Alcoholic extract of white lily flower Lilium candidum L., method for obtaining same, and cosmetic composition containing same

Abstract

The present invention relates to an extract of the white lily flower Lilium candidum L, a process for obtaining it, a cosmetic composition containing it, and various cosmetic uses.

Description

[Technical Field]

[0001] The present invention relates to an extract of the flower of the white lily (Lilium candidum L.), a process for obtaining it, cosmetic compositions containing it, and various cosmetic uses. [Background technology]

[0002] The skin is mainly composed of three layers, namely, the epidermis, the dermis, and the subcutaneous tissue, starting from the outermost layer. The epidermis plays a major role in protecting the skin and maintaining its proper functioning. Skin aging and photoaging and the associated deleterious changes can manifest themselves in a variety of ways, including: - Loss of firmness and elasticity due to tissue loss in the epidermis and / or dermis; - Loss of radiance due to reduced microcirculation and slower cell renewal of the epidermis; - the appearance of pigmented spots; and / or - Dry skin caused by a weakened barrier function of the stratum corneum and slower renewal of the epidermis.

[0003] Therefore, there is a need to provide multifunctional active agents that can act on the multiple causes of adverse skin changes due to aging and / or changes in physiological mechanisms associated with aging. Summary of the Invention [Means for solving the problem]

[0004] The present applicant has now discovered that an alcoholic extract of the flowers of the white lily, Lilium candidum L., obtained by a specific method, exhibits beneficial activity in terms of skin aging by stimulating or inhibiting physiological mechanisms, in particular by providing antioxidant capacity and thus protecting against oxidative stress. Indeed, as demonstrated in the examples, the alcoholic extract of the flowers of the white lily, Lilium candidum L., according to the present invention, possesses the advantageous cosmetic property of being able to activate the biological pathway of the "NRF2-mediated oxidative stress response." Additionally, dermal aging is characterized, in particular, by the progressive differentiation of papillary fibroblasts into reticular fibroblasts, resulting in dermal hardening. However, the alcoholic extract of the flowers of white lily, Lilium candidum L., according to the present invention, can increase the expression of papilla markers CCRL1, NTN, and PDPN in reticular fibroblasts, and decrease the expression of reticular markers CDH2, CNN, and MGP. Furthermore, the extract according to the present invention has an anti-aging effect on the skin, since it is possible to improve the morphology of the epidermis, in particular by promoting epidermal thickening, in a model of aged skin reconstructed with reticular fibroblasts.

[0005] Thus, according to a first aspect, the present invention relates to an alcoholic extract of the flowers of the white lily, Lilium candidum L., comprising at least lilysine glycosides and hopantenic acid.

[0006] According to a second aspect, the present invention also relates to a method for extracting flowers of the white lily, Lilium candidum L., comprising at least the following steps: a) crushing flowers of the white lily, Lilium candidum L.; b) extracting the crushed flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, filtering after each extraction; c) mixing the filtrate from the extraction of 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 monoalcohol solvent from the mixture obtained in d) and final dilution with another alcohol solvent.

[0007] The present invention also relates to an alcoholic extract of flowers of the white lily, Lilium candidum L., obtainable by such a method.

[0008] Additionally, the present invention also relates to a cosmetic composition comprising an alcoholic extract of flowers of the white lily, Lilium candidum L., according to the present invention, in a cosmetically acceptable carrier. The term "cosmetically acceptable carrier" is understood to mean a medium that is compatible with the skin, mucous membranes, and superficial body growths. Preferably, the cosmetic composition according to the present invention is suitable for topical application. [Brief explanation of the drawings]

[0009] [Figure 1] Illustrated is the relative expression (vs. untreated condition) of papilla 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-D3). [Figure 2] Figure 1 illustrates the overall morphology of skin reconstructed with papillary fibroblasts (left) or reticular fibroblasts (center and right) that was untreated (control, left and center) or treated with 0.25% lily extract (right) in n=3 donors (D1-D3). White brackets illustrate the observed differences in viable epidermal thickness. [Figure 3]Shown is the percentage of cells expressing the Ki67 proliferation marker in reconstructed skin containing untreated papillary fibroblasts (control) and papillary fibroblasts treated with control solvent (propanediol) or 0.25% lily extract (n=3 donors D1-D3). DETAILED DESCRIPTION OF THE INVENTION

[0010] The starting material used consists of fresh or dried flowers of the white lily, Lilium candidum L. Lilium candidum L. is a herbaceous plant of the Liliaceae family, also known as the white lily or Madonna lily. The Madonna lily is a bulbous perennial plant of the lily family with fleshy shoot scales that can grow up to 1.8 m in height. Basal leaves appear on the bulbs, forming a rosette. The leaves on sturdy stems are glossy, pointed, alternate and small in size. The spectacular flowering period produces 5-10 trumpet-shaped pure white flowers, 15 cm long, each consisting of six large tepals, a long pistil, and six long stamens. Flowering occurs in June. Madonna lilies are cultivated in France.

[0011] The dry extract of Lilium candidum L., the subject of the present invention, is composed of several families of primary and secondary metabolites of interest. The alcoholic extract of the flowers of the white lily, Lilium candidum L., which is the subject of the present invention, contains in particular at least lilysine glycosides and hopantenic acid. In particular, the alcohol extract of Lilium candidum L. of the present invention has the following properties: - 40 to 70% by weight, preferably 50 to 60% by weight, of sugars, - 1 to 5% by weight, preferably 2.5 to 3.2% by weight, of compounds derived from di-O- and tri-O-glycosylated kaempferol, - 8 to 15% by weight, preferably 10 to 13% by weight, of lilysine glycoside, and 1 to 5% by weight, preferably 2 to 3% by weight, of hopantenic acid Including, Percentages are expressed as % by weight based on the total weight of the extract. Di-O- and tri-O-glycosylated kaempferol derivatives are compounds of the following structure I (corresponding to kaempferol tri-O-glycopyranoside) or II (corresponding to kaempferol di-O-glycopyranoside): [ka] and Liridin glycosides are compounds corresponding to formula III: [ka] and Hopantenic acid, for its part, corresponds to the following structure IV: [ka]

[0012] The sugars present in the lily extract according to the present invention preferably contain glucose, fructose and / or sucrose, and in particular 20-25% by weight of fructose, 17-21% by weight of glucose and 5-11% by weight of sucrose. It will be appreciated that the applicant is responsible for the success in preparing an extract of lily containing, in particular, lilysin glycosides, hopantenic acid and, preferentially, di-O- and tri-O-glycosylated kaempferol derivatives, thereby obtaining its particularity. The alcoholic extract of Lilium candidum L. according to the invention can in particular be obtained by a method for extracting the flowers of the white lily, Lilium candidum L., comprising the following steps: a) crushing flowers of the white lily, Lilium candidum L.; b) extracting the crushed flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, filtering after each extraction; c) mixing the filtrate from the extraction of 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 monoalcohol solvent from the mixture obtained in d) and final dilution with another alcohol solvent.

[0013] Preferably, the white lily flowers used in step a) are fresh or dried flowers. The white lily flowers are in particular crushed or pulverized by any means known to those skilled in the art, for example to a fine particle size of less than 2 cm. In step b), the crushed white lily flowers are subjected to at least one, preferably at least two, extractions, for example with one or more alcoholic solvents selected from: C1-C4 monoalcohols, such as, for example, methanol, ethanol or isopropanol; and diols, such as, for example, propylene glycol, 1,3-propanediol or dipropylene glycol. Preferably, the alcohol solvent is a monoalcohol containing 2 to 4 carbon atoms, more preferentially ethanol. Preferably, the alcohol solvent is used as a mixture with at least 90% water, preferably at least 95% water, and preferably 90% to 99% water, preferably 95% to 99% water. Preferably, ethanol is used as a mixture with at least 90% water, preferably at least 95% water. Preferably, ethanol is used as a mixture with 90% to 99% water, preferably 95% to 99% water. In particular, ethanol is used as a mixture with 96% water.

[0014] Extraction is generally carried out by immersing 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, for a period of 1 hour to 6 hours, with gentle stirring. The extraction step b) is followed by filtration, for example through a 50 μm screen, to remove plant residues. According to a preferred embodiment, step b) comprises carrying out at least two extractions, each of which is followed by a filtration step. At the end of extraction step b), the filtrate is collected and mixed, and the filtrate mixture is then left to stand for at least 6 hours, preferably at least 10 hours.

[0015] The filtrate or filtrate mixture obtained at the end of step c) is then filtered again to remove insoluble matter. This step is step d). Preferably, the filtration of the extract obtained in c) is carried out through a 4 μm filter screen or membrane. Thus, a filtrate is obtained.

[0016] Finally, the solvent present in the filtrate is removed and the remainder of this filtrate is diluted with another alcoholic solvent (step e)). The order of the solvent removal step and the step of diluting the filtrate with another solvent can be reversed. In particular, the solvent present in the filtrate can be removed, and then the remainder of the filtrate can be diluted with another alcoholic solvent. Alternatively, the filtrate can first be diluted with another alcoholic solvent, and then the solvent can be removed. In this case, too, the dilution of the filtrate and the removal of the solvent can be performed simultaneously. The alcoholic solvent used in step e) is different from the alcoholic solvent used in step a), and is therefore understood as "another alcoholic solvent." Taking this restriction into account, the alcoholic solvent is typically selected from the same group as the alcoholic solvent in step a), i.e., C1-C4 monoalcohols and diols. Preferably, the final dilution is carried out in a diol, preferably 1,3-propanediol. Preferably, the removal of the solvent in step e) is carried out by evaporation, in particular vacuum evaporation.

[0017] Preferably, between steps d) and e), a step of decolorizing the filtrate obtained in d) is added. This can be done by adsorbing the pigments present in the filtrate onto activated carbon. This decolorizing step can be followed by a step of filtering the decolorized filtrate obtained, in particular to a 1 μm filter.

[0018] Preferentially, the alcoholic extract of flowers of the white lily, Lilium candidum L., according to the invention can be obtained by a method comprising the following steps: a) crushing flowers of the white lily, Lilium candidum L.; b) extracting the crushed flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, filtering after each extraction; c) mixing at least two filtrates from the two extractions of 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) to obtain a filtrate; - decolorizing the filtrate obtained in d) by adsorption on activated carbon; then - filtering the decolorized filtrate through a 1 μm membrane; and e) removing the ethanol from the obtained filtrate by evaporation, followed by final dilution with 1,3-propanediol.

[0019] Advantageously, the extracts used according to the invention are light in color. The extracts are also in a sufficiently concentrated form to allow them to be used without the formulation problems usually encountered in the concentrations required to obtain activity in cosmetic or dermatological compositions in emulsion form and, when in concentrated form, without the dark color that occurs in plant extracts obtained in the usual way. In particular, after evaporating the ethanol, the extract of white lily, Lilium candidum L. flowers contains 15-25% by weight of dry lily extract and 75-85% by weight of 1,3-propanediol, preferably about 20% by weight of dry lily extract and 80% by weight of 1,3-propanediol. As a result, the extract according to the invention can be used directly to prepare a cosmetic composition.

[0020] According to another aspect, the present invention relates to the cosmetic use of an alcoholic extract of flowers of the white lily, Lilium candidum L., according to the invention, as an antioxidant and / or anti-ageing agent. In yet another aspect, the present invention relates to the cosmetic use of an alcoholic extract of flowers of the white lily, Lilium candidum L., according to the present invention, for preventing and / or reducing adverse skin changes due to aging, in particular as an agent for activating the NRF2-mediated oxidative stress response and / or for promoting acanthosis.

[0021] Advantageously, the alcoholic extract of the flowers of the white lily, Lilium candidum L., according to the invention, has indeed been found to have several activities of interest, in particular with regard to preventive or reparative physiological mechanisms linked to adverse skin changes due to ageing.

[0022] Advantageously, it has been found in particular that the alcoholic extract of flowers of the white lily, Lilium candidum L., according to the invention, is able to increase the expression of papilla markers CCRL1, NTN and PDPN and to decrease the expression of reticular markers CDH2, CNN and MGP in reticular fibroblasts. The extract according to the invention is also able to improve epidermal morphology, in particular by promoting epidermal thickening, in a model of aged skin reconstituted with reticular fibroblasts.

[0023] In yet another aspect, the present invention also relates to a cosmetic composition comprising an alcoholic extract of flowers of the white lily, Lilium candidum L., according to the invention, in a cosmetically acceptable carrier. Preferably, the extract is present in the cosmetic or dermatological composition in a proportion of 0.001 to 10% by weight, in particular 0.01 to 10% by weight, preferably 0.1 to 10% by weight, relative to the total weight of the composition. The cosmetic composition may be particularly suitable for topical application.

[0024] Advantageously, said cosmetic composition may be in the form of a powder, an emulsion, a microemulsion, a nanoemulsion, a suspension, a lotion, a cream, a gel solution (which may be aqueous or aqueous-alcoholic), a foam, a serum, an aerosol solution or dispersion, or a dispersion of lipid vesicles. In the case of emulsions, they may be water-in-oil emulsions or oil-in-water emulsions. The cosmetic composition according to the present invention may also contain a solvent selected according to the various ingredients and the form of administration. Examples include water (preferably demineralized water or floral water) or alcohol such as ethanol. Said cosmetic composition may also comprise, in addition to the extract according to the invention, at least one additive conventional in the art, such as, for example, at least one compound chosen from emollients or moisturizers, gelling and / or thickening agents, surfactants, oils, active agents, dyes, preservatives, antioxidants, active agents, organic or inorganic powders, sunscreens and fragrances; - one or more humectants, such as polyols (glycerol, diglycerol, propylene glycol, propanediol, caprylyl glycol, pentylene glycol, hexanediol), sugars, glycosaminoglycans such as hyaluronic acid and their salts and esters; and polyquaterniums such as Lipidure PMB. The humectant is present in the composition in a content of about 0.1% to 30%, preferably 0.005% to 10%, by total weight of the composition; one or more emollients, which may be chosen, for example, from esters such as jojoba esters, fatty acid esters of fatty alcohols (octyldodecyl myristate, triethylhexanoin, dicaprylyl carbonate, isostearyl isostearate, caprylic / capric triglyceride), butters such as shea butter (Butyrospermum parkii 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 esters), waxes (Acacia decurrens flower wax and Helianthus annuus seed wax, C10-18 triglycerides), vegetable oils, phytosqualane, alkanes (undecane, tridecane). The emollient is present in the composition in a content of about 0.1% to 30%, preferably 0.5% to 10%, by total weight of the composition; one or more gelling agents and / or thickeners for the aqueous phase, chosen, for example, from cellulose derivatives, gums of plant origin (guar, carob, alginates, carrageenans, pectins), gums of microbial origin (xanthan), clays (laponite), hydrophilic or amphiphilic crosslinked or non-crosslinked homo- and copolymers of acryloylmethylpropanesulfonic acid (amps) and / or acrylamide and / or acrylic acid and / or salts or esters of acrylic acid (sold under the names Aristoflex AVC, Aristoflex AVS, Aristoflex HMB, Simulgel NS, Simulgel EG, Simulgel 600, Simulgel 800, Pemulen, carbopol, Sepiplus 400, Seppimax zen, Sepiplus S, Cosmedia SP). The gelling agent and / or thickening agent is present in the composition in a content of about 0.1% to 10% by total weight of the composition; one or more surfactants, in particular * Anionic surfactants such as isethionates, taurates, sarcosinates, glycinates, glutamates, phosphates (C20-22 alkyl phosphates sold under the name Sensanov WR), * Amphoteric surfactants such as betaine derivatives and amphoacetates, * Non-ionic surfactants such as polyglycerin derivatives, sugar derivatives (glucoside or xyloside derivatives sold under the names Montanov 68, Montanov 202, Montanov 82, Montanov L, Easynov), lecithin. The surfactant is present in a content of about 0.1% to 15% by weight, preferably 0.5% to 10% by weight, relative to the total weight of the composition; - for example vitamins such as vitamin C and its derivatives (ascorbic acid glucoside, 3-o-ethyl ascorbic acid, ascorbyl tetraisopalmitate), 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 (licorice (Glycyrrhiza glabra) extract, Centella asiatica (Centella asiatica) asiatica leaf extract, rye seed extract), yeast extract, alpha-hydroxy acids such as glycolic acid or lactic acid, tranexamic acid and its derivatives such as cetyl tranexamate ester, and one or more active agents of natural, biotechnological or synthetic origin having biological activity and skin benefit depending on the biological site. The active agent is present in the composition in an amount of about 0.1% to 10% by weight of the total composition. Other additives conventionally used in cosmetics may also be present in the compositions according to the invention, in particular preservatives, antioxidants or fragrances well known in the art. A person skilled in the art can select from all of these possible additives both the nature and the amounts of them added to the composition so that the composition retains all of its properties. [Example]

[0025] The following examples illustrate the invention in a non-limiting manner.

[0026] Example 1: Preparation of an alcoholic extract of flowers of the white lily, Lilium candidum L., according to the present invention The alcoholic extract of flowers of the white lily, Lilium candidum L., according to the present invention is prepared by a method comprising the following steps: a) 200g of white lily (Lilium candidum L.) flowers crushed into powder; b) Extract the powder in 2000 g of 96° ethanol with stirring at 50° C. for 2 hours; The resulting mixture 1 is screen filtered through a 50 μm screen; filtrate 1 is set aside; Extract the spent grain in 2000 g of 96° ethanol with stirring at 50° C. for 2 hours; The resulting mixture 2 is screen filtered through a 50 μm screen; the spent grain is discarded; c) Add filtrate 2 to filtrate 1 and allow the mixture to settle overnight; d) filtering the supernatant through 4 μm; - Determining the solids in 3 x 5 g extracts placed at 110°C for 3 hours; - Add 20% activated carbon to the dry extract, then decolorize the filtrate at ambient temperature with stirring for 3 hours; - 4μm and 1μm membrane filtration: remove activated carbon; e) Add 180 g of 1,3-propanediol and stir for 30 minutes, then evaporate the ethanol in a rotary evaporator; Final filtration is carried out at 4 μm.

[0027] Example 2: Testing the properties of a lily extract according to the invention against oxidative stress Protocol: Normal human epidermal keratinocytes from two young donors were seeded into 6-well plates and cultured in KGM2-supplemented medium (Lonza) at 37°C and 5% CO2 for 72 hours. Cells were then incubated with 0.25% extract for 24 hours or not (untreated condition). Each condition was performed in duplicate. Total RNA was extracted using the RNeasy 96 Plate Extraction Kit (Qiagen) according to the supplier's recommendations. RNA quantity and quality were assessed using Multiskan Go (Thermo Fischer). Complementary DNA was synthesized, and transcriptomes were generated using the Affymetrix GeneChip Human Transcriptome Array 2.0. Bioinformatics analysis of genes whose expression was regulated by at least twofold (FC: fold change, expression level relative to the untreated condition) was performed using Ingenuity Pathway Analysis software (IPA®, Qiagen).

[0028] Results: 0.25% of the extract was able to modulate the expression of an average of 158 genes (59 of which were inhibited and 99 were stimulated) in normal human keratinocytes 24 hours after treatment. IPA software was used to evaluate the changes in biological activity after treatment with the extract. Maps representing the modulation of expression of various biological pathways were constructed (data not provided). The transcriptome map obtained 24 hours after treatment revealed that many biological functions were modulated by 0.25% of the extract. In particular, Madonna lily can regulate 64 canonical signaling pathways.

[0029] Among the biological pathways significantly activated by 0.25% lily extract was the NRF2 (erythroid transcription factor 2-related transcription 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 nonenzymatic antioxidant proteins, particularly ferritin, which is composed of FTL and FTH1 subunits. Stimulation of this biological pathway for whole-cell detoxification in normal keratinocytes 24 hours after treatment with 0.25% extract confers antioxidant and anti-aging properties (oxidative stress contributes to skin aging).

[0030] [Table 1]

[0031] Example 3: Anti-aging properties of an extract of lily according to the invention in cellular and tissue models of skin aging At the cellular level, the dermis can be divided into two subpopulations of fibroblasts: papillary fibroblasts, located beneath the epidermis at the level of the papilla and possessing a younger phenotype, and reticular fibroblasts, which are deeper and more differentiated, exhibiting an older phenotype. These two subpopulations express specific markers: CCRL1, NTN, and PDPN for papillary fibroblasts, and CDH2, CNN, and MGP for reticular fibroblasts. Dermal aging is particularly characterized by the progressive differentiation of papillary fibroblasts into reticular fibroblasts, resulting in dermal hardening. This aging can be modeled in the laboratory by reconstructing equivalent skin at the cellular level using reticular fibroblasts and at the tissue level using reticular fibroblast-synthesized dermis. These models can therefore be used to identify ingredients with anti-aging properties that can reverse dermal aging. Cell model protocol: Normal human dermal reticular fibroblasts from three donors were seeded into 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 not incubated (untreated condition). Total RNA was extracted using the RNeasy 96 Plate Extraction Kit (Qiagen) according to the supplier's recommendations. RNA quantity and quality were assessed using Experion (Biorad). Complementary DNA was synthesized (iScript kit, Bioorad), and then qPCR reactions were performed using the SYBR Green method (Biorad) according to this program: 95°C for 5 minutes, followed by 35 cycles of 95°C for 20 seconds, 60°C for 20 seconds, and 72°C for 20 seconds, followed by a melting curve. Expression analysis was performed using iQ5 software (Biorad) using the ΔΔCt method with stably expressed reference genes. In this way, the expression of papillary fibroblast-specific biomarkers (CCRL1, NTN, and PDPN) and reticular fibroblast-specific biomarkers (CDH2, CNN, and MGP) was evaluated in the samples. Results: As shown in Figure 1, 0.25% of the extract can increase the expression of papillary markers CCRL1, NTN, and PDPN, and decrease the expression of reticular markers CDH2, CNN, and MGP in reticular fibroblasts after 96 hours of treatment. Therefore, the lily extract has the ability to reverse the senescent phenotype of reticular fibroblasts to a younger phenotype closer to that of papillary fibroblasts.

[0032] Tissue model protocol: Normal human dermal papillary or reticular fibroblasts from three donors were seeded in DMEM medium (HyClone) supplemented with 5% serum and cultured at 37°C and 5% CO2 for 5 days. The cells were then incubated with 0.25% extract or 0.25% propanediol solvent (diluent-only condition) for 4 days, or not (untreated condition). Dermal equivalents were generated with treated or control cells and then re-incubated with 0.25% lily extract or propanediol in DMEM medium supplemented with 5% serum and 1% penicillin-streptomycin (Invitrogen) at 37°C and 5% CO2 for 7 days, or not. Normal human epidermal keratinocytes were then seeded onto the dermal equivalents, and the cultures were maintained for 2 days 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). Skin reconstructed with papillary fibroblasts (Pap) or reticular fibroblasts (Ret) was placed at the air / liquid interface and treated or not for 14 days according to the conditions described above. After that, the tissue sections were fixed, dehydrated, and embedded in paraffin. Histological sections were then cut and used for hematoxylin-eosin-saffron (HES) staining or Ki67 immunolabeling. Hematoxylin-eosin-saffron (HES) staining was performed to observe their overall morphology, especially the thinner epidermal thickness in the model of aged reticular skin (versus "young" papillary skin). 6Immunolabeling for Ki67, a cell proliferation marker, was also performed to count positive cells in the reconstituted skin with papillary fibroblasts. Antigen unmasking was performed in citrate buffer (pH 6) followed by incubation in blocking solution to prevent nonspecific antibody binding. The sections were then incubated overnight at 4°C with a primary antibody specifically targeting Ki67 (MIB-1, Dako) and then with an avidin-biotin-peroxidase system according to the supplier's recommendations (GE Healthcare). Labeling was visualized by adding AEC (3-amino-9-ethylcarbazole), and the sections were counterstained with hematoxylin.

[0033] Results: As shown in Figure 2, 0.25% of the lily extract according to the invention is able to improve epidermal morphology, particularly by promoting epidermal thickening, in a model of aged skin reconstructed with reticular fibroblasts (right side) compared to the untreated condition (center). Thus, skin treated with this extract regained a more youthful morphology, closer to that obtained in skin reconstructed with untreated papillary fibroblasts (left side).

[0034] As shown in Figure 3, 0.25% of the extract can also increase the number of cells expressing the Ki67 proliferation marker in the basal layer of skin reconstituted with papillary fibroblasts, thus promoting cell regeneration.

[0035] Thus, 0.25% lily extract can slow the rate of dermal aging in cell and tissue models by altering the reticular phenotype to a young papillary phenotype, thus demonstrating potent anti-aging properties.

[0036] Example 4: Cosmetic Composition The following compositions can be prepared by methods conventional to those skilled in the art. The amounts given below are expressed as weight percent. Ingredients in capital letters are identified according to their INCI names.

[0037] [Table 2]

[0038] [Table 3]

[0039] These compositions can be applied to the skin daily, morning and / or evening.

Claims

1. An alcoholic extract of flowers of white lily (Lilium candidum L.) containing at least lilysine glycoside and hopantenic acid.

2. - 40 to 70% by weight, preferably 50 to 60% by weight, of sugars, - 1 to 5% by weight, preferably 2.5 to 3.2% by weight, of compounds derived from di-O- and tri-O-glycosylated kaempferol, - 8 to 15% by weight, preferably 10 to 13% by weight, of lilysine glycosides, and 1 to 5% by weight, preferably 2 to 3% by weight, of hopantenic acid Including, 2. The alcohol extract of white lily flowers according to claim 1, wherein said percentage is expressed as a weight percentage based on the total weight of said alcohol extract of white lily flowers.

3. 3. The alcoholic extract of white lily flowers according to claim 1 or 2, wherein the sugars comprise glucose, fructose and / or sucrose, preferably 20 to 25% by weight of fructose, 17 to 21% by weight of glucose and 5 to 11% by weight of sucrose.

4. 1. A method for extracting flowers of white lily (Lilium candidum L.), comprising: a) crushing flowers of white lily (Lilium candidum L.); b) extracting the crushed flowers at least once, preferably at least twice, in the presence of an alcoholic solvent, filtering after each extraction; c) mixing the filtrate from the extraction of 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 monoalcohol solvent from the mixture obtained in d) and then final dilution with another alcohol solvent. A method comprising:

5. 5. The method according to claim 4, characterized in that the white lily flowers used in step a) are fresh or dried flowers.

6. 6. The method according to claim 4 or 5, characterized in that in step a) the white lily flowers are crushed to a size of less than 2 cm.

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

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

9. 9. The method according to any one of claims 4 to 8, characterized in that each extraction of step b) is followed by filtration through a 50 μm screen.

10. 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 filtration screen or membrane.

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

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

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

14. 4. Cosmetic use of an alcoholic extract of flowers of white lily (Lilium candidum L.) according to any one of claims 1 to 3 as an antioxidant and / or for preventing and / or reducing adverse skin changes due to aging and / or for activating an NRF2-mediated oxidative stress response and / or for promoting epidermal thickening.

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

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