction of matrix proteins (such as collagen and elastin) and by increasing the synthesis of essential elements for maintaining the homeostasis of keratinocytes and melanocytes (such as collagens-I, -IV, -VII and -XVII, as well as elastin, laminin and hyaluronic acid). In fact, melanocyte physiology is closely related to signals from neighboring epidermal cells (such as keratinocytes and fibroblasts). Similarly, the peripheral extracellular matrix of these melanocytes forms a microenvironment that significantly affects their production;

[0129] - Inhibits senescence of melanocytes by increasing the length of melanocyte dendrites that distribute melanin to keratinocytes and the number of spindle-shaped melanocytes (which are indicators of the observed youthfulness of the cells), and by reducing the presence of the enzyme SAβ-GAL ("senescence-associated β-galactosidase") that is strongly present in senescent cells, the amount of DDK1, which is identified as a pro-senescence factor in melanocytes, and the production of IL-6 and IL-8 (which are molecules whose secretion is increased in senescent cells compared to young cells), thereby maintaining the proper functioning of melanocytes and inhibiting their entry into senescence.

[0130] The in vivo studies presented below also confirm this particularly attractive and original activity against all aging spots.

[0131] Preferably, the present invention also provides the use of an extract according to the present invention derived from plant cells of the genus Lavandula or a composition according to the present invention containing the extract for at least one treatment selected from the group consisting of:

[0132] - anti-aging treatments; and / or

[0133] - moisturizing treatment; and / or

[0134] - Soothing treatment.

[0135] In the description below, in vitro test results are given, demonstrating beneficial cosmetic activity on the skin and its appendages.

[0136] The extract according to the invention can be combined with one or more other active ingredients in effective concentrations, which can act synergistically or additively to strengthen and achieve the desired effects described for the present invention, such as the following ingredients: filtering radiation, in particular UVA, UVB, IR or problems from blue light, hydration, moisturizing, humectants, calming, muscle relaxants, slimming, restructuring, firming, re-plumping, lifting, smoothing, action on blood microcirculation, inflammation, free radicals, anti-aging, anti-fine lines and wrinkles, brightening, action on skin color, anti-glycation, anti-carbonylation, pro-pigmentation, action on the stratum corneum, action on the dermal-epidermal junction, action on HSP protein production, action on skin firmness, elasticity and tone, action on hair growth or anti-regeneration (including eyelashes and eyebrows), action on the eye contour (dark circles and bags under the eyes), peptides, vitamins, etc.

[0137] More particularly, in order to improve or beautify the general condition of the skin, the extract according to the invention may be combined with one or more of the following active ingredients, without this list being exhaustive:

[0138] -Moisturizing and nourishing, such as Vegesome Moist 24 TM (Sederma): active ingredient containing a powder consisting of hollow granules of Lycopodium clavatum loaded with an extract of Imperata cylindrica, which gradually hydrates the epidermis; and / or

[0139] - Coloring, such as Silverfree TM (Sederma): active ingredients containing the Pal-PP peptide and / or the Pal-PA peptide of the multiple steps of the pigmentation stimulation method and / or the peptides generally with pro-pigmentation activity described in the applicant's patent application WO2014 / 080376, and / or TYR-OL proposed by Sederma and described in patents FR2702766 and WO03 / 017966TM and TYR-EXCEL TM , which are based on oleyltyrosine, and / or one or more norsyringone glycoside derivatives described by the University of Hamburg in patent application WO2017 / 121445, and / or dihydroxyacetone, and / or one or more derivatives of the chroman-4-one family described by MERCK in patent application WO2007 / 087956; and / or compounds based on pyrazoline-4,5-dione described by L'OREAL in patent application WO97 / 35842; and / or

[0140] -Prevents signs of photoaging, such as Venuceane TM (Sederma): contains an active ingredient from a biotechnological extract of Thermus thermophilus, which prevents the visible signs of photoaging (spots, wrinkles, dryness...), protects the cell structure from UV damage and strengthens skin integrity; and / or

[0141] - Skin tensors, such as Feminage TM (Sederma): contains an active ingredient of Engelhardia chrysolepsis extract, which provides elasticity and firming properties to the skin; and / or

[0142] -Anti-pollution products, such as Citystem TM (Sederma): Contains an active ingredient of a biotechnological extract of Marrubium vulgare, which softens and smoothes the skin, improves skin texture, reduces the visibility of blackheads, while leaving the skin radiant and purified.

[0143] Detailed examples are given below in the section on Galenic medicine.

[0144] The composition according to the invention can be applied to the face, body, neckline, scalp, hair, eyelashes, body hair in any form or vehicle known to those skilled in the art, in particular in the form of a solution, dispersion, emulsion, paste or powder, alone or as a premix, or as a vehicle for a premix in a combined form, incorporated or absorbed in a carrier such as macrocapsules, microcapsules or nanocapsules, macrospheres, microspheres or nanospheres, liposomes, oleosomes or chylomicrons, macroparticles, microparticles or nanoparticles or macrosponges, microsponges or nanosponges, microemulsions or nanoemulsions, or absorbed on organic polymer powders, talc, bentonite, spores or exines and other inorganic or organic carriers.

[0145] In cosmetics, mention may be made in particular of use in the skin care range for the face, body, hair and body hair, and in the makeup treatment range, in particular in the eyelash, eyebrow and skin makeup range.

[0146] The composition can be incorporated onto nonwoven or woven materials having natural or synthetic fibers, wool or any material intended to come into contact with the skin and which can be used in clothing including tights and socks, briefs, day or night underwear, tissue, handkerchiefs or fabrics to apply the cosmetic effect of the composition by contact with the skin / textile and to enable sustained topical delivery (cosmetics-textiles).

[0147] Therefore, according to the present invention, there is also provided a woven or non-woven fabric comprising an extract according to the invention for use in non-therapeutic cosmetic treatments.

[0148] Cosmetic preparations can be grouped into different product lines used for personal care and / or beautification products, including skin care, cleansing, makeup, cleansing, sun protection, artificial tanning, pre-shave, mid-shave, or after-shave, moisturizers, humectants, emollients, conditioners, exfoliants, astringents, depilatories, or antiperspirants, deodorants, etc.

[0149] The Personal Care Products Council ("International Cosmetic Ingredient Dictionary & Handbook" published by the Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, DC) describes a non-limiting variety of cosmetic and pharmaceutical ingredients commonly used in the skin care industry that can be used as additional ingredients in the compositions of the present invention, provided they are physically and chemically compatible with the other ingredients in the composition and, in particular, with the active ingredients of the present invention. Furthermore, the nature of these additional ingredients should not unacceptably alter the benefits of the active ingredients of the present invention. These additional ingredients can be synthetic or natural, such as plant extracts or produced by biological fermentation processes.

[0150] Particularly useful additional skin care actives for combination with the compositions can be found in the commercial literature of Sederma and on the website www.sederma.fr, and in the commercial literature of Croda and on the website www.croda.fr.

[0151] Commercially available active substances widely used in cosmetic compositions may also be mentioned as examples: betaine, glycerol, Actimoist Bio 2 TM (Active Organic Matter), AquaCacteen TM (Mibelle AGCosmetics), Aquaphyline TM (Silab), AquaregulK TM (Solabia), Carciline TM (Greentech), Codiavelane TM (BiotechMarine), Dermaflux TM (Arch Chemicals, Inc)、Hydra'Flow TM (Sochibo), Hydromoist L TM (Symrise), RenovHyal TM (Soliance), Seamoss TM (Biotech Marine), Essenskin TM (Sederma), Moist 24 TM (Sederma), Argireline TM (commercial name for acetyl hexapeptide-3 from Lipotec), Gatuline Expression TM The well-known spilanthol or Acmella oleracea extract, sold under the trade name Boswellin TM Well-known Boswellia serrata extract, Deepaline PVB TM (Seppic), Syn-AKE TM (Pentapharm), Ameliox TM 、Bioxilift TM (Silab), PhytoCellTec TM Argan(Mibelle),Papilactyl D TM (Silab), Preventhelia TM (Lipotec), or one or more of the following active ingredients marketed by Sederma: Subliskin TM , Venuceane TM 、Moist 24TM 、Vegesome Moist 24 TM 、Essenskin TM 、Juvinity TM 、Revidrat TM 、Resistem TM 、Chronodyn TM 、Kombuchka TM 、Chromocare TM 、Calmosensine TM 、Glycokin factor S TM 、Biobustyl TM 、Idealift TM 、Ceramide 2 TM 、CeramideA2 TM 、Ceramide HO3 TM 、Legance TM 、Intenslim TM 、Prodizia TM 、Beautifeye TM 、Pacifeel TM 、Zingerslim TM [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Among the plant extracts (in the form of classical plant extracts or prepared by in vitro processes) that can be used as further active substances, mention may be made more particularly of ivy, such as English ivy (Hedera helix) extract, Bupleurum chinensis extract, Bupleurum falcatum extract, Arnica montana L. extract, rosemary (Rosmarinus officinalis N.) extract, marigold (Calendula officinalis) extract, sage (Salvia officinalis L.) extract, ginseng (Panax ginseng) extract, ginkgo (gingko biloba) extract, St. John's wort (Hyperycum perforatum) extract, butcher's clover (Ruscus aculeatus) extract. L.) extract, European gnats (Filipendula ulmaria L.) extract, big-flowered jarva tea (Orthosiphon stamincus Benth.) extract, artichoke (Cynara scolymus) extract, algae (Fucus vesiculosus) extract, birch (Betula alba) extract, green tea, kola nut (Cola nipida) extract, horse chestnut extract, bamboo extract, centella asiatica extract, heather extract, rockweed extract, willow extract, mouse-ear extract, horse chestnut extract, atractylodes extract, golden chamomile (Chrysanthellumindicum) extract, apricot (Armeniacea), Atractylodis platicodon, Sinnomenum, pharbitidis, extracts of plants of the genus Flemingia, Coleus such as C. forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C. xanthantus, and C.barbatus extracts, such as extracts of the root of Coleus forskohlii, Ballote extracts, Guioa extracts, Davallia extracts, Terminalia extracts, Barringtonia extracts, Trema extracts, Antirobia, Cecropia, Argania, Dioscoreae extracts, such as Dioscorea opposita or Mexican yam extracts, Ammi visnaga extracts, Siegesbeckia extracts, in particular Siegesbeckia orientalis extracts, plant extracts of the Ericaceae family, in particular bilberry extracts (Vaccinium angustifollium) or bearberry (Arctostaphylos uva ursi), Aloe vera extracts, vera), plants containing sterols (e.g., phytosterols), Manjistha (extracted from plants of the genus Rubia, particularly Rubia cordifolia), and Guggal (extracted from plants of the genus Commiphora, particularly Commiphora mukul), kola extract, chamomile, red clover extract, Pipermethysticum extract (Kava Kava from Sederma TM ), Bacopa monieri extract (Bacocalmine from Sederma TM ) and sea whip extract, Glycyrrhiza uralensis root extract, z a glabra extract, mulberry extract, melaleuca (tea tree) extract, Larrea divaricata extract, Rabdosia rubescens extract, Euglena gracilis extract, Fibraurea recisa hirudinea extract, Chaparral sorghum extract, sunflower extract, Enantia chlorantha extract, Spermacocea (Mitracarpe) extract, Buchu barosma extract, Lawsonia inermis L. extract, Adiantium capillus-veneris L. extract, Chelidonium majus extract, Luffacylindrica extract, "Japanese Mandari" (Citrus reticulata Blanco var.unshiu) extract, Camelia sinensis extract, Imperata cylindrica extract, Glaucium flavum extract, Cupressus sempervirens extract, Polygonatum multiflorum extract, Lovely hemsleya extract, Sambucus nigra extract, Phaseolus lunatus extract, Centaurium extract, Macrocystis pyrifera extract, Turnera diffusa extract, Anemarrhena asphodeloides extract, Portulaca pilosa extract, Humulus lupulus extract, Coffea arabica extract, Ilex paraguariensis extract, or Globularia odorifera extract cordifolia extract, Oxydendron arboretum extract, Albizzia julibrissin extract, Zingimber zerumbet smith extract, Astragalus membranaceus extract, Atractylodes macrocephalae extract, Plantago lanceolata extract, Leontopodium alpinum (or Leontopodium edulis) extract, Mirabilis jalapa extract, Apium graveolens extract, Summer solstice grass, Buddleja macrophylla, Syringa oleracea, Chinese wolfberry, or orchid extract.

[0153] The compositions of the present invention may comprise peptides, including but not limited to dipeptides, tripeptides, tetrapeptides, pentapeptides and hexapeptides and derivatives thereof. According to a particular embodiment, the concentration of the additional peptide(s) in the composition ranges from 1 x 10 -7 % to 20% by weight, preferably 1x10 -6 % to 10% by weight, preferably 1x10 -5 % to 5% by weight.

[0154] The term "peptide" herein refers to peptides containing 20 amino acids or less, their derivatives, isomers, and complexes with other substances (such as metal ions, for example, copper, zinc, manganese, magnesium, etc.). The term "peptide" refers to both natural peptides and (bio)synthetic peptides. It also refers to compositions containing peptides and existing in nature and / or commercially available.

[0155] Suitable dipeptides for use herein include, but are not limited to, carnosine (β-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM, or PP.

[0156] Suitable tripeptides for use herein include, but are not limited to, RKR, HGG, GHK, GGH, GHG, GKH, KPK, KFK, KavaK, KβAK, KabuK, KacaK, KPK, KMOK, KMO2K (MO2 is dioxysulfoxide methionine), PPL, PPR, SPR, QPA, LPA or SPA.

[0157] Mention may also be made of non-limiting examples of tripeptides including:

[0158] - lysine with proline grafted to its side chain, such as K(P)HG or K(P)GH, K(P);

[0159] - lysine with pyroglutamic acid grafted to its side chain, such as K(Pyr)HG or K(Pyr)GH, K(Pyr);

[0160] - Lysine with acetylated amine functional groups on the side chains, such as K(Ac)HG or K(Ac)GH, K(Ac);

[0161] - Lysine K(Hyp)HG or K(Hyp)GH, K(Hyp) whose side chain is grafted with hydroxyproline as disclosed in WO 2016 / 097965.

[0162] Non-limiting examples of suitable tetrapeptides are KTFK (SEQ ID NO: 1), GQPR (SEQ ID NO: 2), RSRK (SEQ ID NO: 3), KTAK (SEQ ID NO: 4), KAYK (SEQ ID NO: 5), KFYK (SEQ ID NO: 6) or TKPR (SEQ ID NO: 7).

[0163] Non-limiting examples of suitable pentapeptides are KTTKS (SEQ ID NO: 8) and KTSKS (SEQ ID NO: 9) and examples of hexapeptides are GKTTKS (SEQ ID NO: 10) and VGVAPG (SEQ ID NO: 11).

[0164] Other suitable peptides for the use according to the invention may be selected from the non-limiting list: lipophilic derivatives of peptides, preferably palmitoyl (Pal) derivatives or myristoyl (Myr), and metal complexes as described above (e.g. copper complexes of the tripeptides HGG or GHK).

[0165] Preferred dipeptides include, for example, N-palmitoyl-β-Ala-His, N-acetyl-Tyr-Arg-hexadecyl ester (Calmosensine TM Idealift TM ,Sederma), Pal-KT, Pal-RT, Pal-PP and Pal-PA.

[0166] Preferred tripeptide derivatives include, for example, copper derivatives of HGG (Lamin TM ), Pal-GKH and Pal-GHK (from Sederma), Lipospondin (N-oleoyl-KFK) and its conservatively substituted analogs, N-acetyl-RKR-NH2 (peptide CK+), N-Biot-GHK (from Sederma), Pal-KAvaK, Pal-KβAlaK, Pal-KAbuK, Pal-KAcaK or Pal-KMO2K (from Sederma) )、Pal-KVK(DSM's Syn-Coll TM ) and its derivatives.

[0167] Mention may also be made of the anti-aging tripeptide of the general formula X-Pro*-Pro*-Xaa-Y described in WO2015181688, wherein Xaa is selected from Leu, Arg, Lys, Ala, Ser and Asp, and at the N-terminus, X is selected from H, -CO-R 1 and -SO2-R 1 , and at the C-terminal end, Y is selected from OH, OR 1 、NH2、NHR 1 or NR 1 R 2 , R 1 and R 2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy groups, which may be linear, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulphurized, said groups may have heteroatoms, in particular O, S and / or N, in their main chain, and Pro* corresponds to proline, an analogue or derivative thereof; including, for example, Myr-PPL-OH and Myr-PPR-OH.

[0168] Reference may also be made to the pro-pigmentation and / or pro-MEC dipeptides and tripeptides of the general formula X-(Xaa1)n-Pro*-Xaa2-Y disclosed in WO2014 / 080376, wherein n=0, 1 or 2, Xaa1 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, Pro, and analogs and derivatives thereof; or a polar amino acid selected from Ser, Thr, Tyr, Asp, Glu, and analogs and derivatives thereof; and when n=2, the two amino acids Xaa1 are the same or different; Xaa2 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, and analogs and derivatives thereof, or a basic amino acid selected from Arg, Lys, His, and analogs and derivatives thereof; at the N-terminus , X is selected from H, -CO-R1 and -SO2-R1; at the C-terminus, Y is selected from OH, OR1, NH2, NHR1 or NR1R2; R1 and R2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy and aryloxy groups, which can be linear, branched, cyclic, polycyclic, saturated, unsaturated, hydroxylated, carbonylated, phosphorylated and / or sulfurized, and the group may have or not O, S and / or N heteroatoms in its backbone, and Pro* corresponds to proline, an analogue or derivative thereof; including, for example, the following peptides: Pal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPA-OH, Myr-SPA-OH, Pal-PM-OH, Pal-PA-OH and Pal-PP-OH.

[0169] According to the present invention, suitable tetrapeptide derivatives for use as additional peptides include, but are not limited to, Ela-KTAK (SEQ ID NO: 12), Ela-KAYK (SEQ ID NO: 13), Ela-KFYK (SEQ ID NO: 14), Pal-GQPR (SEQ ID NO: 15) or Pal-KTFK (SEQ ID NO: 16).

[0170] Suitable pentapeptide derivatives for use herein as additional peptides include, but are not limited to, Pal-KTTKS (SEQ ID NO: 17) (MATRIXYL TM , Sederma), Pal-KTSKS (SEQ ID NO: 18), Pal-YGGFXaa (SEQ ID NO: 19), wherein Xaa is Trp, Phe, Tyr, Tic, 7-hydroxy-Tic or Tpi or a mixture thereof.

[0171] Suitable hexapeptide derivatives for use herein include, but are not limited to, Pal-HLDIIXaa (SEQ ID NO: 20) (wherein Xaa is Trp, Phe, Tyr, Tic, 7-hydroxy-Tic, or Tpi), Pal-GKTTKS (SEQ ID NO: 21), Pal-VGVAPG (SEQ ID NO: 22) (DERMAXYL TM , Sederma) or a mixture thereof.

[0172] Preferred compositions commercially available and sold by Sederma:

[0173] - Tripeptides or derivatives including Biopeptide-CL containing GHK TM 、Maxi Lip TM or Procapil TM ;

[0174] - tetrapeptide or derivative including RIGIN containing Pal-GQPR (SEQ ID NO: 15) and excipients TM 、Eyeliss TM , Crystalide containing Pal-KTFK (SEQ ID NO: 16) vehicle (solvated in microemulsion) TM ;

[0175] - pentapeptide or derivative, such as Matrixyl containing Pal-KTTKS (SEQ ID NO: 17) TM .

[0176] Also mention:

[0177] -Pal-GHK and Pal-GQPR (SEQ ID NO: 15) mixture (Matrixyl TM 3000), and

[0178] - a mixture of Pal-GHK and Pal-VGVAPG (SEQ ID NO: 22) (Biobustyl TM )

[0179] The following peptides sold as well as further active ingredients may be mentioned:

[0180] -Vialox sold by Pentapharm TM (INCI name = Pentapeptide-3 (synthetic peptide containing alanine, arginine, isoleucine, glycine and proline)), Syn-ake TM (β-Ala-Pro-Dab-NH-Bzl) or Syn-CollTM (Pal-Lys-Val-Lys-OH);

[0181] -Argireline sold by Lipotec TM (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (INCI name = Acetyl Hexapeptide-3) (SEQ ID NO: 23), Leuphasyl TM (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO:24), Aldenine TM (Gly-His-Lys), Trylagen TM (INCI Name=Pseudoalteromonas Ferment Extract, Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Tripeptide-10 Citrulline (Reaction product of citrulline and Tripeptide-10 (a synthetic peptide composed of aspartic acid, isoleucine, and lysine)), Tripeptide-1), Eyeseryl TM (Ac-β-Ala-His-Ser-His) (SEQ ID NO:25), Serilesine TM (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO:26) or Decorinyl TM (INCI name: Tripeptide-10 Citrulline = reaction product of citrulline and tripeptide-10 (a synthetic peptide composed of aspartic acid, isoleucine, and lysine);

[0182] -Collaxyl sold by Vincience TM (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO:27)) or Quintescine TM (Cys-Gly);

[0183] -Cytokinol sold by Les Laboratoires Serobiologiques / Cognis TM LS (casein hydrolysate);

[0184] -Kollaren sold by l'lnstitut Européen de Biologie Cellulaire TM (Gly-His-Lys), IP2000 TM (Pal-Val-Tyr-Val) or Meliprene TM(INCI name = Monofluoroheptapeptide-1: reaction products of acetic acid and a synthetic peptide containing arginine, glycine, glutamic acid, histidine, norleucine, p-fluorophenylalanine and tryptophan);

[0185] -Neutrazen sold by Innovations TM (Pal-His-D-Phe-Arg-NH2); or

[0186] -BONT-L-Peptide sold by lnfinitec Activos TM 、Timp-Peptide TM or ECMModuline TM .

[0187] It is also possible to envisage combining the present invention with one or more cyclic peptides, in particular the cyclic peptides extracted from linseed oil described in the applicant's patent application WO 2019 / 149450.

[0188] The extract according to the invention or the composition containing it may be preferably combined with at least one compound selected from the group consisting of vitamin B3, compounds such as niacinamide or tocopherol, retinoid compounds such as retinol, hexamidine, α-lipoic acid, resveratrol or DHEA, hyaluronic acid, ceramides, peptides (particularly N-acetyl-Tyr-Arg-O-hexadecyl, Pal-VGVAPG (SEQ ID NO: 22), Pal-KTTKS (SEQ ID NO: 17), Pal-KTSKS (SEQ ID NO: 18), Pal-GHK, Pal-KMO2K, Pal-GQPR (SEQ ID NO: 15) and Pal-K(P)HG (MATRIXYL TM Morphomics TM , Sederma)), said compound is a classic active agent used in topical cosmetic or dermo-pharmaceutical compositions.

[0189] The extract or composition according to the present invention may be applied topically to the target area.

[0190] The effective amount of the extract according to the present invention, i.e. its dosage, depends on the intended use of the composition. It depends on a variety of factors, such as the patient's age, condition, severity of the disorder or disease, and the mode of administration. An effective amount is a non-toxic amount sufficient to achieve the desired effect.

[0191] In a cosmetic composition according to the invention containing at least an extract according to the invention, in order to be present in an effective amount, the extract is generally present in an amount ranging from 0.000001% (0.01 ppm) to 15% (150,000 ppm), preferably in the range of 0.00001% (0.1 ppm) to 10% (100,000 ppm), based on the total weight of the composition, depending on the use of the composition and the desired effect, which is more or less pronounced. More preferably, the effective amount is between 0.0001% (1 ppm) and 0.01% (100 ppm), based on the total weight of the composition, which is much lower than current market standards.

[0192] All percentages and ratios used herein are by weight of the total composition and all measurements made are at 25°C, unless otherwise specified.

[0193] For reference, for cosmetic facial treatments, the European standard dosage for creams is 2.72mg / cm 2 / day / person, and for cosmetic body treatments, the European standard dosage of lotion is 0.5mg / cm 2 / day / man.

[0194] According to other characteristics, the cosmetic treatment method according to the invention can be combined with one or more other treatment methods for the skin, such as luminotherapy, heat or aromatherapy treatments.

[0195] A device with multiple compartments or a kit may be provided for applying the above-described method, which may comprise, for example and without limitation: a first compartment containing a composition comprising an extract according to the invention and, in a second compartment, a composition containing another active ingredient and / or excipients, in which case the compositions contained in the first and second compartments are considered a combined composition for simultaneous, separate or stepwise use in time, in particular in one of the above-described treatment methods.

[0196] Details

[0197] The present invention will be better understood based on the description of the following embodiments, studies, and drawings described below.

[0198] 1. Description of the accompanying drawings

[0199] [ Figure 1 ]: It represents a chromatogram monitoring secondary metabolites produced from the cell biomass of Mentha serrata just before the addition of naringenin.

[0200] [ Figure 2 ]:It means as [ Figure 1] but seven days after the addition of naringenin, illustrating the biotransformation of naringenin into a set of its glycosylated derivatives.

[0201] [ Figure 3 ]:It indicates the effect of luteolin on the cell biomass of lavender a few minutes after addition of luteolin. Figure 1 ] monitoring chromatogram in .

[0202] [ Figure 4 ]:It means as [ Figure 3 ] but eight days after the addition of luteolin, illustrating the biotransformation of luteolin into a set of its glycosylated derivatives.

[0203] [ Figure 5 ]: It represents a graph illustrating the production kinetics of glycosylated flavonoids and rosmarinic acid during cell culture of Mentha piperita according to the method of the present invention.

[0204] [ Figure 6 ]: This is a graph showing the relationship between skin deformation and time under the action of mechanical deformation.

[0205] 2. Preparation Example of the Extract According to the Present Invention

[0206] 2.1. From the American mint plant

[0207] 2.1.1. Establishment of cell lines

[0208] Selected peppermint leaves were collected, washed and cut into small pieces of a few millimeters to prepare a large number of explants. After a series of decontamination treatments, the leaf samples were placed on a set of agar media to induce callogenesis (callus formation).

[0209] After an appropriate period of time, aggregates of dedifferentiated cells, called calli, are formed. These are transferred to a larger volume of fresh medium to allow them to proliferate. A certain number of subcultures (transfers to fresh medium) are performed to stabilize the cell line, i.e., until it exhibits a satisfactory and constant proliferation rate, phenotype retention, and a constant content of the target bioactive compounds (primary and secondary metabolites).

[0210] The cell line then undergoes a selection step consisting of culturing the cells for an appropriate period of time, taking the cell aggregates formed and plating them in liquid culture medium for a period of time, allowing the cells to proliferate in small clusters and / or individually. The culture medium used is of the Murashige and Skoog type.

[0211] The best cell line is one that can yield as quickly and reproducibly as possible a satisfactory biomass with an optimal content of selected metabolites, optimal biological activity and a homogeneous phenotype. Rosmarinic acid is one of the selected target secondary metabolites.

[0212] 2.1.2. Obtaining Dedifferentiated Monarda Cell Biomass, Including Desired Secondary Metabolites As starting material, a cell line prepared as described above or a pre-existing and preserved line is used.

[0213] First, the peppermint cell line was propagated to obtain a biomass with sufficient numbers of dedifferentiated cells to perform a large-scale production step.

[0214] Perform the following steps:

[0215] a. The selected cell line is inoculated in a liquid culture medium and cultured for a sufficient time to obtain an amount of biomass that is doubled 2-fold or even 3-fold compared to the original biomass;

[0216] b. Optionally, the suspension obtained in a) is transferred to a fresh liquid culture medium and cultured again for a sufficient time to double or even triple the amount of biomass compared to the original biomass;

[0217] c. Optionally, repeat step b);

[0218] Steps a) to c) constitute the said pre-cultivation step. The culture medium used is of the Murashige and Skoog type.

[0219] The cell suspension obtained in stages a) to c) is transferred to a bioreactor containing a specific hormone-deficient production medium, where the biomass will grow, particularly during the exponential phase. Once the desired biomass level is reached, 20 to 90 mg / L of naringenin and 0.1 to 15 mg / L of methyl jasmonate are added as inducers in the middle of the exponential phase. The culture medium used in this stage is type B5 from Gamborg. This type of culture medium significantly increases the amount of rosmarinic acid produced by the plant cells. Under these conditions, the culture is carried out for a sufficient time to obtain a cell biomass containing sufficient amounts of the target secondary metabolites (i.e., the glycosylated derivatives of naringenin and rosmarinic acid, which are the subject of the present invention). This step d) is referred to as the bioreactor culture step.

[0220] Bioreactor:

[0221] Volume: 5 to 100 times larger than the volume of the biomass used as inoculum; the inner surface of the bioreactor is smooth and uniform.

[0222] Growing conditions:

[0223] Culture medium: A medium containing mineral salts (solutions of macro- and trace elements), vitamins, plant hormones, and sucrose. For the cell line establishment procedure, plant agar is added to the solid medium.

[0224] Temperature: between 15°C and 35°C, preferably between 20°C and 30°C, and even more preferably at 25°C.

[0225] Duration: between 7 and 21 days, while stirring the biomass to allow for optimal aeration, preferably between 10 and 14 days.

[0226] Stirring of the biomass: The biomass is optimally aerated and simultaneously stirred internally or externally. When the biomass is large, low and efficient stirring is maintained in the final step. For the purposes of the present invention, suitable internal stirring is a propeller rotating at between 0.10 and 0.75 m / s, preferably at 0.21 m / s, or external stirring is an orbital stirring preferably between 40 and 200 rpm and preferably at about 110 rpm.

[0227] Oxygenation: This is typically achieved using sterile air or a gas mixture containing 10% to 100% oxygen by volume.

[0228] 2.1.3. Recovery of Dedifferentiated Cell Biomass

[0229] Biomass consists of cells—partially lysed cells or whole cells—in suspension in the form of clumps or individuals.

[0230] Filtration was performed to eliminate the remaining culture medium (supernatant) and recover the cell biomass.

[0231] Optionally, prior to the filtration step, an antioxidant is added to the biomass to protect secondary metabolites from oxidation.

[0232] The recovered cell biomass can be characterized by HPLC / UV.For this purpose, the cells are extracted in an ethanol / water mixture (70 / 30 by volume) for analysis.

[0233] HPLC / UV analysis of the extract showed that the cell biomass contained 200 to 500 ppm by weight of the glycosylated flavonoid derivatives, 1000 to 2000 ppm by weight of rosmarinic acid, relative to the total weight of the extract.

[0234] 2.1.4. Processing of the biomass of dedifferentiated cells to obtain the extract according to the invention

[0235] The contents of the cells are extracted by osmotic diffusion with the addition of glycerol, and the liquid and solid phases are separated by centrifugation or filtration or equivalent protocols to obtain a cell extract free of cell debris.

[0236] Optionally, extraction of cell contents can also be performed by grinding, mechanical or chemical lysis of the cells.

[0237] The peppermint extract according to the invention then comprises the intracellular contents of dedifferentiated plant cells, free of cell fragments thereof.

[0238] The active compounds contained in the obtained extract were analyzed and characterized by chromatography. The results of the analysis are given in point 5 of the description below.

[0239] The following steps are optional:

[0240] 1. Drying of the extract, in particular by freeze drying, zeodratation or nebulization, to allow for better stability of the target compound, improving long term storage without the need for added preservatives.

[0241] 2. High pressure homogenization of cell biomass: allows to reduce the size of cell aggregates.

[0242] 3. Purification of the cell extract to increase the content of glycosylated flavonoids and rosmarinic acid, for example by an additional ethanol / water extraction (70 / 30 by volume).

[0243] 2.2. From the lavender plant or the desert verbena plant

[0244] The same method steps as described above for Mentha serrata were performed, except that the aglycone flavonoid precursor used here was luteolin. The amount of luteolin added was between 200 mg / L and 1000 mg / L.

[0245] In the same way as for Mentha piperita, at the end of the process an extract of plant origin is obtained, characterized by the presence of glycosylated derivatives of luteolin in the intracellular content of the harvested plant cells.

[0246] The active compounds contained in the obtained extract were analyzed and characterized by chromatography. The results of the analysis are given in point 5 below.

[0247] 3. Preparation Examples of Active Ingredients for Cosmetic Use

[0248] The extract according to the invention, as prepared according to the above examples, may constitute such an active ingredient, depending on the concentration of its secondary metabolites, with glycerol, for osmotic diffusion, advantageously constituting a physiologically acceptable medium.

[0249] To form the active ingredient, the extract can also be diluted in a physiologically acceptable medium to reduce the concentration of its secondary metabolites. This medium can be the same as the medium used for osmotic diffusion (here glycerol) or different, such as butylene glycol alone or mixed with glycerol.

[0250] The composition thus formed may comprise, for example, 20% by weight of a fresh biomass extract of dedifferentiated cells in glycerol (approximately 20 to 80%), said composition containing 0.002% to 4% by weight of said glycosylated flavonoid derivatives and 0.002% to 4% by weight of rosmarinic acid relative to the weight of said composition.

[0251] This ingredient can then be used to prepare cosmetic formulations such as those described below. An effective amount of this ingredient corresponds to 0.3% to 15%, preferably 1% to 5%, more preferably 2% to 4% and typically 3% by weight of the formulation.

[0252] 4. Examples of Cosmetic Preparations

[0253] Examples of cosmetic preparations containing the extracts according to the invention as described above are given below. In addition, these preparations may also contain additional cosmetic active ingredients, which are used in each case to support and / or supplement the activity of the active ingredients according to the invention. These ingredients can be divided into any category according to their function(s), application site (body, face, neck, chest, hands, etc.), desired end effect and target consumer, such as anti-wrinkle, moisturizing, anti-dark circles, firming, anti-glycation, plumping, soothing, muscle relaxant, anti-redness, detoxification, etc.

[0254] Cream form

[0255] [Table 1]

[0256]

[0257] plan:

[0258] Swell the carbomer in water (phase A) and heat. Weigh and heat B. Melt C and let it cool. Add D to C, then add C+D to A and mix. To C+D+A, add B, then E, then F, then G and homogenize.

[0259] Essence form

[0260] [Table 2]

[0261]

[0262] plan:

[0263] Weigh and mix A. Weigh and mix B. Add B to A while stirring, mix. Weigh and mix C. Add C to B+A while stirring, mix. Weigh and mix D. Add D, then E, then F to B+A+C, homogenize.

[0264] Melt C and let it cool. Add D to C, then add C+D to A and mix. To C+D+A, add B, then E and homogenize.

[0265] Examples of ingredients that can be added to these formulations (sold by SEDERMA):

[0266] VENUCEANE TM : Contains an active ingredient based on a biotechnological extract of Thermus thermophilus, which prevents the visible signs of photoaging (spots, wrinkles, dryness, etc.), protects the cell structure from damage caused by UV radiation and strengthens skin integrity; AQUALANCE TM : Penetrating protective moisturizing active ingredient; CALMOSENSINE TM : Calming active ingredient; CRYSTALIDE TM : Active ingredient that soothes the epidermis more specifically.

[0267] 5. Analysis and testing

[0268] The extracts used for the analytical tests were the extracts described above in point 2.1.3. The cells were extracted by mixing in an ethanol / water solution (70 / 30 by volume), which was then filtered and then diluted before injection into the HPLC / UV.

[0269] The assay was performed on a Waters HSS C18 column with a 15 min elution gradient consisting of a 50 mM aqueous ammonium formate phase at native pH, water, and acetonitrile.

[0270] It does characterize the intracellular contents, i.e., the substances produced by plant cells.

[0271] 5.1. Comparison during the method, before adding the precursor and after its metabolism

[0272] The goal was to demonstrate biotransformation in plant cells. For this purpose, for the two plant examples given above—menthol and lavender—chromatograms were analyzed for extracts prepared from biomass samples taken from the bioreactor just before and after the addition of the precursors, as well as for extracts prepared from a 7-day biomass sample. For each plant, the two chromatograms were compared.

[0273] result

[0274] 5.1.1. American Mentha piperita

[0275] [ Figure 1 ]: represents a chromatogram monitoring secondary metabolites produced from the cell biomass of Mentha serrata just before the addition of naringenin.

[0276] [ Figure 2 ]:Indicates [ Figure 1 ] but seven days after the addition of naringenin, illustrating the biotransformation of naringenin into a set of its glycosylated derivatives.

[0277] exist[ Figure 1 In the chromatogram in FIG, a peak of rosmarinic acid was observed at 3.2 minutes. This is consistent with the expectation from the cell culture of Mentha serrata, in which rosmarinic acid is known to be a secondary metabolite.

[0278] exist[ Figure 2 ], the peak corresponding to rosmarinic acid (3.2 min) was still observed, but a peak between 3.5 and 5.5 min was also observed in [ Figure 1 ] were absent from the chromatogram of . It was determined that they corresponded to a group of glycosylated derivatives of naringenin.

[0279] In addition, the retention time of naringenin is 8 minutes. It can be seen that the peak is [ Figure 1 ]or[ Figure 2 ] does not appear in .

[0280] This clearly shows that dedifferentiated plant cells of Mentha serrata incorporated naringenin added to the culture medium and then metabolized it into a group of glycosylated derivatives of naringenin.

[0281] 5.1.2. Lavender

[0282] [ Figure 3 ]:Indicates the effect of luteolin on the cell biomass of lavender a few minutes after addition of luteolin. Figure 1 ] monitoring chromatogram in .

[0283] [ Figure 4 ]:Indicates [ Figure 3 ] but eight days after the addition of luteolin, illustrating the biotransformation of luteolin into a group of its glycosylated derivatives.

[0284] exist[ Figure 4 ], a peak at 3.10 minutes corresponding to rosmarinic acid was observed, and several peaks corresponding to the pool of glycosylated derivatives of luteolin between 3.30 and 6.00 minutes and a final peak at 6.50 minutes corresponding to luteolin were also observed.

[0285] exist[ Figure 3], there are only peaks of rosmarinic acid and luteolin. The peak corresponding to luteolin is in [ Figure 3 ] is 0.0125 arbitrary units and in [ Figure 4 ] is 0.005 arbitrary units.

[0286] The dedifferentiated plant cells of the lavender extract according to the present invention incorporate and metabolize most of the luteolin added to the biomass into a group of glycosylated derivatives of luteolin.

[0287] It can also be seen in this case that the cells of lavender are able to metabolize very large amounts of luteolin, which advantageously makes it possible to obtain an extract very concentrated in secondary metabolites.

[0288] 5.2 Effects of Adding Aglycone Flavonoids and / or Inducers on the Extracts Obtained After Metabolism

[0289] 5.2.1. American Mentha piperita

[0290] The obtained peppermint extracts were tested by in vitro cell culture under different conditions, using only one type of growth medium, such as Murashige and Skoog.

[0291] The products produced by the method were measured in order to compare the contents of glycosylated derivatives of naringenin and rosmarinic acid obtained.

[0292] - Extract 1: extract according to the invention obtained with the addition of naringenin;

[0293] - Extract 2: extract according to the invention obtained with the addition of naringenin and methyl jasmonate (inducer);

[0294] - Extract 3: extract with added methyl jasmonate but without aglycone flavonoids;

[0295] - Extract 4: an extract to which neither aglycone flavonoids nor inducers were added.

[0296] result

[0297] Content of rosmarinic acid and glycosylated naringenin derivatives in different extracts of American mint:

[0298] [Table 3]

[0299]

[0300] LOD: Limit of Detection

[0301] The results show that the extracts obtained without the addition of naringenin (extracts 3 and 4) do not exhibit glycosylated flavonoids, compared to the extracts according to the invention obtained with the addition of naringenin (extracts 1 and 2). An inductive effect of methyl jasmonate (extract 3) is also visible, with a significant increase in the level of rosmarinic acid (91 ppm to 302 ppm), and this effect is also visible for the extract obtained with the addition of naringenin (extract 2) compared to the extracts obtained without the addition of naringenin (extracts 1 and 4).

[0302] It is also noted that in the extract according to the invention obtained with the addition of naringenin and methyl jasmonate (Extract 2), a strong increase in the level of rosmarinic acid (20 to 280 ppm) and a very strong increase in the level of glycosylated flavonoids (54 to 334 ppm) was observed compared to the extract according to the invention obtained with the addition of naringenin alone (Extract 1). This shows that the addition of naringenin to the process according to the invention induces advantages not only in the level of rosmarinic acid, but also in the level of glycosylated flavonoids.

[0303] 5.2.2. Lavender

[0304] The obtained lavender extracts were also tested in vitro by cell culture under different conditions, using only one type of growth medium, Murashige and Skoog.

[0305] The products produced by the method were measured in order to compare the contents of glycosylated derivatives of luteolin and rosmarinic acid obtained.

[0306] - Extract 1: extract according to the invention obtained with the addition of luteolin;

[0307] - Extract 2: extract according to the invention obtained with the addition of luteolin and methyl jasmonate (inducer);

[0308] - Extract 3: extract with added methyl jasmonate but without aglycone flavonoids;

[0309] - Extract 4: an extract to which neither aglycone flavonoids nor inducers were added.

[0310] result

[0311] Contents of rosmarinic acid and glycosylated luteolin derivatives in different lavender extracts:

[0312] [Table 4]

[0313]

[0314] The results showed that the extracts obtained without the addition of luteolin (Extracts 3 and 4) did not show glycosylated flavonoids, compared to the extracts according to the invention obtained with the addition of luteolin (Extracts 1 and 2). An inductive effect of methyl jasmonate (Extract 3) was also observed, with a significant increase in the level of rosmarinic acid (1450 ppm to 3584 ppm) compared to the extracts obtained without the addition of luteolin (Extracts 1 and 4), and this effect was also observed for the extract obtained with the addition of luteolin (Extract 2).

[0315] It was also observed that the extract according to the invention obtained with the addition of luteolin and methyl jasmonate (Extract 2) showed a strong increase in the level of rosmarinic acid (1293 to 3933 ppm) and a very strong increase in the level of glycosylated flavonoids (1572 to 2109 ppm) compared to the extract according to the invention obtained with the addition of luteolin alone (Extract 1). This shows that the addition of methyl jasmonate to the method according to the invention induces advantages not only in the level of rosmarinic acid but also in the level of glycosylated flavonoids.

[0316] 5.2.3. Desert Verbena

[0317] The resulting Verbena deserticola extract was also tested in vitro using different cell culture conditions. Here, only one type of growth medium, Murashige and Skoog, was used. The product produced by the described method was measured to compare the content of glycosylated derivatives of luteolin obtained.

[0318] - Extract 1: extract according to the invention obtained with the addition of luteolin;

[0319] - Extract 2: an extract to which neither aglycone flavonoids nor inducers were added.

[0320] result

[0321] Content of glycosylated derivatives of luteolin in different desert verbena extracts:

[0322] [Table 5]

[0323] Glycosylated derivatives of luteolin Extract 1 according to the present invention (+ luteolin) 1764ppm Extract 2 (containing neither luteolin nor methyl jasmonate) <LOD

[0324] The results show a very strong increase in glycosylated flavonoids in the extract according to the invention obtained with the addition of luteolin (Extract 1) compared to the extract without glycosylated flavonoids (Extract 2) obtained without the addition of luteolin.

[0325] 5.2.4. Conclusion

[0326] Thus, the addition of aglycone flavonoids to biomass can produce glycosylated flavonoids that were not present in the plant cells of the original biomass. The addition of methyl jasmonate can increase the amount of rosmarinic acid and the amount of glycosylated derivatives of the precursor flavonoids.

[0327] 5.3 Kinetics of target molecule production during the same cell culture period

[0328] The aim was to compare the amounts of aglycone flavonoids and their glycosylated derivatives over time before and after addition of aglycone flavonoids during the preparation of peppermint extract obtained by in vitro cell culture according to the present invention.

[0329] result

[0330] [ Figure 5 The bar graph of FIG1 illustrates the production kinetics of glycosylated derivatives of rosmarinic acid and naringenin during cell culture, with the content in dry biomass on the ordinate and different culture times (in days) on the abscissa. All percentages are given by weight relative to the total weight of the dry extract.

[0331] The graph shows that after adding methyl jasmonate and naringenin at T = 7 days of culture, the extract obtained contained only rosmarinic acid, which increased from 1.7% before addition to 2.3% after addition.

[0332] A few hours after the addition of naringenin (T = 7 days of culture + 5-6 hours after addition), glycosylated derivatives of naringenin appeared (around 1.1%). The amount then increased (1.35% at T = 8 days of culture), reached a plateau (1.32% at T = 9 days of culture), and then decreased (1.2% at T = 10 days of culture and 0.9% at T = 14 days of culture).

[0333] As for rosmarinic acid, it increased over time after the addition of methyl jasmonate (from 1.7% before addition at T = 7 days of culture to 3.3% at T = 14 days of culture).

[0334] This kinetic monitoring can also show that the addition of aglycone flavonoids to the culture medium leads to the production by the plant cells of glycosylated derivatives of these aglycone flavonoids that were not present in the initial biomass.

[0335] It can be seen that the addition of the inducer increases the amount of rosmarinic acid, a molecule already present in the initial biomass, ie before the addition of the aglycone flavonoids and the inducer.

[0336] 5.4 Mechanism of Target Molecules Produced by Peppermint Extract

[0337] principle

[0338] The aim of this study was to explain the mechanism of secondary metabolite production in P. americana cells, particularly the production of glycosylated derivatives.

[0339] A number of hypotheses were proposed:

[0340] 1) Aglycone flavonoids can reveal specific metabolic pathways that do exist but have not yet been utilized due to a lack of precursors. If the enzyme responsible for converting aglycone flavonoids is naturally present in the cell, then the enzyme will be found outside the cell when the cells are ground. Therefore, the enzyme can convert aglycone flavonoids into glycosylated derivatives whether inside or outside the cell. Therefore, this phenomenon can be observed in intact cells (the first condition) and in disrupted cells (the third condition), which have already converted naringenin into glycosylated derivatives of aglycone flavonoids. In the supernatant (the second condition), no glycosylated derivatives should be found because the enzyme can only be present in the cell and not directly in the supernatant.

[0341] 2) Aglycone flavonoids can play the role of epigenetic probes and serve as precursors. This epigenetic probe can send a signal to the cell DNA, so it can generate and / or mobilize certain specific enzymes that can metabolize the probe. Precursors are molecules that are used to convert. Contrary to the first hypothesis, the enzyme cannot be generated and / or mobilized outside the cell because the DNA is inactive outside the cell. Therefore, aglycone flavonoids cannot be converted into glycosylated derivatives in broken cells (the second condition), but can only be converted into glycosylated derivatives in intact cells (the first condition). In the supernatant (the second condition), glycosylated derivatives should not be found because the enzyme can only be present in the cell and cannot be directly present in the supernatant.

[0342] 3) aglycone flavonoids can serve as epigenetic probes but not as precursors. This epigenetic probe can unlock some DNA reading zones for at least one pathway metabolism, which is different from the second hypothesis. In this case, aglycone flavonoids only serve as probes, but not as precursors, because this pathway metabolism is not induced. The same as the second hypothesis, the enzyme can't be generated and / or mobilized extracellularly because DNA is inactive extracellularly. But, contrary to the second hypothesis, aglycone flavonoids only serve as epigenetic probes, so other molecular families except glycosylated derivatives will occur. Therefore, in supernatant (the second condition), glycosylated derivatives should not be found either. In the case of intact cells (the first condition), aglycone flavonoids can be converted into glycosylated derivatives, and fragmented cells (the third condition) trigger another chemical composition by opening other pathway metabolisms.

[0343] Summarize:

[0344] - The first hypothesis is confirmed if both intact cells (first condition) and disrupted cells (third condition) convert aglycone flavonoids into glycosylated derivatives.

[0345] - If only intact cells (first condition) convert aglycone flavonoids into glycosylated derivatives, the second hypothesis is confirmed.

[0346] - If intact cells (first condition) convert aglycone flavonoids into glycosylated derivatives and disrupted cells (third condition) trigger another chemical composition by opening other metabolic pathways, the third condition is confirmed.

[0347] plan

[0348] Comparative experiments were conducted on precultures of Mentha serrata obtained by in vitro cell culture according to the present invention. Three conditions were tested:

[0349] For the first condition, the biomass, consisting of suspended cells in the form of clumps or individuals - partially lysed cells or intact cells - and the culture medium (supernatant) were recovered and analyzed separately.

[0350] For the second condition, the biomass was filtered to recover the cells and the supernatant was removed. Only the cells were analyzed.

[0351] For the third condition, the biomass is filtered to recover the cells and the supernatant is removed. The cells are transferred to new culture medium and then disrupted, for example, by sonication and / or freezing.

[0352] For each condition, the recovered material was divided into two vials, where the contents of one vial were subsequently stimulated with naringenin and methyl jasmonate, while the contents of the other vial were left unstimulated and thus constituted a control group.

[0353] The contents of each vial were dosed for rosmarinic acid and naringenin derivatives according to the following:

[0354] - For vials with stimulated contents: after recovery of cells, supernatant or disrupted cells, and before stimulation (T=0), 24 hours after stimulation (T=24 hours) and 48 hours after the first stimulation and just after a new stimulation (T=48 hours).

[0355] - For vials without stimulated contents: after recovery of cells, supernatant or disrupted cells (T=0), 24 hours later (T=24 hours) and 48 hours later (T=48 hours).

[0356] result

[0357] Contents of glycosylated derivatives of rosmarinic acid and naringenin under different conditions:

[0358] [Table 6]

[0359]

[0360] The results show:

[0361] - For the first condition, before stimulation, cells contained a small amount of rosmarinic acid. 24 hours after stimulation with naringenin and methyl jasmonate, stimulated cells contained glycosylated derivatives of naringenin and a higher amount of rosmarinic acid.

[0362] - For the second condition, the supernatant contained no rosmarinic acid before stimulation. 24 hours after stimulation, the supernatant stimulated with naringenin and methyl jasmonate contained no glycosylated derivatives of naringenin and no rosmarinic acid. However, naringenin added during stimulation was still present in the supernatant 24 hours after stimulation, indicating that it had not been degraded and / or converted. This is explained by the absence of enzymes capable of converting naringenin into glycosylated derivatives of naringenin in the supernatant.

[0363] In the third condition, before stimulation, the disrupted cells contained no rosmarinic acid. This is because rosmarinic acid is not a stable molecule outside the cell and is easily oxidized and degraded if not protected by a solvent. Twenty-four hours after stimulation, disrupted cells stimulated with naringenin and methyl jasmonate contained no glycosylated derivatives of naringenin and no rosmarinic acid, but only the added naringenin. This is because, when cellular material is outside the cell, enzymes capable of converting naringenin into glycosylated naringenin derivatives are absent, and any potentially produced rosmarinic acid is rapidly degraded.

[0364] In summary, it has been demonstrated that, according to the present invention, naringenin acts as an epigenetic probe in cells, sending signals to DNA to produce and / or mobilize specific enzymes capable of converting naringenin into glycosylated derivatives of naringenin.

[0365] 6. In vitro efficacy testing

[0366] A number of biological activity tests were performed on the extracts according to the invention prepared as described in point 2. They showed a number of potential cosmetic activities.

[0367] ANOVA and Student's t-test for paired series were performed for each test to determine the significance of the results.

[0368] 6.1 Peppermint extract from the United States

[0369] 6.1.1. Anti-aging activity on skin

[0370] 6.1.1.1. Prevention of oxidative stress

[0371] Oxidative stress plays a central role in the skin's response to various stresses. Free radicals (H2O2, OH·, O2-, O2, 1 O2…) causes damage to proteins, lipids and DNA, leading to premature aging of the skin and its appendages.

[0372] plan

[0373] Normal human fibroblasts (HNF) are grown to confluence in their culture medium. The cells are then contacted with a composition according to the present invention for 24 hours, and then receive a fluorescent probe intended to label ROS produced in the cells. After introducing 30 min and flushing, the cells are again treated with a composition according to the present invention, and do not receive any substance or receive a medicament intended to form ROS (oxidative stress). The amount of intracellular ROS is estimated by fluorescence readings (e.g., 490 nm / cm:520 nm). The cell number is estimated using the Hoechst 33258 method (DNA staining), with weighting of the data obtained.

[0374] For melanocytes, an equivalent protocol was used.

[0375] result

[0376] Changes in ROS production in fibroblasts (n=3) and melanocytes (n=5) with or without oxidative stress. Effect of 0.32% of the extract according to the invention compared to the control:

[0377] [Table 7]

[0378]

[0379] These results show that the extract according to the invention significantly reduces the intracellular content of ROS in fibroblasts or melanocytes subjected to or not to oxidative stress.

[0380] Therefore, the peppermint extract according to the present invention has strong antioxidant capacity and can effectively combat premature aging of the skin.

[0381] 6.1.1.2. Protecting the dermal-epidermal junction (DEJ)

[0382] The DEJ ensures adhesion between the dermis and the epidermis. During aging, a decrease in the synthesis of its components (especially collagen and laminin) is observed. Aging of the DEJ has a significant adverse effect on the skin's resilience and loss of its vitality.

[0383] 6.1.1.2.1. Stimulation of collagen VII and laminin synthesis

[0384] plan

[0385] Human keratinocytes (HK) were cultured at subconfluence and either contacted with the extract according to the invention or not (for control). After this contact, the collagen VII content of the culture supernatant and the cell layer was measured, respectively, and laminin was evaluated using an ELISA kit.

[0386] The cell number was estimated using the Hoechst method and used to normalize the results.

[0387] result

[0388] Changes in the production of collagen-VII (n=4) and laminin (n=4) in keratinocytes. Effect of 0.66% of the extract according to the invention compared to the control:

[0389] [Table 8]

[0390]

[0391] These results show that the extract according to the invention stimulates the production of collagen-VII and laminin, essential elements of DEJ.

[0392] 6.1.1.2.2. Stimulation of collagen-IV and -XVII synthesis

[0393] plan

[0394] During 7 days, gel comprising extract according to the present invention was applied daily to the skin explant surface. Then, skin was cut and collagen-IV and collagen-XVII were synthesized by immunohistochemical method (using a special primary antibody marker section to be measured, and showing this marker by a special fluorescent secondary antibody of this primary antibody). Then, the intensity of quantitative labeling was carried out by image analysis of the photograph taken.

[0395] In addition, 0.5% of the extract according to the invention or a placebo (control) was applied to cultured human keratinocytes and collagen VII synthesis was measured by ELISA on cell extracts after disruption. The results were converted to cell number, which was evaluated by contrast labeling of cell nuclei with the fluorescent dye Hoechst 33258, which labels DNA.

[0396] result

[0397] Changes in the synthesis of collagen IV and collagen XVII on skin explants after 7 days (n=3). Effect of 0.66% of the extract according to the invention compared to the control:

[0398] [Table 9]

[0399]

[0400]

[0401] *AUF: Arbitrary Fluorescence Unit

[0402] These results show that the extract according to the invention stimulates the production of collagen-IV and -XVII, which are also essential elements of DEJ.

[0403] All these results show that the peppermint extract according to the invention has a direct effect on strengthening JDE by stimulating the collagen and laminin proteins that constitute it. The extract according to the invention can act on skin aging associated with JDE structural damage by counteracting the loss of softness and elasticity caused by this structural damage. Furthermore, collagens IV and XVII are known to be involved in correct melanocyte anchoring. Stimulating their synthesis by the extract according to the invention can prevent premature aging of melanocytes, which leads to the appearance of pigmentation defects and loss of skin tone uniformity.

[0404] 6.1.1.3. Protection of the dermal extracellular matrix

[0405] Elastase and MMPs (matrix metalloproteinases: dermal matrix proteases) are proteases that break down extracellular matrix proteins, such as elastin and various collagens. Their production increases with age, and acute or chronic stress amplifies this phenomenon. Excessive production of MMPs leads to a decrease in dermal strength through a loss of dermal density and finesse. Excessive elastase production leads to a loss of skin elasticity.

[0406] plan

[0407] Fibroblasts were grown to confluence in their culture medium. The cells were exposed to solar radiation once daily for four days. At the end of the exposure period, the cells were exposed to the extract according to the present invention. Total RNA was extracted for analysis of elastase, MMP-2, and MMP-3 by RT-qPCR.

[0408] result

[0409] Changes in the amount of elastase (n=4), MMP2 (n=4) and MMP3 (n=3) in irradiated fibroblasts. Effect of 0.16% of the extract according to the invention compared with the control:

[0410] [Table 10]

[0411]

[0412]

[0413] The results showed that the extract according to the present invention significantly reduced the amount of elastase, MMP2 and MMP3. By limiting the production of proteases, the extract reduced the overall harmful activity of these proteases on extracellular matrix (ECM) proteins (such as collagen and elastin), thereby contributing to the good health of the dermis and the protection of the microenvironment in which cells reside.

[0414] 6.1.1.4. Stimulation of dermal extracellular matrix molecular synthesis

[0415] 6.1.1.4.1. Stimulation of elastin synthesis

[0416] Elastin is a protein found in the extracellular matrix of the dermis. During aging, the amount of elastin produced decreases, leading to improper assembly of elastin fibers and changes in the elasticity of the skin.

[0417] plan

[0418] Human dermal fibroblasts were grown to confluence in their culture medium. The cells were immersed in buffer and illuminated using a sun lamp (UV Technology; Honle; 60 mJ / cm 2 *) at 30 mJ / cm 2 * were exposed to UV light once daily for 8 consecutive days. After each exposure, the cells received culture medium containing the extract according to the invention. At the end of this exposure, the cell layer was rinsed, fixed, and labeled with an anti-elastin antibody. The labeling was visualized using a fluorescent secondary antibody, and photographs were captured under a microscope. Image analysis of these photographs allowed quantification of elastin production. Cell nuclei were contrast-labeled using the fluorescent dye HOESCHT 33258, which labels DNA, to assess cell populations and thus weight the acquired fluorescence data.

[0419] result

[0420] Changes in elastin production in fibroblasts exposed or not to sunlight (n=3). Effect of 0.16% of the extract according to the invention compared to the control:

[0421] [Table 11]

[0422] Non-irradiated fibroblasts Irradiated fibroblasts comparison Unexposed references UV exposed reference Change (%); significance +196％；p<0.01 +524％；p<0.01

[0423] The results showed that the extract according to the invention significantly increased elastin production in fibroblasts, both under stress and without stress. In both cases, these increases were substantial and significant. This stimulation is particularly attractive, as elastin production decreases with age, and this phenomenon is particularly pronounced during exposure to the sun.

[0424] 6.1.1.4.2. Stimulation of collagen-I synthesis

[0425] Collagen I is the most abundant protein in the dermis. With age, dermal fibroblasts produce less supportive proteins, including collagen I. Therefore, it is crucial for beautiful, firm skin.

[0426] plan

[0427] The same protocol as described in point 6.1.1.2.2 above.

[0428] result

[0429] Changes in the amount of collagen-I on skin explants after 7 days (n=4). Effect of 0.66% of the extract according to the invention compared with the control:

[0430] [Table 12]

[0431]

[0432] The results showed that the extract according to the present invention clearly and significantly stimulated collagen-I production.

[0433] Elastin and collagen-I production ensure good mechanical properties, firmness, resilience and elasticity of the skin and thus, for example, prevent and / or treat skin sagging, wrinkles and fine lines.

[0434] 6.1.1.5. Preventing glycation

[0435] Protein glycation caused by reducing sugars in the skin is the cause of premature skin aging.

[0436] Once glycated, the enzymatic and / or structural properties of the protein are altered, which interferes with the proper functioning of the cells or organisms involved. This alters the mechanical and elastic properties of the dermis, which becomes less flexible and stiffer, but also more relaxed and less reactive, which also leads to a duller complexion.

[0437] plan

[0438] The test uses a model protein, serum albumin (which serves as a target), and edible reducing sugars from fruit. In the presence or absence of an extract according to the invention, the protein is gradually glycated (bound to sugars) in an irreversible manner. Changes in glycation are monitored by fluorescence.

[0439] result

[0440] Changes in saccharification (n=2). Effect of 0.66% of the extract according to the invention compared to the control:

[0441] [Table 13]

[0442]

[0443] These results show the strong anti-glycation potential of the active agents according to the invention, which can help combat skin aging and loss of radiance of the complexion.

[0444] 6.1.1.6. Anti-plaque activity

[0445] Melanocytes are the skin's pigment cells. Keeping melanocytes alive and healthy ensures high-quality pigmentation and limits the formation of dark and white spots that are common during skin aging.

[0446] 6.1.1.6.1. Protection of melanocyte dendrites

[0447] Melanocytes produce large amounts of melanin and distribute it to neighboring keratinocytes via branching extensions called dendrites. Keratinocytes absorb melanin, helping to protect the cells and their DNA from the sun's harmful effects. During aging, dendrites become smaller and melanocytes become larger. These dendrites accumulate melanin and are unable to distribute it, which can lead to toxicity.

[0448] plan

[0449] Human melanocytes in culture and at low density were contacted with the extract according to the invention and stressed by H2O2, a molecule strongly produced under UV stress.The length of melanocyte dendrites was quantified by image analysis.

[0450] result

[0451] Changes in the length of dendrites of melanocytes stressed by H2O2 (n=4). Effect of 0.50% of the extract according to the invention compared with the control:

[0452] [Table 14]

[0453]

[0454] The results show that the extract according to the invention strongly protects melanocytes from H2O2-induced stress, as the retraction of dendrites is less pronounced, compared to the control condition (placebo).

[0455] 6.1.1.6.2. Increased number of young melanocytes

[0456] The presence of spindle-shaped melanocytes is a good indicator of the youthfulness of the observed cell population. When the cell population is older, the melanocytes are larger.

[0457] plan

[0458] Human melanocytes were forced into premature aging. They were then cultured for 19 consecutive days in the presence of the extract according to the invention, demonstrating its safety for these cells, which are considered very fragile. The cell culture medium was replaced every 2 to 3 days. The cells were then photographed, and the number of spindle cells was quantified and compared to the total number of cells.

[0459] result

[0460] Changes in the number of spindle cells in melanocyte cultures (n=3). Effect of 0.16% of the extract according to the invention compared with the control:

[0461] [Table 15]

[0462]

[0463] The results show that the extract according to the invention significantly increases the number of spindle cells in the total melanocyte population, which means that the melanocyte population is less senesced and therefore younger.

[0464] 6.1.1.6.3. Increased hyaluronic acid synthesis in melanocytes

[0465] Hyaluronic acid is produced by fibroblasts, by keratinocytes and also by melanocytes. Hyaluronic acid interacts with keratinocytes and melanocytes in the epidermis via receptors.

[0466] plan

[0467] The human melanocytes in culture and connected received the extract according to the present invention for 24 hours. After rinsing, the cells were exposed to UVB in a buffer solution and then cultured in a culture medium containing the extract according to the present invention. The cells were then peeled off from the culture medium and extracted. Hyaluronidase was inactivated by heating, and the hyaluronic acid content around the cells was estimated by ELISA. The Hoechst method was used to estimate cell number and to standardize the results.

[0468] result

[0469] Changes in the amount of hyaluronic acid surrounding melanocytes after UVB exposure (n=4). Effect of 0.32% of the extract according to the invention.

[0470] [Table 16]

[0471]

[0472] The results showed that the extract according to the present invention can significantly increase the production of hyaluronic acid around melanocytes when the cells are irradiated, which is very attractive in preventing the occurrence of melanocyte aging.

[0473] 6.1.1.6.4. Reduction of senolytic enzyme activity

[0474] The enzyme SA β-galactosidase (senescence-associated β-galactosidase) is very active in senescent cells, whereas it is inactive or only slightly active in young cells.

[0475] plan

[0476] Human melanocytes were forced into premature aging. They were then cultured for five consecutive days in the presence of an extract according to the invention. Fresh culture medium was added on the third day to maintain cell survival. The cells were labeled with an indolegalactose derivative, which allows for quantification of SA-β-galactosidase enzyme activity. Accumulation of the enzyme in the lysosomes of senescent cells is visualized in blue.

[0477] result

[0478] Changes in SA-β-galactosidase in melanocyte cultures (n=5). Effect of 0.16% of the extract according to the invention compared with the control:

[0479] [Table 17]

[0480]

[0481] The results showed that the extract according to the present invention significantly reduced the activity of SA-β-galactosidase accumulated in senescent cells. Therefore, the extract according to the present invention can reduce the number of senescent cells.

[0482] 6.1.1.6.5. Reduction of DKK-1 Production

[0483] Overproduction of DKK1 protein in the dermis leads to melanocyte senescence and, therefore, to a complete cessation of melanin production.

[0484] plan

[0485] Fibroblasts were grown to confluence in their culture medium. The cells were then contacted with the extract according to the invention. At the end of this contact period, the fibroblasts were exposed to UVB stress and then again contacted with the extract according to the invention. The culture supernatant was assayed for DKK-1, a molecule identified as a pro-senescent molecule in melanocytes.

[0486] The cell number was estimated using the Hoechst method and used to normalize the results.

[0487] result

[0488] Changes in the amount of DDK-1 in UVB-stressed fibroblasts (n=3). Effect of 0.66% of the extract according to the invention compared with the control:

[0489] [Table 18]

[0490]

[0491] The results show that the extract according to the invention significantly reduces DKK-1 production in fibroblasts. This strengthens the attractiveness of the extract according to the invention for reducing the progression or development of the senescent phenotype in melanocytes.

[0492] 6.1.1.6.6. Reduction of inflammatory markers

[0493] IL-6 (interleukin-6) and IL-8 (interleukin-8) are part of the increased secretion of senescent cells. In addition, IL-6 is known for its pro-inflammatory aspects and as a player in DNA fragmentation, and IL-8 is a molecule that recruits and stimulates macrophages, which in turn release other cytokines and matrix proteases.

[0494] plan

[0495] Melanocytes were irradiated with UVB once daily for two days to induce senescence. Between each irradiation period (i.e., for a total of 48 hours), the cells were exposed to an extract according to the present invention. The IL-6 and IL-8 production in the culture medium was then dosed using the Hoechst method and standardized.

[0496] result

[0497] Changes in the amount of IL-6 (n=6) and IL-8 (n=6) in melanocytes after UVB irradiation. The effect of 0.32% of the extract according to the invention compared with the control:

[0498] [Table 19]

[0499]

[0500] The results showed that the extract according to the present invention significantly reduced the amount of IL-6 and IL-8 in melanocytes.

[0501] 6.1.1.6.7. Conclusion

[0502] All these results in points 6.1.1.6.1 to 6.1.1.6.6 show that the extract according to the invention can slow down the aging of melanocytes and, therefore, the appearance of pigmentary disorders such as white and / or black age spots.

[0503] 6.1.2. Moisturizing activity and epidermal protection from external aggressions

[0504] 6.1.2.1. Stimulation of hyaluronic acid synthesis

[0505] Hyaluronic acid is a major component of the epidermis, contributing to its barrier function and helping to maintain satisfactory hydration. It can absorb 1,000 times its weight in water. It takes the form of a watery, nourishing gel that fills the spaces between keratinocytes. It prevents dryness, which can alter the skin's texture and leave it feeling rough.

[0506] plan

[0507] Human keratinocytes were cultured at subconfluence and then exposed to or not exposed to the extract according to the invention (for control conditions). After this exposure, the synthesis of hyaluronic acid was determined using the ELISA method. Cell concentration was estimated using the Hoechst method.

[0508] result

[0509] Changes in hyaluronic acid production in keratinocytes (n=4). Effect of 0.32% of the extract according to the invention compared to the control:

[0510] [Table 20]

[0511]

[0512] These results show that the extract according to the invention significantly increases hyaluronic acid synthesis by keratinocytes.

[0513] 6.1.2.2. Strengthening the skin barrier

[0514] Keratinocytes migrate from the basal layer (the deeper layer of the epidermis) to the stratum corneum (the outermost layer) and differentiate into corneocytes. The stratum corneum is a semipermeable protective layer that prevents water loss and maintains skin hydration. Better differentiation of keratinocytes leads to a stronger skin barrier and thus helps to better protect the epidermis from external aggressions and helps to maintain better hydration.

[0515] plan

[0516] Human keratinocytes were cultured at subconfluence and then contacted with the extract according to the invention or not (for control case).Differentiation was monitored visually by observing the phenotype of the keratinocytes over 4 days.

[0517] result

[0518] [Table 21]

[0519]

[0520] The evaluation was performed visually. In the control, untreated cells showed a continuous layer of keratinocytes with well-defined cell outlines. On the other hand, in the presence of the extract according to the invention, the cells shrank and the intercellular contacts decreased until spaces appeared between the cells, an aspect characteristic of the late differentiation of keratinocytes.

[0521] These results show that peppermint extract contributes to better protection of the epidermis and better hydration of the skin.

[0522] 6.1.3. Antiseborrheic activity

[0523] Oily skin is associated with excessive sebum production by sebocytes. Excess sebum leads to changes in the properties of the skin and scalp, for example by increasing the formation of pimples and blackheads and clogging pores, which then become dilated and more visible, leading to uneven skin texture and / or overgrowth of dandruff-causing bacteria (such as Malassezia fungi on the scalp).

[0524] Anti-seborrheic cosmetic active ingredients will counteract this development by reducing sebum production, which will have the effect of tightening skin pores, smoothing the skin and reducing the oily / shiny appearance and irregular texture typical of oily skin and / or making the scalp healthier with less dandruff and the associated itching.

[0525] plan

[0526] Sebocytes were inoculated in their growth medium. At confluence, the cells were exposed to or not exposed to the extract according to the present invention (control) for 48 hours. After removal of the culture medium, the monolayer was incubated with a Nile Red marker for intracellular lipids, which estimates the amount of lipids in the cells. Viability was estimated in parallel on the same layer using a fluorescent dye.

[0527] result

[0528] Changes in lipid synthesis of sebaceous gland cells (n=3). Effect of 0.66% of the extract according to the invention compared with the control:

[0529] [Table 22]

[0530] Change (%); significance comparison refer to 0.66% of the extract according to the invention -61％；p<0.01

[0531] These results show that exposure of sebocytes to the extract according to the invention reduces the amount of lipids in sebum-producing cells.

[0532] The extract according to the invention can be used to treat skin disorders associated with oily skin.

[0533] 6.1.4. Weight Loss Activity

[0534] 6.1.4.1. Lipolysis

[0535] Glycerol is a product of triglyceride hydrolysis. Therefore, stimulating its production in the presence of the active ingredient reflects an increase in lipolysis. Therefore, increasing lipolysis will reduce the size of adipocytes and, therefore, reduce adipose tissue.

[0536] plan

[0537] Human preadipocytes are seeded and induced to differentiate with a specific cocktail of inducers. In order to obtain mature adipocytes loaded with triglycerides, the cells are contacted with the extract according to the invention in a maintenance medium.

[0538] The supernatant was then recovered.The amount of glycerol released from intracellular triglyceride hydrolysis was measured daily for 4 days using a commercial kit from Sigma.

[0539] Viability assays were performed in parallel by Hoechst staining to quantify cell numbers.

[0540] Visual inspection was performed before each recovery to verify the absence of toxicity.

[0541] result

[0542] Changes in the concentration of glycerol released by adipocytes after 4 days of contact with the extract according to the invention (n=3). Effect of 0.66% of the extract according to the invention compared with the control:

[0543] [Table 23]

[0544] Change (%); significance comparison refer to 0.66% of the extract according to the invention +152％；p<0.01

[0545] These results show that the extract according to the invention increases lipolysis in adipocytes.

[0546] 6.1.4.2. Anti-adipogenic effect

[0547] Glycerol-3-phosphate dehydrogenase (G3PDH) is a fat storage enzyme whose expression is strongly increased during the differentiation of preadipocyte fibroblasts into adipocytes. Therefore, its inhibition will reduce the amount of adipocytes and, therefore, adipose tissue.

[0548] plan

[0549] A hormone mixture was used to form mature human adipocytes from an immature state. During this phase, a series of extracts according to the present invention were administered. The increase in fat reserves was compared with a control by visual inspection and by measuring the activity of the fat storage enzyme (G3PDH). Viability tests were performed in parallel.

[0550] result

[0551] Changes in G3PDH activity in adipocytes during differentiation (n=3). Effect of 0.66% of the extract according to the invention compared with the control:

[0552] [Table 24]

[0553]

[0554]

[0555] These results show that the extract according to the invention significantly slows down the differentiation of adipocytes.

[0556] In conclusion, advantageously, the extract according to the invention is doubly effective, on the one hand increasing lipolysis and on the other hand slowing down lipogenesis. Thus, the extract has a strong slimming effect.

[0557] 6.1.5. Skin soothing activity

[0558] Inflammatory mediators such as IL-6 and PGE2 are often present in micro-inflammatory episodes. Reducing the presence of these mediators has the effect of reducing the discomfort felt by sensitive and reactive skin.

[0559] plan

[0560] Normal human dermal fibroblasts are grown in their culture medium until confluence is obtained. The cells are then contacted with the extract according to the present invention for 24 hours, and then UVB radiation is carried out to the cell mat (cellmat), and again contacted with the extract according to the present invention for 24 hours. The amount of PGE2 and IL-6 synthesized is measured in the culture supernatant by ELISA.

[0561] Cell number was assessed to weight the data obtained.

[0562] result

[0563] Changes in the amount of IL-6 and PGE2 in UVB-irradiated fibroblasts (n=3). The effect of 0.66% of the extract according to the invention was compared with the control:

[0564] [Table 25]

[0565]

[0566] Advantageously, the extract according to the invention strongly and significantly reduced both pro-inflammatory messengers tested.

[0567] Therefore, the extract according to the present invention can be used to relieve skin discomfort such as redness, tightness, etc. of sensitive skin.

[0568] 6.1.6. Conclusion

[0569] All the in vitro results presented above show that the peppermint extract prepared according to the process of the present invention advantageously possesses a very broad spectrum of biological activities and is particularly attractive in cosmetics.

[0570] The peppermint extract according to the present invention limits the reduction of the youthfulness of the cells by limiting the effects of oxidative stress induced from the outside. By limiting the production of proteases (elastase and MMP), and by promoting the production of the elements of the JDE (which are collagens-IV, VII, XVII, hyaluronic acid and laminin), the microenvironment of the cells is also protected. In addition, the peppermint extract according to the present invention acts by stimulating the production of elastin and collagen-I, which are elements in the dermal matrix that are known to be strongly affected by natural aging. This makes it possible to maintain and enhance the microenvironment of the cells near the DEJ and avoid disorders after the weakening of the DEJ.

[0571] Finally, the peppermint extract according to the invention helps keep the melanocytes alive and in good health, making it possible to guarantee high-quality pigmentation and thus limit the formation of dark and white spots that are common in the skin aging process.

[0572] 6.2. From lavender extract

[0573] The test protocol was the same as described for U.S. mint in point 6.1 above.

[0574] 6.2.1. Anti-aging activity on the skin

[0575] 6.2.1.1. Prevention of oxidative stress

[0576] result

[0577] Changes in ROS production in fibroblasts (n=3) with or without oxidative stress. Effect of 0.05% of the extract according to the invention compared to the control:

[0578] [Table 26]

[0579]

[0580] The results showed that the extract according to the present invention could significantly reduce the intracellular content of reactive oxygen species in fibroblasts that were subjected to or not subjected to oxidative stress.

[0581] Therefore, the lavender extract according to the present invention has strong antioxidant capacity and can effectively combat premature aging of the skin.

[0582] 6.2.1.2. Preventing glycation

[0583] result

[0584] Changes in saccharification. The effects of 0.05% of the extract according to the invention were compared with the control group:

[0585] [Table 27]

[0586]

[0587] These results show the strong anti-glycation potential of the active agents according to the invention, which can help combat skin aging and loss of complexion radiance.

[0588] 6.2.2. Moisturizing activity

[0589] 6.2.2.1. Hyaluronic acid

[0590] result

[0591] Changes in hyaluronic acid production in keratinocytes (n=5). Effect of 0.01% of the extract according to the invention compared to the control:

[0592] [Table 28]

[0593] Change (%); significance comparison refer to 0.01% of the extract according to the invention +185％；p<0.01

[0594] These results show that the extract according to the invention significantly increases hyaluronic acid synthesis by keratinocytes.

[0595] 6.2.3. Skin soothing activity

[0596] 6.2.3.1. Proinflammatory Messengers

[0597] result

[0598] Changes in the production of PGE2, IL-6 and IL-8 in UVB-irradiated fibroblasts. The effect of 0.0125% of the extract according to the invention was compared with the control:

[0599] [Table 29]

[0600]

[0601] Advantageously, the extract according to the invention strongly and significantly reduces the three pro-inflammatory messengers tested.

[0602] 6.2.3.2. Inhibition of cannabinoid 2 receptor (CB2)

[0603] Cannabinoid receptor 2 (CB2) is expressed in the skin, and its activation is associated with anti-inflammatory, antioxidant, seboregulatory, and immunomodulatory effects, but not psychoactive effects.

[0604] plan

[0605] An agonist with specific affinity for CB2 is radiolabeled. The extract according to the invention and the radiolabeled agonist are placed in the presence of a membrane containing CB2 receptors. Binding of the extract to the CB2 receptor is evaluated by comparing the radioactivity (binding of the agonist) in a control condition with that in the presence of the extract.

[0606] result

[0607] Changes in the inhibition of CB2 receptor binding. The effect of 0.05% of the extract according to the invention was compared with the control:

[0608] [Table 30]

[0609]

[0610] The results showed that the extract according to the invention binds to the CB2 receptor, but not the specific control.

[0611] Therefore, the extract according to the present invention can be used to relieve skin discomfort such as redness, tightness, etc. of sensitive skin.

[0612] 7. In vivo efficacy testing

[0613] As an example, the tests conducted involved the original cosmetic activity of peppermint extract prepared by in vitro cell culture according to the method of the present invention in treating aging spots.

[0614] 7.1. Principles and Protocols of In Vivo Testing

[0615] Skin spots (sometimes brown and sometimes white) are one of the first visible signs of skin aging. As explained above, the origin of these spots lies in the pigment cells of the skin: the melanocytes.

[0616] Brown spots are caused by an overproduction of melanin.

[0617] In contrast, vitiligo is caused by the absence of melanin. Furthermore, these spots have a degenerated matrix in the dermis and DEJ and a flattened surface, which makes the skin less flexible and more rigid.

[0618] Tested products

[0619] Cream as described in point 4. above.

[0620] plan

[0621] The effectiveness of the cream was evaluated in two studies conducted over a period of 56 days on a total of 52 volunteers compared to a placebo. These studies allowed the evaluation of the effect of the cream on various types of brown and white spots present on the volunteers' skin.

[0622] Specific inclusion criteria

[0623] The first study, conducted on a small group of 27 women (mean age 58 years [range 49-67]), included testing using a multispectral camera and testing using a photographic table.

[0624] The second study was conducted on a second group of 25 women (mean age 59 years [range 47-69]), including Test.

[0625] For both recruitment groups, the following criteria also had to be met: being in menopause or perimenopause, phototype II to IV, and having imperfect skin, i.e., distinct brown spots on the face and arms, and distinct white spots on the arms.

[0626] Study type, duration, administration

[0627] For two months, volunteers applied the cream according to the invention twice daily to the face and forearms and a placebo cream to the opposite sides.

[0628] statistics

[0629] For quantification of plaques, statistical studies were performed using the Student t-test or, if necessary, the nonparametric Wilcoxon test for paired series.

[0630] In the case of expert evaluation, the Khi2 test was used to compare response frequencies.

[0631] 7.2. Evaluation of brown and white spots using a multispectral camera

[0632] plan

[0633] Using Antera A multispectral LED camera provides three-dimensional images when exposed to different wavelengths of visible light. Facial photos are taken in cross-polarization mode to eliminate any parasitic light and improve the sharpness of the spots.

[0634] The system allows multiple analyses in parallel and thus provides multiple parameters:

[0635] - Color parameters L*, a* and b* (CIELAB color space);

[0636] - the relative amounts of chromophores such as hemoglobin and melanin;

[0637] - Different regional anatomical parameters such as wrinkles, texture or atrophic and hypertrophic volumes.

[0638] For brown spots, maximal pigmentation was studied, and pigmentation heterogeneity was monitored to quantify intramacular pigmentation variations.

[0639] The volume and maximum depth of the white patches were studied. In fact, the white patches form slightly concave depressions with a smooth surface and lack of a network of microdepressions. These characteristics are associated with the destruction of the underlying skin.

[0640] Of the panelists in the first group above, only 23 women had vitiligo comprising these features (ie, dimples and sufficiently noticeable flattening in the skin).

[0641] result

[0642] Changes in maximum pigmentation and unevenness of brown spots of the skin. Effects of the cream according to the invention:

[0643] [Table 31]

[0644]

[0645] Changes in the maximum depth and volume of white skin spots. Effects of the cream according to the invention:

[0646] [Table 32]

[0647]

[0648] The results of both tables show that the cream according to the invention significantly reduces the pigmentation and unevenness of the brown spots, as well as the depressions formed by the white spots, making both types of spots less noticeable and allowing a more even skin tone.

[0649] 7.3. Brown spot evaluation by photographic workbench

[0650] plan

[0651] A V05 photography stand (Orion Concept, France) was used for the facial photographs of the volunteers, which were taken in cross-polarization mode to eliminate any parasitic light. A panel of 6 expert judges evaluated these photographs according to the following criteria: more even skin and less pigmented.

[0652] result

[0653] The results showed a 64% favorable response to the cream according to the invention, which was significantly higher than the 35% response to the placebo cream.

[0654] 7.4 Evaluation of viscoelastic parameters of leukoplakia

[0655] plan

[0656] use (Courage & Khazaka) measures the viscoelastic parameters of the skin. This device measures the deformation of an area of ​​skin subjected to repeated mechanical suction pressure and its recovery force. The device provides a graph of the relationship between skin deformation and time, as shown in [ Figure 6 ]. Figure 6 ], the parameters Uf, Ue and Ua are indicated, representing the total elongation, immediate elongation and elongation recovered after stress, respectively.

[0657] The measurements were performed on areas with white spots. Three independent acquisitions were performed at T0 and T=56 days.

[0658] result

[0659] Changes in total elongation (Uf), immediate elongation (Ue) and recovery elongation (Ua) of white spots on the skin (N=25). Effects of the cream according to the present invention:

[0660] [Table 32]

[0661]

[0662]

[0663] *nsd: non-significant data

[0664] The results showed that all three parameters were better after 56 days of application of the cream containing the extract according to the invention.

[0665] In the case of the cream containing the extract according to the invention, the increase in Uf combined with the increase in Ue shows a better elongation of the skin, which results in a less hard, more deformable, more flexible skin at the level of white spots.

[0666] The concomitant increase in Ua shows that if the skin is deformed more, it also recovers better to its original state, which proves good elasticity.

[0667] Thus, the results show that after 56 days of application of the cream containing the extract according to the invention, the hypopigmented areas softened and recovered better.

[0668] The cream according to the invention makes it possible to improve the quality of the skin with regard to white spots, which will be less noticeable.

[0669] 7.5. Conclusion

[0670] All the in vivo results presented above show that the active ingredient of Mentha scabra according to the invention advantageously treats age spots. In fact, the active ingredient according to the invention acts on both types of spots (which are less noticeable) and allows a more even skin tone.

Claims

1. A method for obtaining a plant-derived extract by in vitro plant culture, comprising the following steps carried out sequentially from an undifferentiated or dedifferentiated plant cell line: - a pre-cultivation step aimed at amplifying the biomass of said plant cells; - a cultivation step of the biomass in a bioreactor, comprising at least one multiplication phase, and - a processing step of the harvested biomass to prepare said extract comprising the secondary metabolites of interest, wherein in the culturing step in the bioreactor, at least one aglycone flavonoid is added to the culture medium.

2. The method according to claim 1, wherein the aglycone flavonoid is selected from aglycone flavanones and / or aglycone flavones.

3. The method according to claim 2, wherein the aglycone flavanone is selected from naringenin, eriodictyol, buteflavone or a mixture thereof.

4. The method according to claim 2, wherein the aglycone flavonoid is selected from luteolin and apigenin or a mixture thereof. 5 . The method according to claim 1 , wherein the addition of the at least one aglycone flavonoid is carried out during the exponential phase of proliferation of the biomass.

6. Method according to one of the preceding claims, wherein induction is initiated during the cultivation step in the bioreactor.

7. The method according to claim 6, wherein the induction is chemical induction using an inducer of biological origin selected from the group consisting of chitosan, methyl jasmonate, jasmonic acid and salicylic acid.

8. The method according to claim 1 or 2, wherein the treating step comprises removing the culture medium.

9. Method according to any of the preceding claims, wherein the biomass treatment comprises a step of releasing intracellular contents outside the plant cells.

10. The method according to claim 9, wherein said step of releasing said intracellular contents is performed by osmotic diffusion or by cell lysis.

11. The method according to claim 9 or 10, wherein the treating step comprises eliminating cell debris after releasing the intracellular contents.

12. The method according to any one of the preceding claims, wherein the cell line of plant cells is prepared extemporaneously.

13. A method according to any one of the preceding claims, wherein it is applied to a plant of the Lamiaceae family.

14. The method according to claim 13, wherein the plant is selected from the group consisting of Monarda and / or Lavandula.

15. The method according to claim 14, wherein the plant is mint and / or lavender.

16. The method according to any one of claims 1 to 12, wherein it is applied to a plant of the family Mirabilis jalapa.

17. The method according to claim 16, wherein the plant is selected from the genus Scutellaria.

18. The method according to claim 17, wherein the plant is Verbena deserticola.

19. An extract of plant origin obtainable by a process according to any one of the preceding claims.

20. The plant-derived extract according to claim 19, wherein it is obtained from a plant of the Lamiaceae family, and wherein it contains glycosylated flavonoids and rosmarinic acid as secondary metabolites.

21. The extract according to claim 20, obtained from Mentha piperita, comprising glycosylated derivatives of naringin as secondary metabolites.

22. The extract according to claim 20, obtained from lavender, comprising a glycosylated derivative of luteolin as a secondary metabolite.

23. The plant-derived extract according to claim 19, wherein it is obtained from a plant of the family Mirabilis jalapa and wherein it comprises glycosylated flavonoids.

24. The extract according to claim 23, obtained from Verbena deserticola, comprising glycosylated derivatives of luteolin as secondary metabolites.

25. Cosmetic composition comprising as active ingredient an extract according to claims 17 to 24 and a physiologically acceptable medium.

26. Use of an extract according to any one of claims 17 to 24 or a composition according to claim 24 for non-therapeutic cosmetic treatments.

27. Use of the extract according to claim 26, wherein the non-therapeutic cosmetic treatment is topical.

28. Use of the extract according to claim 26 or 27, wherein the extract is a Mentha spp. extract and the treatment is selected from: - anti-aging treatments; and / or - anti-seborrheic treatment; and / or - moisturizing treatment; and / or - treatments to strengthen the skin barrier; and / or - weight loss treatment; and / or - Soothing treatment.

29. Use according to claim 28, wherein the anti-aging treatment is suitable for treating white and / or brown age spots on the skin.

30. Use according to claim 26 or 27, wherein the extract is a lavender extract and the treatment is selected from: - anti-aging treatments; and / or - moisturizing treatment; and / or - Soothing treatment.

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