Alcohol extract of white lily holy lilium, method for obtaining same, and cosmetic composition containing same

Abstract

The present invention relates to an extract of Lilium sieboldii Holy, a method for obtaining the extract and a cosmetic composition containing the extract, as well as different cosmetic uses.

Description

Technical Field

[0001] The present invention relates to an extract of Madonna lily (Lilium candidum L.), to a method for obtaining said extract, to a cosmetic composition containing said extract, and also to various cosmetic uses. Background Art

[0002] The skin mainly consists of three layers, namely, starting from the shallowest surface layer, the epidermis, the dermis, and the subcutaneous tissue.

[0003] The epidermis plays an important role in protecting the skin and maintaining its normal functions.

[0004] The aging and photoaging of the skin and the associated harmful changes can manifest in various ways, including:

[0005] - Loss of firmness and elasticity due to epidermal and / or dermal tissue loss;

[0006] - Loss of luster due to reduced microcirculation and slowed cell renewal in the epidermis;

[0007] - Appearance of pigment spots; and / or

[0008] - Skin dryness due to decreased stratum corneum barrier function and slowed epidermal renewal.

[0009] Therefore, there is a need to provide a multifunctional active agent that can act on a series of causes of harmful skin changes resulting from aging and / or changes in physiological mechanisms associated with aging. Summary of the Invention

[0010] In fact, the present applicant has now found that an alcoholic extract of Madonna lily (Lilium candidum L.) obtained by a specific method exhibits favorable activity in skin aging by stimulating or inhibiting physiological mechanisms, in particular having antioxidant potential through its protective properties against oxidative stress. Indeed, as demonstrated in the examples, the alcoholic extract of Madonna lily according to the present invention has favorable cosmetic properties: it can activate the biological pathway of "NRF2-mediated oxidative stress response". In addition, dermal aging is characterized in particular by the progressive differentiation of papillary fibroblasts into reticular fibroblasts, leading to dermal sclerosis. However, the alcoholic extract of Madonna lily according to the present invention can increase the expression of papillary markers CCRL1, NTN, and PDPN in reticular fibroblasts, while decreasing the expression of reticular markers CDH2, CNN, and MGP.

[0011] The extract according to the present invention also has an anti-skin aging effect because it can improve the morphology of the epidermis, especially by promoting epidermal thickening in an aged skin model reconstructed using reticular fibroblasts.

[0012] Thus, according to a first aspect, the present invention relates to an alcoholic extract of Lilium candidum, which alcoholic extract contains at least lilidine glycoside and pantothenic acid.

[0013] According to a second aspect, the present invention also relates to a method for extracting Lilium candidum, which method comprises at least the following steps:

[0014] a) Grinding Lilium candidum;

[0015] b) Extracting the ground flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, with a filtration step after each extraction;

[0016] c) Mixing the extracted liquid filtrates from step b), and allowing the resulting mixture to stand for at least 6 hours, preferably at least 10 hours;

[0017] d) Filtering the mixture obtained in c);

[0018] e) Removing the monohydric alcohol solvent from the mixture obtained in d), and performing a final dilution in another alcoholic solvent.

[0019] The present invention also relates to an alcoholic extract of Lilium candidum, which alcoholic extract can be obtained by such a method.

[0020] Furthermore, the present invention also relates to a cosmetic composition, which cosmetic composition contains the alcoholic extract of Lilium candidum according to the present invention in a cosmetically acceptable carrier. The term "cosmetically acceptable carrier" should be understood to mean a medium that is compatible with the skin, mucous membranes, and body surface growth. Preferably, the cosmetic composition according to the present invention is suitable for topical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Shows the relative expression (relative to the untreated condition) of papillary markers (CCRL1, NTN, PDPN) and reticular markers (CDH2, CNN, MGP) in reticular fibroblasts (ret) treated with 0.25% lily extract for 96 hours (n = 3 donors, D1 to D3).

[0022] Figure 2Shows the overall morphology of skin reconstructed with papillary fibroblasts (left) or reticular fibroblasts (middle and right), which were untreated (control, left and middle) or treated with 0.25% lily extract (right) (n = 3 donors, D1 to D3). White brackets indicate the differences in the observed viable epidermis thickness.

[0023] Figure 3 Shows the percentage of cells expressing the Ki67 proliferation marker in skin reconstructed with untreated papillary fibroblasts (control), and papillary fibroblasts treated with the control solvent (propylene glycol) or 0.25% lily extract (n = 3 donors, D1 to D3). Detailed implementation mode

[0024] The starting materials used consisted of fresh or dried flowers of white lily (Lilium candidum).

[0025] Lilium candidum is a herbaceous plant of the lily family, known as white lily or Madonna lily.

[0026] Madonna lily is a bulbous perennial of the lily family, with fleshy scales and can grow up to 1.8 meters tall.

[0027] The bulb produces basal leaves, forming a rosette.

[0028] These leaves are supported by a sturdy stem, are shiny, pointed, alternate and gradually decrease in size.

[0029] The spectacular flowering produces 5 to 10 trumpet-shaped, immaculate white flowers, 15 cm long, consisting of 6 large tepals, a long pistil and 6 long stamens. It flowers in June.

[0030] Madonna lily grows in France.

[0031] The dry extract of Lilium candidum, which is the subject of the present invention, consists of several families of primary and secondary metabolites of interest.

[0032] The alcoholic extract of the white lily Lilium candidum, which is the subject of the present invention, particularly contains at least lilicoside and pantothenic acid.

[0033] In particular, the alcoholic extract of Lilium candidum of the present invention contains:

[0034] - 40% to 70% by weight, preferably 50% to 60% by weight of saccharides,

[0035] - 1% to 5% by weight, preferably 2.5% to 3.2% by weight of compounds derived from di-O- and tri-O-glycosylated kaempferol,

[0036] - 8% to 15% by weight, preferably 10% to 13% by weight, of lilicoside, and

[0037] - 1% to 5% by weight, preferably 2% to 3% by weight, of pantethine,

[0038] The percentages are expressed by weight based on the total weight of the extract.

[0039] The di - O - and tri - O - glycosylated kaempferol derivatives are compounds having the following structure I (corresponding to kaempferol tri - O - glucopyranoside) or II (corresponding to kaempferol di - O - glucopyranoside):

[0040]

[0041] Lilicoside is a compound corresponding to the following formula III:

[0042]

[0043] Pantethine per se corresponds to the following structure IV:

[0044]

[0045] The saccharides present in the lily extract according to the invention preferably include glucose, fructose and / or sucrose, and in particular, 20% to 25% by weight of fructose, 17% to 21% by weight of glucose and 5% to 11% by weight of sucrose.

[0046] It is commendable that the applicant has successfully prepared a lily extract which particularly contains lilicoside, pantethine and derivatives of di - O - and tri - O - glycosylated kaempferol, thus conferring its specificity.

[0047] The alcoholic extract of Lilium candidum of the present invention can be particularly obtained by a method for extracting the white lily Lilium candidum, which method comprises the following steps:

[0048] a) Grinding the white lily Lilium candidum;

[0049] b) Extracting the ground flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, followed by a filtration step after each extraction;

[0050] c) Mixing the extracted liquid filtrates from step b), and allowing the resulting mixture to stand for at least 6 hours, preferably at least 10 hours;

[0051] d) Filtering the mixture obtained in c);

[0052] e) Remove the monohydric alcohol solvent from the mixture obtained in d), and perform a final dilution in another alcohol solvent.

[0053] Preferably, the white lily flowers used in step a) are fresh or dried flowers.

[0054] In particular, it is ground or crushed into pieces by any means known to those skilled in the art, for example, to a fineness of less than 2 cm.

[0055] In step b), the ground white lily flowers are extracted at least once, preferably at least twice, with one or more alcohol solvents, which are, for example, selected from:

[0056] - C1 - C4 monohydric alcohols, such as methanol, ethanol or isopropanol; and

[0057] - diols, such as propylene glycol, 1,3 - propanediol, or dipropylene glycol.

[0058] Preferably, the alcohol solvent is a monohydric alcohol containing 2 to 4 carbon atoms, more preferably ethanol.

[0059] Preferably, the alcohol solvent is used as a mixture with water at at least 90%, preferably at least 95%. Preferably, the alcohol solvent is used as a mixture with water in the range of 90% to 99%, preferably in the range of 95% to 99%.

[0060] Preferably, ethanol is used as a mixture with water at at least 90%, preferably at least 95%. Preferably, ethanol is used as a mixture with water in the range of 90% to 99%, preferably in the range of 95% to 99%.

[0061] In particular, ethanol is used as a 96% mixture with water.

[0062] The extraction is usually carried out by immersing the white lily flowers in one or more of the above - mentioned solvents at a temperature of 40°C to 60°C, preferably 45°C to 55°C, and gently stirring for a period of 1 hour to 6 hours.

[0063] After the extraction step b), filtration is carried out, for example, filtering through a 50 - μm sieve to remove plant residues.

[0064] According to a preferred embodiment, step b) is carried out with at least two extractions. In this case, a filtration step is carried out after each extraction.

[0065] At the end of the extraction step b), the liquid filtrates are recovered and mixed. Then the filtrate mixture is allowed to stand for at least 6 hours, preferably at least 10 hours.

[0066] The filtrate or filtrate mixture obtained at the end of step c) is then filtered again to remove insoluble matter: this is step d). Preferably, the filtration of the extract obtained in c) is carried out on a 4 μm filter sieve or filter membrane. A liquid filtrate is thus obtained.

[0067] Finally, the solvent present in the liquid filtrate is removed and the remaining part of the filtrate is diluted with another alcohol solvent (step e)).

[0068] The order of the steps of removing the solvent and diluting the filtrate with another solvent is interchangeable. In particular, the solvent present in the liquid filtrate can be removed and the remaining part of the filtrate is then diluted with another alcohol solvent. Alternatively, the liquid filtrate is first diluted with another alcohol solvent and then the solvent is removed. Furthermore, alternatively, the dilution of the filtrate and the removal of the solvent can be simultaneous. The alcohol solvent used in step e) is referred to as "other alcohol solvent" because it is different from the alcohol solvent used in step a). Taking this limitation into account, the alcohol solvent is typically selected from the same group as step a), i.e., selected from C1-C4 monohydric alcohols and diols.

[0069] Preferably, the final dilution is carried out in a diol, preferably in 1,3-propylene glycol. Preferably, the removal of the solvent in step e) is carried out by evaporation, in particular by vacuum evaporation.

[0070] Preferably, between steps d) and e), a step of decolorizing the filtrate obtained in d) is added. The decolorization can be performed by adsorption of the pigment present in the filtrate by activated carbon. The decolorization step can be followed by a step of filtering the decolorized filtrate obtained, in particular filtering to 1 μm.

[0071] Preferably, the alcohol extract of Lilium can be obtained by a method comprising the following steps:

[0072] a) Grind white lily flowers (Lilium Madonna);

[0073] b) subjecting said ground flower to at least one, preferably at least two, extractions in the presence of an alcoholic solvent, each extraction being followed by a filtration step;

[0074] c) mixing at least two liquid filtrates from the two extractions of step b) and leaving the resulting mixture to stand for at least 6 hours, preferably at least 10 hours;

[0075] d) filtering the mixture obtained in c) to obtain a filtrate;

[0076] - decolorizing the filtrate obtained in d) by adsorption on activated carbon; then

[0077] - filtering the decolorized filtrate on a 1 μm membrane; and

[0078] e) Ethanol is removed from the resulting filtrate by evaporation and then final dilution is carried out with 1,3 - propanediol.

[0079] Advantageously, the extract used according to the invention is light - colored.

[0080] Furthermore, the extract is in a sufficiently concentrated form so as not to cause formulation problems (which usually occur at the concentrations required to obtain activity in emulsion - type cosmetic or dermatological compositions) when used, and is not dark - colored, which is different from plant extracts obtained by conventional methods when in concentrated form.

[0081] In particular, after evaporation of the ethanol, the extract of Lilium candidum contains from 15% to 25% by weight of the dry extract of lily and from 75% to 85% by weight of 1,3 - propanediol, preferably approximately 20% by weight of the dry extract of lily and 80% by weight of 1,3 - propanediol.

[0082] Thus, the extract according to the invention can be used directly for the preparation of cosmetic compositions.

[0083] According to another aspect, the invention relates to the cosmetic use of the alcoholic extract of Lilium candidum according to the invention as an antioxidant and / or anti - aging agent.

[0084] According to yet another aspect, the invention relates to the cosmetic use of the alcoholic extract of Lilium candidum (Lilium candidum) according to the invention for the prevention and / or reduction of harmful skin changes caused by aging. Furthermore, the extract is particularly used as a reagent for activating the NRF2 - mediated oxidative stress response and / or as a reagent for promoting epidermal thickening.

[0085] In fact, it has been advantageously found that the alcoholic extract of Lilium candidum according to the invention has a plurality of interesting activities related to physiological mechanisms associated with the prevention or repair of harmful skin changes (especially those caused by aging).

[0086] In particular, it has been advantageously found that the alcoholic extract of Lilium candidum according to the invention can increase the expression of the papillary markers CCRL1, NTN and PDPN in reticular fibroblasts while decreasing the expression of the reticular markers CDH2, CNN and MGP.

[0087] The extract according to the invention can also improve the morphology of the epidermis, in particular by promoting epidermal thickening in an in vitro model of aged skin reconstructed using reticular fibroblasts.

[0088] According to another aspect, the present invention also relates to a cosmetic composition comprising an alcoholic extract of *Lilium candidum* according to the present invention in a cosmetically acceptable carrier. Preferably, the extract is present in the cosmetic or skin composition in a proportion of from 0.001% to 10% by weight, especially from 0.01% to 10% by weight, preferably from 0.1% to 10% by weight, relative to the total weight of the composition. The cosmetic composition is particularly suitable for topical application.

[0089] Advantageously, the cosmetic composition may be in the form of a powder, an emulsion, a microemulsion, a nanoemulsion, a suspension, a lotion solution, a cream, a gel (which may be aqueous or hydroalcoholic), a foam, a serum, an aerosol solution or dispersion, or a lipid vesicle dispersion.

[0090] In the case of an emulsion, it may be a water-in-oil or an oil-in-water emulsion.

[0091] The cosmetic composition according to the present invention may also comprise a solvent selected according to the various ingredients and forms of application.

[0092] By way of example, mention may be made of water (preferably demineralized water or floral water) or an alcohol such as ethanol.

[0093] In addition to the extract according to the present invention, the cosmetic composition may also comprise at least one additive commonly used in the art, such as at least one compound selected from the following: emollients or humectants, gelling agents and / or thickening agents, surfactants, oils, active agents, dyes, preservatives, antioxidants, active agents, organic or inorganic powders, sunscreens and fragrances;

[0094] - one or more humectants such as polyols (glycerol, diglycerol, propylene glycol, propanediol, octyldecanol, pentylene glycol, hexylene glycol), sugars, glycosaminoglycans such as hyaluronic acid, and their salts and esters; and polyquaternium salts such as Lipidure PMB.

[0095] The humectant will be present in the composition in an amount of from about 0.1% to 30%, preferably from 0.005% to 10%, by weight of the total weight of the composition;

[0096] - One or more emollients, which may be selected from, for example, esters (such as jojoba esters, fatty acid esters of fatty alcohols (octyldodecyl myristate, glyceryl triethylhexanoate, dicaprylyl carbonate, isostearyl isostearate, triglycerin caprylate / caprate)), butters such as shea butter (butyrospermum parkii fruit butter extract, shea butter ethyl ester, sold under the names Lipex Sheasoft, Lipex Shea-U, Lipex Shea, Lipex Shealight, Lipex Shea Tris) or moringa butter (moringa oil / hydrogenated moringa oil ester), waxes (acacia flower wax and sunflower seed wax, C10-18 triglycerides), vegetable oils, phytosqualene, alkanes (undecane, tridecane).

[0097] The emollient(s) will be present in the composition in an amount of about 0.1% to 30%, preferably 0.5% to 10%, based on the total weight of the composition;

[0098] - One or more gelling agents and / or thickening agents for the aqueous phase, which are selected from, for example, cellulose derivatives, gums of plant origin (guar gum, carob gum, alginates, carrageenans, pectin), gums of microbial origin (xanthan gum), clays (laponite), acryloyl methylpropanesulfonic acid (amps) and / or acrylamide and / or acrylic acid and / or salts or esters of acrylic acid, hydrophilic or amphiphilic crosslinked or non-crosslinked homopolymers and copolymers (sold under the names Aristoflex AVC, Aristoflex AVS, Aristoflex HMB, Simulgel NS, Simulgel EG, Simulgel 600, Simulgel 800, Pemulen, carbopol, Sepiplus400, Seppimax zen, Sepiplus S, Cosmedia SP).

[0099] The gelling agent(s) and / or thickening agent(s) will be present in the composition in an amount of about 0.1% to 10%, based on the total weight of the composition;

[0100] - One or more surfactants, especially

[0101] * Anionic surfactants such as hydroxyethyl sulfonates, taurates, sarcosinates, glycinate, glutamate, phosphates (C20-22 alkyl phosphates, sold under the name Sensanov WR),

[0102] * Amphoteric surfactants such as betaine derivatives, amphoacetates,

[0103] Non-ionic surfactants, such as polyglycerol derivatives, sugar derivatives (glucoside or xyloside derivatives, sold under the names Montanov 68, Montanov 202, Montanov 82, Montanov L, Easynov), lecithin.

[0104] The surfactant will be present in an amount of about 0.1% to 15% by weight, preferably 0.5% to 10% by weight, relative to the total weight of the composition;

[0105] - One or more active agents of natural, biotechnological or synthetic origin that are biologically active and effective on the skin via biological sites, such as selected from: vitamins such as vitamin C and its derivatives (ascorbic acid glucoside, 3-o-ethyl ascorbic acid, tetrahexyldecyl ascorbate), vitamin A and its derivatives, vitamin E and its derivatives, vitamin B3 or niacinamide, panthenol, trace elements, allantoin, adenosine, peptides (palmitoyl tetrapeptide-7, palmitoyl tripeptide-1, palmitoyl pentapeptide-4, acetyl dipeptide-1 cetyl ester, acetyl tetrapeptide-5, sold under the names NP Rigin, Matrixyl 3000, Idealift, Eyeseryl), plant extracts (Glycyrrhiza glabra extract, Centella asiatica leaf extract, Secale cereale seed extract), yeast extract, alpha-hydroxy acids such as glycolic acid or lactic acid, tranexamic acid and its derivatives such as cetyl tranexamate, etc.

[0106] The active agent will be present in the composition in an amount of about 0.1% to 10% by weight of the total weight of the composition;

[0107] Other additives commonly used in cosmetics may also be present in the composition according to the invention, in particular preservatives, antioxidants or fragrances well known in the art.

[0108] A person skilled in the art is able to select both the nature and the amount of those additives to be added to the composition from all these possible additives such that the composition retains all its properties.

[0109] The present invention is illustrated in a non-limiting manner by the following examples.

[0110] Example 1: Preparation of an ethanol extract of Lilium candidum L. according to the present invention

[0111] An alcoholic extract of Lilium candidum according to the invention is prepared by a method comprising the following steps:

[0112] a) Grind 200 g of Lilium candidum into a powder;

[0113] b) Extract the powder with 2000 g of 96° ethanol with stirring at 50 °C for 2 hours;

[0114] Screen the resulting mixture 1 through a 50-μm sieve: reserve filtrate 1;

[0115] Extract the spent brewer's grains with 2000 g of 96° ethanol while stirring at 50 °C for 2 hours;

[0116] Screen the resulting mixture 2 through a 50-μm sieve: discard the spent brewer's grains;

[0117] c) Add filtrate 2 to filtrate 1 and let the mixture stand overnight;

[0118] d) Filter the supernatant through a 4-μm filter;

[0119] - Take 3 x 5 g of the extract and place it at 110 °C for 3 hours for solid determination;

[0120] - After adding 20% activated carbon relative to the dry extract, decolorize the filtrate with stirring at ambient temperature for 3 hours;

[0121] - Filter through 4-μm and 1-μm membranes: remove the activated carbon;

[0122] e) Add 180 g of 1,3-propanediol and stir for half an hour; and evaporate the ethanol in a rotary evaporator;

[0123] Perform a final filtration through a 4-μm filter.

[0124] Example 2: Testing of the lily extract according to the present invention for antioxidant stress properties

[0125] Protocol: Inoculate normal human epidermal keratinocytes from two young donors into 6-well plates and culture them in a medium supplemented with KGM2 (Lonza) at 37 °C and 5% CO2 for 72 hours. Then incubate the cells with 0.25% extract for 24 hours, or without incubation (untreated condition). Each condition is performed in duplicate. According to the supplier's recommendations, use the RNeasy 96 Plate Kit (Qiagen) to extract total RNA. Evaluate the quantity and quality of the RNA by MultiskanGo (Thermo Fischer). Synthesize complementary DNA and generate a transcriptome on the Affymetrix GeneChip Human Transcriptome Array 2.0. Perform bioinformatics analysis on genes that are expressed at least 2-fold regulated (FC: fold change, expression level relative to the untreated condition) using Ingenuity Pathway Analysis software ( Qiagen).

[0126] Results: After 24 hours of treatment with 0.25% extract, the extract was able to modulate the expression of 158 genes in normal human keratinocytes on average (the expression of 59 genes was inhibited and the expression of 99 genes was stimulated).

[0127] IPA software was used to evaluate the changes in biological activity after treatment with the extract. A map representing the regulation of the expression of various biological pathways was established (data not shown). The transcriptome map obtained after 24 hours of treatment showed that 0.25% extract modulated many biological functions. In particular, Lilium candidum was able to modulate 64 canonical signaling pathways.

[0128] Among the biological pathways significantly activated by 0.25% lily extract was the NRF2 (nuclear factor-erythroid 2-related factor-2)-mediated oxidative stress response pathway. NRF2 is a transcription factor that, once activated, can increase the expression of antioxidant enzymes such as GCLM, GPX, GST, NQO1, SOD, and TXNR. NRF2 also activates the transcription of non-enzymatic antioxidant proteins (such as ferritin, especially ferritin composed of FTL and FTH1 subunits). After treatment with 0.25% extract for 24 hours, this biological pathway for overall cellular detoxification was stimulated in normal keratinocytes, endowing them with antioxidant and anti-aging properties, as oxidative stress causes skin aging.

[0129] Table I: List of genes involved in the "NRF2-mediated oxidative stress response" biological pathway, whose expression was significantly stimulated by 0.25% lily extract. NRF2: nuclear factor-erythroid 2-related factor-2, FC: expression level relative to the untreated condition

[0130]

[0131] Example 3: Anti-aging properties of the lily extract according to the present invention in cellular and tissue models of skin aging Properties

[0132] At the cellular level, the dermis can be divided into two fibroblast subpopulations: papillary fibroblasts (located at the papillary layer level under the epidermis and having a younger phenotype) and reticular fibroblasts (deeper and more differentiated, presenting an older phenotype). These two subpopulations express specific markers: CCRL1, NTN, and PDPN for papillary fibroblasts, while CDH2, CNN, and MGP for reticular fibroblasts. The aging characteristics of the dermis are particularly characterized by the progressive differentiation of papillary fibroblasts into reticular fibroblasts, resulting in dermal sclerosis. This aging can be modeled in the laboratory, at the cellular level using reticular fibroblasts, and at the tissue level by reconstructing equivalent skin with dermis synthesized by reticular fibroblasts. Therefore, these models can be used to identify components with anti-aging properties that can reverse skin aging.

[0133] Cell model protocol: Normal human dermal reticular fibroblasts from three donors were seeded in 6-well plates and cultured in DMEM medium supplemented with 5% serum at 37 °C and 5% CO2 for 72 hours. The cells were then incubated with 0.25% extract in DMEM medium supplemented with 2.5% serum for 96 hours, or without incubation (untreated condition). Total RNA was extracted using the RNeasy 96-well plate extraction kit (Qiagen) according to the supplier's recommendations. The quantity and quality of the RNA were evaluated by Experion (Biorad). Complementary DNA was synthesized (iScript kit, Biorad), and then qPCR reactions were performed using the SYBR Green method (Biorad) according to the following program: 5 minutes at 95 °C, followed by 35 cycles of 20 seconds at 95 °C, 20 seconds at 60 °C, and 20 seconds at 72 °C, followed by generation of a melting curve. Expression analysis of stably expressed reference genes was performed using the ΔΔCt method with iQ5 software (Biorad). Thus, the expression of biomarkers specific for papillary fibroblasts (CCRL1, NTN, and PDPN) and biomarkers specific for reticular fibroblasts (CDH2, CNN, and MGP) in the samples was evaluated.

[0134] Results: As Figure 1 shown, after treatment with 0.25% extract for 96 hours, the extract was able to increase the expression of papillary markers CCRL1, NTN, and PDPN in reticular fibroblasts and was also able to decrease the expression of reticular markers CDH2, CNN, and MGP. Thus, the lily extract has the ability to reverse the senescent phenotype of reticular fibroblasts to a younger phenotype (close to that of papillary fibroblasts).

[0135] Tissue model protocol: Normal human dermal papillary or reticular fibroblasts from three donors were seeded and cultured in DMEM medium supplemented with 5% serum (HyClone) at 37 °C and 5% CO2 for 5 days. The cells were then incubated for 4 days with 0.25% extract or 0.25% propylene glycol solvent (diluent condition only), or without incubation (untreated condition). Dermal equivalents were generated with the treated or control cells, and these dermal equivalents were then incubated again for 7 days at 37 °C and 5% CO2 in DMEM medium supplemented with 5% serum and 1% penicillin-streptomycin (Invitrogen) with 0.25% lily extract or 0.25% propylene glycol, or without incubation (untreated condition). Then, normal human epidermal keratinocytes were seeded onto the dermal equivalents, and the cultures were maintained in DMEM / Ham's F12 (3:1) medium supplemented with 5% serum, 1.1 μM hydrocortisone, 1 μM isoproterenol, 0.1 μM insulin (Sigma-Aldrich) and 1% penicillin-streptomycin (Invitrogen) for 2 days. The skin reconstructed with papillary fibroblasts (Pap) or reticular fibroblasts (Ret) was placed at the air / liquid interface and re-treated according to the above conditions for 14 days or not treated, after which it was fixed, dehydrated and then embedded in paraffin. Histological sections were then cut and used for hematoxylin-eosin-saffron (HES) staining or Ki67 immunolabeling.

[0136] Hematoxylin-eosin-saffron (HES) staining was performed to observe their overall morphology, especially the epidermal thickness, which becomes thinner in the aged reticular skin (relative to the "young" papillary skin) model6. Immunolabeling of the cell proliferation marker Ki67 was also performed to count the positive cells in the skin reconstructed with papillary fibroblasts. Antigen demasking was performed in citrate buffer at pH 6, followed by incubation in a blocking solution to prevent non-specific binding of the antibody. The sections were then incubated overnight at 4 °C with a primary antibody (MIB-1, Dako) specifically targeting Ki67 according to the supplier's (GE Healthcare) recommendations, and then incubated with an avidin-biotin-peroxidase system. Labeling was visualized by adding AEC (3-amino-9-ethylcarbazole), and the sections were counterstained with hematoxylin.

[0137] Results: As Figure 2As shown, in an aged skin model reconstructed with reticular fibroblasts, compared to the untreated condition (middle), 0.25% of the lily extract according to the present invention is able to improve the morphology of the epidermis, particularly by promoting epidermal thickening (right). Thus, the skin treated with the extract resumes a younger morphology, approaching that presented by the skin reconstructed with untreated papillary fibroblasts (left).

[0138] As Figure 3 shown, 0.25% of the extract is also able to increase the number of cells expressing the Ki67 proliferation marker in the basal layer of the skin reconstructed with papillary fibroblasts. Thus, it promotes cell renewal.

[0139] Therefore, 0.25% of the lily extract is able to slow down dermal aging in cell and tissue models by changing the reticular phenotype to a younger papillary phenotype. Thus, it has strong anti-aging properties.

[0140] Example 4: Cosmetic composition

[0141] The following compositions can be prepared in a conventional manner by those skilled in the art. The amounts indicated below are expressed as weight percentages. The ingredients indicated in capital letters are identified according to the INCI name.

[0142] A – oil / water gel emulsion

[0143]

[0144] b – oil / water cream emulsion

[0145]

[0146]

[0147] These compositions can be applied to the skin every morning and / or evening.

Claims

1. An alcoholic extract of white lily (Lilium candidum), said alcoholic extract containing at least lilicoside and pantothenic acid.

2. The alcoholic extract of white lily according to claim 1, said alcoholic extract containing: - from 40% to 70% by weight, preferably from 50% to 60% by weight, of saccharides, - from 1% to 5% by weight, preferably from 2.5% to 3.2% by weight, of compounds derived from di-O- and tri-O-glycosylated kaempferol, - from 8% to 15% by weight, preferably from 10% to 13% by weight, of lilicoside, and - from 1% to 5% by weight, preferably from 2% to 3% by weight, of pantothenic acid, the percentages being expressed by weight based on the total weight of the extract.

3. The alcoholic extract of white lily according to any one of the preceding claims, wherein said saccharides comprise glucose, fructose and / or sucrose, preferably from 20% to 25% by weight of fructose, from 17% to 21% by weight of glucose and from 5% to 11% by weight of sucrose.

4. A method for extracting white lily (Lilium candidum), said method comprising the following steps: a) grinding white lily (Lilium candidum); b) performing at least one, preferably at least two, extractions on the ground flowers in the presence of an alcoholic solvent, each extraction being followed by a filtration step; c) mixing the extracted liquid filtrates from step b) and allowing the resulting mixture to stand for at least 6 hours, preferably at least 10 hours; d) filtering the mixture obtained in c); e) removing the monohydric alcohol solvent from the mixture obtained in d) and then performing a final dilution in another alcoholic solvent.

5. The method according to claim 4, wherein The white lily used in step a) is fresh or dried flowers.

6. The method according to claim 4 or 5, characterized in that, In step a), the white lilies are ground to a fineness of less than 2 cm.

7. The method according to any one of claims 4 to 6, characterized in that The alcoholic solvent in step b) is a monohydric alcohol containing 1 to 4 carbon atoms, preferably ethanol.

8. The method according to any one of claims 4 to 7, characterized in that The extraction in step b) is carried out for a period of 1 to 6 hours at a temperature of 40°C to 60°C, preferably 45°C to 55°C.

9. The method according to any one of claims 4 to 8, characterized in that After each extraction in step b), filtration is carried out on a 50 μm sieve.

10. The method according to any one of claims 4 to 9, characterized in that The filtration carried out in step d) is carried out on a 4 μm filter sieve or filter membrane.

11. The method according to any one of claims 4 to 10, characterized in that, Between steps d) and e), a step of decolorizing the filtrate obtained in d), preferably by adsorption on activated carbon, is added, followed by a step of filtering the obtained decolorized filtrate, preferably filtering to 1 μm.

12. The method according to any one of claims 4 to 11, wherein The removal in step e) is carried out by evaporation and then the final dilution is carried out in 1,3-propanediol.

13. An alcohol extract of Madonna lily (Lilium candidum), characterized in that The alcoholic extract can be obtained by the method according to any one of claims 4 to 12.

14. Cosmetic use of the alcoholic extract of white lily (Lilium candidum) according to any one of claims 1 to 3 as an antioxidant, and / or for preventing and / or reducing harmful skin changes caused by aging, and / or as a reagent for activating the NRF2-mediated oxidative stress response, and / or as a reagent for promoting epidermal thickening.

15. A cosmetic composition comprising the alcoholic extract of white lily (Lilium candidum) according to any one of claims 1 to 3 in a cosmetically acceptable carrier.

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