Anti-aging cosmetic composition

Abstract

The present invention relates to a cosmetic skincare composition, preferably anti-aging, for improving the superficial visual appearance of the skin, giving it a more youthful appearance, comprising an extract of Primrose and an extract of dedifferentiated cells of Pale Iris. The invention also relates to the use of such a combination to inhibit the expression of MMP-2 and MMP-9 in epidermal keratinocytes.

Description

Title of the invention: Anti-aging cosmetic composition

[0001] The present invention relates to a cosmetic skincare composition, preferably anti-aging, for improving the superficial visual appearance of the skin, giving it a more youthful appearance, comprising an extract of Primrose and an extract of dedifferentiated cells of Pale Iris. The invention also relates to the use of such a combination to inhibit the expression of MMP-2 and MMP-9 in epidermal keratinocytes. Previous Art

[0002] Cortisol is a steroid hormone that plays a major role in the body's metabolic response to stress. It is secreted by the adrenal cortex, located at the periphery of the adrenal glands situated above the kidneys. Its secretion is under the control of the hypothalamic-pituitary-adrenal (HPA) axis, which results from the interaction between the hypothalamus and the pituitary glands of the brain; and the adrenal glands (De Morrow, 2018; Sapolsky et al., 2000).

[0003] Cortisol release is triggered by physical or psychological stress and has long been considered a biomarker of stress (McEwen, 1998). Cortisol's function is to prepare the body to act in response to stress, and it is an essential component of many bodily functions, such as the regulation of homeostasis, metabolism, the cardiovascular system, the central nervous system, and inflammatory and immune responses (Oakley et al., 2013). Cortisol secretion follows a natural 24-hour cycle. In healthy individuals, peak levels are reached after waking, then decline throughout the day, with the lowest levels produced at the onset of sleep (Clow et al., 2010; Fries et al., 2008).

[0004] Prolonged exposure to stressors can lead to overstimulation of the HPA axis, resulting in fluctuations in cortisol levels. This dysregulation and the resulting disruption of the 24-hour cycle have significant health consequences that can lead to chronic diseases such as cardiovascular and metabolic disorders (Jones and Gwenin, 2020). They also cause significant deficiencies in the skin barrier function (Choe et al., 2017; Evers et al., 2010) and lead to epigenetic abnormalities in dermal fibroblasts (Pratt et al., 1978; Leung et al., 2022).

[0005] Cortisol dysregulation is observed during aging (Piazza et al., 2018). Aging is correlated with increased diurnal cortisol levels and flattened cortisol slopes (Nater et al., 2013; Gafey et al., 2016). The reason for this is still unknown, but a plausible explanation is that the effect of factors of Stress accumulated throughout life can lead to constant and persistent changes in the functioning of the HPA axis and interfere with the body's ability to respond to cortisol (Gafey et al., 2016).

[0006] The majority of body cells possess cortisol receptors; therefore, it plays an important role in regulating and supporting various bodily functions (Jones and Gwenin, 2020). Cortisol acts by binding to the glucocorticoid receptor, which undergoes a conformational change, dissociates from the heat shock protein binding complex, travels to the nucleus, and affects gene expression via binding domains on gene promoter regions or direct interactions with transcription factors such as the AP-1 (activating protein 1) complex and nuclear factor-κB (NF-κB) (Pratt, 1993; Ray, 1994). The glucocorticoid receptor is ubiquitously expressed on all skin cells, playing a role in regulating the immune and inflammatory response, the proliferation, differentiation, and survival of epidermal and hair follicle cells, and the barrier function.The skin and its appendages are not only targets of key stress mediators, they also constitute a local source of these factors, which induce various immune and inflammatory responses. In addition to its production by the adrenal glands, cortisol is produced by keratinocytes, melanocytes, and fibroblasts (Cirillo, 2011; Chen and Lyga, 2014).

[0007] The skin is an immediate sensor of stress and a target of stress responses. As the body's largest organ, it plays a crucial role as a physical and immune barrier, maintaining homeostasis between the external environment and internal tissues. Thanks to the presence of receptors responsible for transmitting external signals to the spinal cord and then to the brain (Schmelz, 2011), the skin is the primary organ for detecting external stressors. Cutaneous sensory fibers also transmit changes in temperature, pH, and inflammatory mediators to the central nervous system. Nerve endings are often associated with receptors, indicating a close interaction (Slominski et al., 2012). The brain responds to these signals, which in turn influence the skin's stress responses.

[0008] The skin is composed of two main layers: the epidermis and the dermis, separated by a thin layer of extracellular matrix called the dermo-epidermal junction or epidermal basal lamina (Rousselle et al., 2022). Largely populated with keratinocytes, the epidermis is subject to constant renewal because the epidermal stem cells of the basal layer, which have a high proliferative potential, constantly generate new daughter cells or transient amplifying cells. After a few division cycles, the daughter cells begin a process of Terminal differentiation leads to the formation of the stratum corneum, which is composed of dead, flattened corneocytes embedded in a lipid matrix. The dermis is composed of fibroblasts and extracellular matrix, which provide it with tone, elasticity, and tensile strength (Wong et al., 2016). The dermo-epidermal junction compartmentalizes the dermis and epidermis and provides essential structural support to both tissues by regulating cell behavior through signaling activities that influence growth, guide cell and molecular movements, and support cell survival (Yurchenco, 2011; Jayadev, 2017). The integrity and renewal of the epidermis depend on the balance between proliferation and differentiation of the basal layer of keratinocytes in contact with the dermo-epidermal junction.Furthermore, various growth factors, morphogens, and other regulatory macromolecules such as matrix metalloproteinases (MMPs) provide signals involved in the regulation of keratinocyte adhesion, differentiation, stratification, and survival (Mouw et al., 2014; Michopoulou, 2015). Growth factors and MMPs produced by basal keratinocytes play an essential role in maintaining dermal and dermo-epidermal junction homeostasis by regulating the synthesis and assembly of matrix molecules such as collagen, elastin, and glycoproteins.

[0009] MMPs are multidomain, zinc-containing endopeptidases involved in homeostatic processes, such as tissue remodeling and repair. These enzymes are produced in a latent form called zymogens, which require a proteolytic activation process to exert their full enzymatic activity. Collagenases (MMP-1) and gelatinases (MMP-2 and MMP-9) are expressed by epidermal keratinocytes during the early phases of tissue repair; they allow cells to detach from their adhesion substrate to migrate, proliferate, and regenerate the epidermis, and play an essential role in the maturation of extracellular matrix proteins at the dermo-epidermal junction and in the dermis (Michopoulou et al., 2015; Rousselle et al., 2019).These enzymes can be responsible for tissue destruction under pathological conditions, including acute and chronic inflammation, skin lesions resulting from the natural aging process, and following exposure to acute stressors such as UV radiation or chronic stressors. Normally absent in the epidermis under physiological conditions, MMP-9 and MMP-2 are overexpressed in the basal layer of the epidermis during skin aging (Nikolakis et al., 2013). Matrix ligands of these two MMPs include a wide range of proteins such as fibrillar collagens, collagens IV and VII, fibronectin, fibrillin, elastin, laminin 332, and numerous growth factors (Morrison et al., 2009). Thus, the increased expression of MMPs 2 and . 9 by the basal keratinocytes of the epidermis participates in an important way in the destruction of proteins of the dermo-epidermal junction, such as collagen IV, but also in the degradation of collagen fibers, elastic fibers and fibrillin microfibrils in the superficial dermis located just below the dermo-epidermal junction.

[0010] The Applicant has surprisingly identified a combination of active ingredients allowing the specific inhibition of the expression of MMP-2 and MMP-9 in the keratinocytes of the epidermis, making it possible to counteract the increase in their expression and their proteolytic activity induced by cortisol, allowing an anti-aging cosmetic effect. Description of the invention

[0011] Thus, according to a first aspect, the invention relates to a cosmetic composition for the skin care of a healthy subject, comprising an extract of Primevare and an extract of dedifferentiated cells of Iris Pale.

[0012] According to another aspect, the invention relates to the cosmetic use in healthy subjects of a cosmetic composition comprising an extract of Primrose and an extract of dedifferentiated cells of Pale Iris.

[0013] According to another aspect, the invention relates to a cosmetic skin care method for a healthy subject, comprising a step of applying to the skin of said subject a cosmetic composition comprising an extract of Primevare and an extract of dedifferentiated cells of Iris Pale.

[0014] According to another aspect, the invention relates to a kit for preparing an anti-aging cosmetic composition, comprising: a. an extract from Primevère; b. an extract of dedifferentiated cells from Pale Iris.

[0015] According to one embodiment, said cosmetic composition is for the anti-aging care of the skin of a healthy subject.

[0016] According to one embodiment, cosmetic use in healthy subjects is carried out to inhibit the expression of MMP-2 and MMP-9 in the keratinocytes of the epidermis.

[0017] According to one embodiment, cosmetic use in healthy subjects is carried out for anti-aging skin care.

[0018] According to one embodiment, cosmetic use in healthy subjects allows the reduction of visible signs of skin aging.

[0019] The embodiments that follow apply equally to the cosmetic composition according to the invention, and to the uses, processes and kits according to the invention.

[0020] According to one embodiment, said cosmetic composition comprises between 0.001 and 0.010% by weight of the Primevère extract composition, preferably between 0.003% and 0.007%, even more preferably 0.005%.

[0021] These percentages are in dry weight extract of Primrose extract.

[0022] According to one embodiment, said cosmetic composition comprises between 0.01% and 0.2% by weight of the composition of dedifferentiated cell extract of Pale Iris, preferably between 0.05% and 0.15%, even more preferably 0.1%.

[0023] These percentages are in dry weight extract of dedifferentiated cells of Pale Iris.

[0024] According to one embodiment, said cosmetic composition comprises between 0.001 and 0.010% by weight of the Primevère extract composition and between 0.01% and 0.2% by weight of the dedifferentiated Iris Pale cell extract composition.

[0025] According to one embodiment, the dedifferentiated cells are obtained from bark, leaves, buds, flowers or fruit skin.

[0026] Even more preferably, in the context of the invention, the extracts are extracts of dedifferentiated cells from Pale Iris leaves.

[0027] According to one embodiment, said at least one extract of Primula is chosen from the group formed by extracts of Primula veris, Primula yetis, Primula Pulgaris, Primula sikkimensis and their mixtures.

[0028] Preferably, said at least one extract of Primula veris is an extract of Primula veris, preferably of Primula veris flowers.

[0029] According to one embodiment, the healthy subject is over 50 years of age, and is preferably of female sign.

[0030] According to one embodiment, the cosmetic composition according to the invention comprises a cosmetically acceptable medium.

[0031] According to one embodiment, said cosmetic composition comprises at least one other cosmetically acceptable agent.

[0032] According to one embodiment, said at least one other cosmetically acceptable agent is chosen from among soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents that enhance the firmness, elasticity and / or barrier effect of the skin, antioxidants, opacifiers, thermal waters, mattifying agents, chemical or mineral filters, trace elements, stabilizing agents, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, perfumes, filters, essential oils, coloring materials, pigments, hydrophilic or lipophilic active ingredients,Lipid vesicles encapsulating one or more active ingredients and / or preservatives.

[0033] According to one embodiment, said cosmetic composition is formulated in the form of a cream, an ointment, a balm, a mask, a lotion, of a lotion, a serum, a bi-phase serum comprising an oily phase and an aqueous phase, a spray, a paste, a foam, an aerosol, a stick, a shampoo, a conditioner, patches, an oil-in-water or water-in-oil or multiple emulsion, an aqueous or oily gel, an anhydrous liquid, paste or solid product, and / or an oil dispersion in an aqueous phase using spherules, these spherules being able to be polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

[0034] According to one embodiment, the cosmetic composition according to the invention is administered by topical application to an area of ​​the skin.

[0035] According to one embodiment, an amount of about 0.1 to 50 mg / cm2, preferably about 0.25 to 25 mg / cm2, even more preferably about 2 to 10 mg / cm2, of cosmetic composition according to the invention is applied to the area of ​​skin.

[0036] According to one embodiment, the cosmetic composition according to the invention is applied once or twice a day, preferably twice a day, for at least 7 days, preferably at least 15 days, even more preferably for at least one month, and particularly preferably for at least 2 months.

[0037] Preferably, according to the invention, said area of ​​skin is the face.

[0038] According to one embodiment, the cosmetic composition according to the invention is a non-therapeutic cosmetic composition. Figures

[0039] [Fig. 1] Impact of cortisol on collagen degradation by epidermal keratinocytes (A) Immunofluorescence analysis of gelatin degradation by primary human keratinocytes. Keratinocytes were spread on fluorescent gelatin immobilized in the absence or presence of cortisol (0.1, 1, and 10 µM) for 48 h, fixed, and stained with phalloidin-FITC. The images show the areas of gelatin degradation (black holes) and the actin cytoskeleton of the cell that carried out this degradation. 20 µm bar (B) Quantification of the number of digested areas / cell, the average size of the areas (µm²), and the % of digested areas / cells in keratinocytes from 2 donors. Each scatter plot includes measurements from 8 independent experiments using distinct cortisol sources (134 < n > 220). ****p<0.0001 compared to the control, ANOVA test.

[0040] [Fig. 2] Assay of MMP-9 and MMP-2 expressed and secreted by human keratinocytes treated or not with cortisol. Homogeneous time-resolved fluorescence (HTRF) assay of MMP-9 (A) and MMP-2 (B) secreted by primary human keratinocytes in culture supernatant treated or not with the concentrations indicated cortisol levels. Each scatter plot includes measurements from 4 independent experiments performed with 2 distinct keratinocyte strains (12 <n>81). ***p<0.001; **p<0.01; *p<0.05 relative to the control, ANOVA test. (C) In cellulo ELISA assay of MMP-9 expressed in primary human keratinocytes treated or not with the indicated concentrations of cortisol. Each scatter plot includes measurements from 4 independent experiments performed with 2 distinct keratinocyte strains and one cortisol source (10 < n > 22). ****p<0.0001; ***p<0.001; *p<0.05 relative to the control, ANOVA test.

[0041] [Fig. 3] Extracts of dedifferentiated pale iris cells and extracts of primrose inhibit the secretion of MMP-9 and MMP-2 by primary keratinocytes. Homogeneous time-resolved fluorescence (HTRF) assay of MMP-9 (A) and MMP-2 (B) secreted by primary human keratinocytes in culture supernatant treated or untreated with the indicated concentrations of dedifferentiated pale iris cell extracts and extracts of primrose. Each scatter plot includes measurements from independent experiments performed with 1 or 2 keratinocyte strains (3 < n > 12). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 relative to the control, ANOVA test.

[0042] [Fig. 4] Extracts of dedifferentiated pale iris cells inhibit cortisol-induced MMP-9 and MMP-2 secretion in primary keratinocytes. Homogeneous time-resolved fluorescence (HTRF) assays of MMP-9 (A) and MMP-2 (B) secreted by primary human keratinocytes in culture supernatant treated or untreated with cortisol (5 pM) and then with the indicated concentrations of dedifferentiated pale iris cell extracts. Each scatter plot includes measurements from independent experiments performed with 2 keratinocyte strains (6 < n > 18). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05 relative to the control, ANOVA test.

[0043] [Fig. 5] Primevare extracts inhibit cortisol-induced MMP-9 and MMP-2 secretion in primary keratinocytes. MMP-9 (A) and MMP-2 (B) secreted by primary human keratinocytes treated or not with cortisol (5 pM) were measured by an in-cell ELISA assay, followed by the indicated concentrations of Primevare extracts. Each scatter plot comprises measurements from independent experiments performed with two keratinocyte strains (12 <n>16). ****p<0.0001 ; ***p<0.001 ; **p<0.01 ; *p<0.05 compared to the control, anova test.

[0044] [Fig. 6] Impact of dedifferentiated cell extracts of Pale Iris, Primrose extracts, and their combination on collagen degradation by epidermal keratinocytes treated with cortisol. (A, B) Quantification of the number of digested zones / cell (A) and the % of digested zones / cells (B) in keratinocytes after in situ zymography analysis. Primary human keratinocytes were treated with the cortisol (5pM) and by the compounds alone or in combination shown on the graph. The scatter plot includes cell measurements from 2 independent experiments (50 < n > 92). ****p<0.0001; ***p<0.001; **p<0.01 compared to the control shown on the graph, ANOVA test.

[0045] [Fig.7] The association of extracts of dedifferentiated cells of Pale Iris with the Primevare extracts inhibit cortisol-induced MMP-9 (A) and MMP-2 (B) secretion in primary keratinocytes superiorly and complementaryly. MMP-9 (A) and MMP-2 (B) secreted by primary human keratinocytes treated with cortisol (5 pM) and then with the indicated concentrations of dedifferentiated pale iris cell extracts, primevare extracts, and their combination were measured by an in-cell ELISA assay. Each scatter plot includes measurements from two independent experiments using 1 keratinocyte strain (12 < n > 24). ****p<0.0001; ***p<0.001 relative to the indicated control, ANOVA test. Definitions

[0046] The primrose, also known as "Primula," is a perennial herbaceous plant belonging to the Primulaceae family. It is widespread in Europe and Asia and is distinguished by its colorful flowers that appear in early spring. Extracts are generally obtained from its flowers and leaves. The extracts are obtained by maceration or infusion processes in solvents, such as water or alcohol. A primrose extract according to the invention can be obtained by any extraction or purification method known to those skilled in the art. In particular, solid-liquid extraction processes in alcoholic (especially ethanolic) media, aqueous media, as well as in media using solvents such as ketones, esters, ethers, polyols, chlorinated solvents, and mixtures of at least two of the aforementioned solvents, such as hydroalcoholic media, may be mentioned.Plant extracts of the genus Primula can be used as is, in liquid or powder form, unpurified or purified.

[0047] By primrose we mean all species of the genus Primula, such as for example Primula veris, Primula yeris, Primula vulgarts, Primula sikkimensis.

[0048] The term "primrose extract" means an extract or a mixture of extracts from plants of the genus Primula, in the family Primulaceae. More preferably, it is an extract or a mixture of extracts from Primula veris cells. This cellular material can be obtained by in vitro or in vivo culture. In vitro culture refers to all techniques known to those skilled in the art that allow, artificially, the production of a plant or part of a plant. Thus, the extract may be an extract or a mixture of extracts from organs (root, stem, leaf), or even organ cells, of at least one plant of the genus Primula sp. in the family Primulaceae. preferably Primula veris, or an extract of undifferentiated cells from at least one such plant.

[0049] The pale iris, or Iris pallida, is a perennial plant belonging to the Iridaceae family, prized for its delicate flowers of pale blue to light violet color and its captivating fragrance.

[0050] The term "extract of dedifferentiated cells of Pale Iris" means an extract or a mixture of extracts of dedifferentiated cells of this plant.

[0051] The term "dedifferentiated cells" means any cell that does not exhibit any of the characteristics of a particular specialization and is capable of living by itself and not in dependence on other cells.

[0052] These dedifferentiated cells can be obtained from plant material from the whole plant or from parts of the plant such as leaves, stems, flowers, petals, roots, fruits, their skin, the covering protecting them, seeds, anthers, sap, thorns, buds, bark, berries, and mixtures thereof.

[0053] The dedifferentiated Pale Iris cells usable according to the invention can be obtained from plants obtained by in vivo culture or from in vitro culture.

[0054] The term "in vivo culture" means any culture of a classic type, that is to say in soil in the open air or in a greenhouse or even without soil or in a hydroponic environment.

[0055] The term "in vitro culture" refers to all techniques known to those skilled in the art that allow the artificial production of a plant or part of a plant. The selection pressure imposed by the physicochemical conditions during the growth of plant cells in vitro makes it possible to obtain standardized plant material, free from contamination and available throughout the year, unlike plants cultivated in vivo.

[0056] The dedifferentiated cells usable according to the invention can be obtained by any method known in the prior art. In this regard, the methods described by E.F. George and P.D. Sherrington in Plant Propagation by Tissue Culture, Handbook and Directory of Commercial Laboratories (Exegetics Ltd 1984) may be cited.

[0057] Preferably, these extracts of dedifferentiated Pale Iris cells are obtained by: a. harvesting leaves from the plant b. cell dedifferentiation c. production of active cells d. filtration e. obtaining the extract.

[0058] By "anti-aging skin care" it is understood that the composition according to the invention allows the reduction of visible signs of skin aging. The signs of Skin aging signs include: wrinkles, fine lines, loss of firmness, sagging skin, age spots, dull complexion, loss of elasticity, dryness, rough texture, enlarged pores, loss of volume, dehydration, hyperpigmentation, thinning of the skin, loss of radiance, appearance of visible veins, redness, sagging of facial contours, weakening of the skin barrier, increased fragility, solar elastosis, and decreased production of collagen and elastin.

[0059] The cosmetic composition according to the invention improves the appearance of the skin, preferably facial skin, and thus makes it possible to hydrate, plump, nourish, revitalize, smooth, regenerate, firm, smooth, and / or detoxify the skin, preferably facial skin, and / or brighten the complexion, reduce signs of aging, and / or smooth and / or reduce the visibility of wrinkles and fine lines. Said cosmetic composition according to the invention also makes it possible to lift the eye contour, smooth fragile areas around the eyes and lips, harmonize and / or brighten the complexion, refresh the eyes, and / or reduce under-eye bags and / or dark circles. Said cosmetic composition according to the invention has an anti-aging effect.

[0060] For the purposes of this invention, "topical application" means application to the skin (including the scalp) and mucous membranes.

[0061] For the purposes of this invention, "cosmetically acceptable" means something that is useful in the preparation of a cosmetic composition, that is generally safe, non-toxic and neither biologically nor otherwise undesirable, and that is acceptable for cosmetic use, in particular by topical application to the skin.

[0062] For the purposes of this invention, "non-therapeutic" means a cosmetic application not intended to treat a patient. In fact, the cosmetic composition according to the invention does not act as a drug for the treatment of pathologies, but rather improves the superficial visual appearance of the skin, giving it a more youthful appearance and reducing the visible signs of aging.

[0063] By "cosmetically acceptable medium" is meant a medium compatible with the skin of the face and / or body. In other words, the medium used has a pleasant color, odor, and feel and does not generate unacceptable discomfort (such as, in particular, tingling, tightness, redness) that might deter the consumer from using this composition.

[0064] In this description, "approximately" means that the value in question may be 10% lower or higher, in particular 5%, in particular 2%, more particularly 1%, than the value indicated.

[0065] By "healthy subject" is meant a human individual not presenting with dermatological diseases that would be treated by the administration of the composition according to the invention, which only allows for the improvement of the superficial visual appearance of the skin, preferably of the face, in order to give it a more youthful appearance.

[0066] In the description and the following examples, unless otherwise indicated, percentages are percentages by weight, and value ranges expressed as "between ... and ..." include the specified lower and upper bounds. The examples below are provided by way of illustration and are not intended to limit the scope of the invention. Examples

[0067] Example 1:

[0068] Materials and methods:

[0069] Cells:

[0070] For all tests, freshly isolated normal human keratinocytes were used. The human keratinocytes were obtained from foreskin biopsy (surgical waste, Pavilion T-Bis, Edouard Herriot Hospital). The culture medium was the KGM-Gold defined medium for keratinocyte culture (containing: Bovine pituitary extract 35 mg, hEGF 10 ng / ml, insulin 5 pg / ml, hydrocortisone 0.5 pg / ml, transferrin 0.1%, epinephrine 0.1%) manufactured by Clonetics and marketed by Lonza, containing 0.15 mM CaC12, pH 7.2 to 7.4. Keratinocytes were obtained using the technique described by Boyce and Ham (Cultivation, frozen storage, and clonal growth of normal human epidermal keratinocytes in serum free-media, Tiss Cuit. Meth. 1985, 9:83-93). The skin fragments, after careful rinsing in PBS buffer containing antibiotics, were freed of the fatty tissue located beneath the dermis using sterile instruments.The skin was then cut into 3 mm² pieces, which were placed in a sterile 0.25% trypsin solution in PBS for 16 hours at 4°C. The dermis / epidermis was separated using fine forceps in a Petri dish containing culture medium to stop the enzymatic action of trypsin. The epidermal fragments were aspirated and repeatedly pipetted to detach the free basal cells. The resulting cell suspension was centrifuged for 5 minutes at 1000 rpm, and the pellet was suspended in a known volume of KBM-2 for live cell counting using trypan blue exclusion dye. 3 x 10⁴ live cells per cm² were inoculated onto 25 cm² tissue culture plates (Corning, Polylabo, France). Keratinocytes were cultured at 37°C in a CO2 incubator (5% CO2, 95% air and 98% humidity).The medium was changed every two days. Subculture occurred when the cells reached subconfluence. The cell mat was then rinsed with PBS and subsequently covered with a 0.05–0.02% trypsin-EDTA solution. After a short incubation at 37°C, . The cells detached from the plastic support. The cells were seeded into 75 cm2 culture dishes. The cells (3 to 5 million per ampoule) were frozen in the culture medium used, in the presence of 10% Dimethyl sulfoxide (DMSO) and 20% calf serum in a volume of 1 ml.

[0071] In situ zymography:

[0072] In situ zymography was performed using the "QCM™ Gelatin Invadopodia Assay (Red)" kit from Merck Millipore (Burlington, Massachusetts, USA), which allows for the fixation of a thin layer of gelatin coupled to the fluorochrome Cyanine 3 (Cy3), enabling rapid detection of matrix degradation by MMPs. Sterile, degreased 12 mm diameter glass coverslips were incubated in a poly-L-lysine solution. After rinsing with sterile PBS, 5% glutaraldehyde was applied to the coverslips for 15 min, and then the coverslips were rinsed with sterile PBS. A PBS solution containing gelatin coupled with Cy3 was applied to the treated coverslips for 2 minutes before being aspirated and removed by washing with sterile PBS. The plates are protected from light and disinfected using 70% ethanol.Once the coverslip treatments are complete, the keratinocytes intended for use in situ zymography are detached from the plate using the trypsin / EDTA solution, centrifuged, and counted. The keratinocytes are then loaded into each of the wells containing the gelatin-coated coverslips, at a density of 35 x 10³ cells / well in complete KBM-Gold, in humid air at 37°C composed of 5% CO₂ and 95% atmospheric air. Two hours after inoculation, the culture medium is aspirated, and 1 ml of untreated KBM-Gold is added to each well. The cells are thus either left untreated for 48 hours in untreated KBM-Gold alone, or treated for 48 hours with cortisol-containing medium at the concentration indicated in the graphs.The following four cortisol sources were tested: Hydrocortisone Supelco C-106, Hydrocortisone H6909, Hydrocortisone HO888, and Hydrocortisone Certified pharmaceutical grade PHR1014 (Sigma Aldrich, Saint Quentin-Falavier, France). Extracts were added at the indicated concentrations 2 hours after the addition of cortisol. The media were then aspirated after 48 hours of treatment, and the cells were fixed with 4% PBS-Paraformaldehyde (PFA) for 20 minutes at room temperature, protected from light, and with shaking. After aspirating the fixative, a permeabilization and saturation step was performed using 500 qL / well of a solution composed of 0.25% PBS-Triton (Triton X-100) and 2% SVF. The slides are stirred for 30 minutes at room temperature and protected from light with this blocking solution, which is then aspirated. Phalloidin coupled with FITC is added for 1 hour and 30 minutes, and the coverslips are washed three times with PBS.Blades. Annotated SuperFrost glass slides (Cari Roth, Karlsruhe, Germany) are used to hold the coverslips in place with ProLong mounting fluid containing DAPI (ProLong™ Gold Antifade Mounting with DAPI, Invitrogen, Eugene, Oregon, USA). The slides are then observed using an inverted confocal microscope (Zeiss LSM800). Images of the different fields of view are acquired using Zeiss ZEN software, and the photographs are analyzed using ImageJ software.

[0073] Assay of MMP-9 and MMP-2 produced by cells by homogeneous time-resolved fluorescence (HTRF, Rewity)

[0074] This method allows for the quantification of soluble MMPs present in the culture medium. HTRF involves the use of two fluorophores, a donor and an acceptor. When biomolecules interact, their proximity allows for fluorescence resonance energy transfer (FRET) from the donor fluorophore to the acceptor fluorophore. The use of long-lived fluorescent europium cryptates as donors enables time-resolved detection in HTRF assays, reducing the brief background fluorescence of compounds and other materials. Time-resolved FRET detection (TR-FRET) with a microplate reader allows for the reading of the biomolecular bond. The organic fluorophore d2 is used as the acceptor.

[0075] The HTRF assay for MMP-9 and MMP-2 is designed to measure the concentrations of MMP-9 or MMP-2 (precursor and active forms) in human cell culture supernatants. These two MMPs play an important role in tissue remodeling by degrading the structural components of the extracellular matrix, such as collagen, gelatin, and elastin. The assay is based on a TR-FRET sandwich immunoassay involving two specific human anti-MMP-9 (or MMP-2) antibodies, one labeled with europium cryptate (Eu3+) (donor) and the other with d2 (acceptor). Both antibodies bind to human MMP, and the donor-acceptor proximity allows for a fluorescent TR-FRET signal. The signal intensity is proportional to the concentration of human MMP present in the sample. The assay is performed in a low-volume (20 qL final) white plate.The samples and a standard curve are dispensed directly into the plate, and antibodies labeled with HTRF fluorophores are added. No washing step is required. Fluorescence reading is performed on a reader specifically equipped with the necessary filters for FRET. Various measurements are taken to extract the specific signal generated by the interaction, providing a highly sensitive and antigen-specific response. The standard curve allows for the precise determination of the assay for the tested samples. Results are expressed in ng / ml.

[0076] Evaluation of MMP-9 and MMP-2 in cellulo

[0077] Normal human keratinocytes (NHKs, PI or P2 maximum passage) were seeded in 96-well microclear plates (Bio-One microclear plates, Greiner) in KBM-2 (Lonza, Belgium) at a density of 10⁴ cells / well. After 24 hours, cortisol (with or without treatment by molecules) or buffers (1 control per cortisol concentration) were added to KBM-2 without supplements. After 48 hours of contact at 37°C, the culture media were removed, the wells were washed twice with sterile PBS maintained at 37°C, and then the cells were fixed with a 2% paraformaldehyde PBS solution for 10 minutes. Two more PBS washes were performed, and a 1% PBS-BSA solution was added for 1 hour. After aspiration, PBS-BSA and the polyclonal antibody directed against MMP-9 (Calbiochem, France) or MMP-2 (Péprotech, Invitrogen, France) are added and brought into contact for 3 hours.After three 5-minute PBS washes, the fluorescent conjugate (FITC) is applied for 2 hours in the dark (Fluorescein (FITC) conjugated AffiniPure Goat anti-mouse IgG, Jackson ImmunoResearch). Two 5-minute PBS washes are performed in the dark, and the wells are dried before their contents are solubilized with 20 mM ammonium hydroxide (NH4O2H20). After shaking for 5 minutes in the dark, fluorescence is detected using an FLX800 reader (Microplate Fluorescence Reader, BIO-TEK INSTRUMENTS, wavelength: excitation 485 nm, emission 528 nm). Each condition is performed in triplicate. Each experiment was performed at least twice.

[0078] A control without primary antibody is performed each time to ensure the absence of non-specific binding of the conjugate. A control without antibody (neither primary nor secondary) is also performed in each experiment to ensure the absence of cell autofluorescence.

[0079] Each results sheet shows the fluorescence intensity of the treated wells (as a percentage of the untreated control). To eliminate potential bias caused by an effect on cell proliferation, all results are normalized using an XTT test performed systematically in parallel.

[0080] Cell proliferation assay (XTT)

[0081] The chemical reaction of the test is based on the production of NADPH by living cells, which reduces yellow XTT tetazolium salts to orange formazan salts. Absorbance is measured at 490 nm using an ELISA-Reader plate reader. Cells were seeded in 96-well plates in KBM-2 culture medium. After 24 hours of culture at 37 °C in the presence of 5% CO2, the culture media were removed and replaced with KBM-2 medium containing cortisol (+ / - treatment with extracts) or buffers (1 control per cortisol concentration). Two days later, the media were removed and replaced with The test reagent. The plates are placed in an incubator at 37°C, and absorbance readings are taken at 3 and 5 hours. Controls without cortisol (using the cortisol solubilization buffer) were prepared on the same plate. The results are presented as the percentage of viability of treated cells compared to untreated controls. Cell viability was calculated using the formula: % viability = (Abs. treated cells / Abs. control cells) x 100.

[0082] Statistical evaluation

[0083] Unpaired t-tests and one-tailed analysis of variance (ANOVA) were used to evaluate the data; p < 0.05 was considered statistically significant. The number of repeated experiments is indicated in the legend of each graph; the number of experimental replicates is denoted by n. Individual data values ​​are presented systematically, especially in experiments where n < 6. Statistical analyses were performed using GraphPad Prism software, version 8.4.2. All results are presented as mean ± SD.

[0084] Results:

[0085] Cortisol stimulates the proteolytic activity of primary human keratinocytes j.

[0086] To visualize and quantify the functional proteolytic activity of MMPs in the context of cultured primary human keratinocytes, in situ zymography on gelatin was performed. Keratinocytes were seeded onto fluorescent gelatin, and the gelatin degradation zones generated by the keratinocytes were visualized by confocal microscopy as black spots beneath the cells (Figure IA). Since keratinocytes adhere to and organize their cytoskeleton on this substrate, concomitant actin staining was performed to identify cell outlines. When quantifying the digested black zones, an average of 19 spots / cell was measured under untreated conditions and an average of 45 to 48 spots in keratinocytes treated with cortisol (Figure IB).Furthermore, the average size of the digestion zones is 0.5 pm² in untreated cells, whereas they reach an area of ​​0.66 to 0.7 pm² when the cells have been treated with cortisol. Since primary keratinocytes in culture exhibit heterogeneity in size, evaluating the % digested area per cell proved to be a more reliable measure, increasing from a degradation rate of 0.6% in control cells to 1.5–1.7% in treated cells.

[0087] A cell viability test showed that cortisol did not cause either proliferation or death of keratinocytes.

[0088] These results show for the first time that cortisol stimulates matrix proteolysis activity of human epidermal keratinocytes.

[0089] Cortisol stimulates the expression and synthesis of MMP-9 and MMP-2 by primary human keratinocytes:

[0090] To identify the enzymes responsible for cortisol-induced extracellular matrix degradation in the in situ zymography experiment, quantitative assays for measuring MMP-9 and MMP-2 were developed ([Fig. 2]), MMP-9 and MMP-2 being the two major soluble MMPs produced by keratinocytes. A time-resolved homogeneous fluorescence (HTRF) assay performed on culture supernatants of keratinocytes treated with different concentrations of cortisol shows a significant increase in MMP-9 (6.5 to 7 ng / ml vs. 4.8 ng / ml for the untreated control; Figure 2A) and MMP-2 (14.2 to 13.5 ng / ml vs. 11.4 ng / ml for the untreated control; Figure 2B) secreted by keratinocytes treated with cortisol concentrations of 10 and 5 pM, respectively.An in-cell ELIS A assay performed directly on cells using an anti-MMP-9 antibody confirms an increase in MMP-9 expression of 269%, 202% and 169% by keratinocytes treated with cortisol at concentrations of 10, 5 and 1 pM respectively compared to the untreated control (Figure 2C).

[0091] Taken together, these results demonstrate that cortisol stimulates the synthesis, secretion and activation of MMP-9 and MMP-2 by primary human keratinocytes.

[0092] Identification of inhibitory components of MMP-9 and MMP-2 production by primary human keratinocytes

[0093] A homogeneous time-resolved fluorescence (HTRF) assay performed on culture supernatants of keratinocytes treated with different concentrations of two plant compounds, extracts of dedifferentiated Pale Iris cells (Figure 3A) and Primrose extracts (Figure 3B), revealed a significant decrease in the expression of MMP-9 and -2 by primary keratinocytes under basal conditions without prior stimulation, while the diluent, glycerin, had no effect. A cell viability assay showed that none of the tested compounds induced either proliferation or death of keratinocytes.

[0094] Extracts of dedifferentiated cells of Pale Iris and extracts of Primrose inhibit the secretion of cortisol-induced MMP-9 and MMP-2.

[0095] The effect of extracts from dedifferentiated Pale Iris cells and extracts of Primrose on the cortisol-induced increase in MMP-9 and -2 expression in keratinocytes was evaluated on two distinct keratinocyte strains using the time-resolved homogeneous fluorescence (HTRF, [Fig. 4]) and in-cell ELIS A ([Fig. 5]), respectively. Assays were performed on supernatants and on cultures of cortisol-treated (5 pM) keratinocytes to which decreasing concentrations of plant extracts were added as described.

[0096] The results report a significant, specific and controlled inhibition of the increase in expression of both MMPs generated by cortisol.

[0097] The combination of extracts from dedifferentiated cells of Pale Iris and extracts of Primrose inhibits the proteolytic activity of MMPs in a way that is very significantly greater than that of each compound used alone.

[0098] The proteolytic activity of two effective concentrations of each of the two compounds was evaluated by the in situ zymography assay ([Fig. 6]). The inhibitory activity of the two compounds was thus confirmed at the functional level, since Pale Iris cells (0.5% and 0.125%) and Primrose extracts (0.25% and 0.125%) significantly inhibited the degradation of gelatin by MMPs produced by keratinocytes (significant reduction in the number of lysis spots / cell and reduction in the total % of surface area degraded / cell compared to cells treated with cortisol). The combination of Pale Iris cell extracts (0.125%) and Primrose extracts (0.125%) generated greater inhibition than that produced by the compounds used alone.The number of damaged areas, 19 and 13 for cells treated with Pale Iris cells (0.125%) and Primrose extracts (0.125%), respectively, drops to 5.7 when the two compounds are combined (Figure 6A, C). Taking cell size into account, the total percentage of damaged surface area per cell decreases from 0.40% when the compounds are used individually to 0.22% when they are combined (Figure 6B, C). These results suggest that the inhibitory mechanisms of Pale Iris dedifferentiated cell extracts and Primrose extracts are different, which would explain their complementary activity.

[0099] The assay of MMP-9 by the ELISA in cellulo test ([Fig.7]) confirms these results and shows that the combinations of dedifferentiated cell extracts of pale iris 0.25% / extracts of primrose (0.125%) and of dedifferentiated cell extracts of pale iris 0.125% / extracts of primrose (0.125%) reduce the expression of cortisol-induced MMP-9 significantly more than the reduction obtained with each compound alone. References

[0100] Chen Y, Lyga J. Brain-skin connection: stress, inflammation and skin aging. Inflamm Allergy Drug Targets. 2014;13:177-90. doi:10.2174 / 18715281136661405 22104422.

[0101] Choe SJ, Kim D, Kim EJ, Ahn JS, Choi EJ, Son ED, Lee TR, Choi EH. Psychological Stress Deteriorates Skin Barrier Function by Activating 11[3-Hydroxysteroid Dehydrogenase 1 and the HPA Axis. SciRep. 2018;8:6334. doi:10.1038 / s41598-018-24653-z.

[0102] Cirillo N, Prime SS. Kératinocytes synthesize and activate cortisol. J Cell Biochem. 2011;112:1499-505. doi: 10.1002 / jcb.23081. PMID: 21344493.

[0103] Clow A, Hucklebridge F, Stalder T, Evans P, Thom L. The cortisol awakening response: more than a measure of HPA axis function. Neurosci Biobehav Rev. 2010;35:97-103. doi: 10.1016 / j.neubiorev.2009.12.011.

[0104] DeMorrow S. Rôle of the Hypothalamic-Pituitary-Adrenal Axis in Health and Disease. Int J Mol Sci. 2018;19:986. doi: 10.3390 / ijms 19040986.

[0105] Evers AW, Verhoeven EW, Kraaimaat FW, de long EM, de Brouwer SJ, Schalkwijk J, Sweep FC, van de Kerkhof PC. How stress gets under the skin: cortisol and stress reactivity in psoriasis. Br J Dermatol. 2010;163:986-91. doi: 10.1111 / j. 1365-2133.2010.09984.x.

[0106] Fries E, Dettenbom L, Kirschbaum C. The cortisol awakening response (CAR): facts and future directions. Int J Psychophysiol. 2009;72:67-73. doi: 10.1016 / j.ijpsycho.2008.03.014.

[0107] Gaffey AE, Bergeman CS, Clark LA, Wirth MM. Aging and the HPA axis: Stress and resilience in older adults. Neurosci Biobehav Rev. 2016;68:928-945. doi: 10.1016 / j.neubiorev.2016.05.036.

[0108] Jayadev R, Sherwood DR. Basement membranes. Curr Biol. 2017 Mar 20;27(6):R207-R211. doi: 10.1016 / j.cub.2017.02.006.

[0109] Jones C, Gwenin C. Cortisol level dysrégulation and its prevalence-Is it nature's alarm clock? Physiol Rep. 2021 Jan;8(24):el4644. doi: 10.14814 / phy2.14644.

[0110] Leung CS, Kosyk O, Welter EM, Dietrich N, Archer TK, Zannas AS. Chronic stress-driven glucocorticoid receptor activation programs key cell phenotypes and functional epigenomic patterns in human fibroblasts. iScience. 2022 Aug 17;25(9): 104960. doi: 10.1016 / j.isci.2022.104960.

[0111] Michopoulou A, Rousselle P. How do epidermal matrix metalloproteinases support re-epithelialization during skin healing? Eur J Dermatol. 2015;25 Suppl 1:33-42. doi: 10.1684 / ejd.2015.2553. PMID: 26083673.

[0112] Morrison CJ, Butler GS, Rodriguez D, Overall CM. Matrix metalloproteinase proteomics: substrates, targets, and therapy. Curr Opin Cell Biol. 2009;21:645-53. doi: 10.1016 / j.ceb.2009.06.006.

[0113] Mouw JK, Ou G, Weaver VM. Extracellular matrix assembly: a multiscale deconstruction. Nat Rev Mol Cell Biol. 2014;15:771-85. doi: 10.1038 / nrm3902.

[0114] Nater UM, Hoppmann CA, Scott SB. Diumal profiles of salivary cortisol and alpha-amylase change across the adult lifespan: evidence from repeated daily life assessments. Psychoneuroendocrinology. 2013 Dec;38(12):3167-71. doi: 10.1016 / j.psyneuen.2013.09.008.

[0115] Nikolakis G, Makrantonaki E, Zouboulis CC. Skin mirrors human aging. Horm Mol Biol Clin Investig. 2013;16:13-28. doi: 10.1515 / hmbci-2013-0018.

[0116] Oakley RH, Cidlowski JA. The biology of the glucocorticoid receptor: new signaling mechanisms in health and disease. J Allergy Clin Immunol. 2013;132:1033-44. doi: 10.1016 / j.jaci.2013.09.007.

[0117] Piazza JR, Dmitrieva NO, Charles ST, Almeida DM, Orona GA. Diurnal cortisol profiles, inflammation, and functional limitations in aging: Findings from the MIDUS study. Health Psychol. 2018 Sep;37(9):839-849. doi: 10.1037 / hea0000629.

[0118] Pratt WB. The mechanism of glucocorticoid effects in fibroblasts. J Invest Dermatol. 1978;71:24-35. doi: 10.1111 / 1523-1747.epl2543774.

[0119] Pratt WB. The rôle of heat shock proteins in regulating the function, folding, and trafficking of the glucocorticoid receptor. J Biol Chem. 1993;268:21455-8. PMID: 8407992.

[0120] Ray A, Prefontaine KE. Physical association and functional antagonism between the p65 subunit of transcription factor NF-kappa B and the glucocorticoid receptor. Proc Natl Acad Sci USA. 1994;91:752-6. doi: 10.1073 / pnas.91.2.752.

[0121] Roig-Rosello E, Rousselle P. The Human Epidermal Basement Membrane: A Shaped and Cell Instructive Platform That Aging Slowly Alters. Biomolecules. 2020;10:1607. doi: 10.3390 / bioml0121607. PMID: 33260936; PMCID: PMC7760980.

[0122] Rousselle P, Braye F, Dayan G. Re-epithelialization of adult skin wounds: Cellular mechanisms and therapeutic strategies. Adv Drug Deliv Rev. 2019;146:344-365. doi: 10.1016 / j.addr.2018.06.019.

[0123] Rousselle P, Laigle C, Rousselet G. The basement membrane in epidermal polarity, stemness, and régénération. Am J Physiol Cell Physiol. 2022;323:C1807-C1822. doi: 10.1152 / ajpcell.00069.2022.

[0124] Sapolsky RM, Romero LM, Munck AU. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and préparative actions. EndocrRev. 2000;21:55-89. doi: 10.1210 / edrv.21.1.0389.

[0125] Schmelz M. Neuronal sensitivity of the skin. Eur J Dermatol. 2011 May;21 Suppl 2:43-7. doi: 10.1684 / ejd.2011.1265.

[0126] Slominski AT, Zmijewski MA, Skobowiat C, Zbytek B, Slominski RM, Steketee JD. Sensing the environment: régulation of local and global homeostasis by the skin's neuroendocrine System. Adv Anat Embryol Cell Biol. 2012;212:v, vii, 1-115. doi: 10.1007 / 978-3-642-19683-6_ 1.

[0127] Wong R, Geyer S, Weninger W, Guimberteau JC, Wong JK. The dynamic anatomy and patterning of skin. Exp Dermatol. 2016;25:92-8. doi: 10.1111 / exd. 12832.

[0128] Yurchenco PD. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb Perspect Biol. 2011;3:a004911. doi: 10.1101 / cshperspect.a004911.< / n> < / n>

Claims

Demands

1. Cosmetic composition for the anti-aging care of the skin of a healthy subject, comprising an extract of Primrose and an extract of dedifferentiated cells of Pale Iris.

2. Cosmetic composition according to any one of the preceding claims, comprising between 0.001 and 0.010% by weight of the Primevère extract composition.

3. Cosmetic composition according to any one of the preceding claims, comprising between 0.01% and 0.2% by weight of the composition of dedifferentiated cell extract of Pale Iris.

4. Cosmetic composition according to any one of the preceding claims, comprising between 0.001 and 0.010% by weight of the Primevare extract composition and between 0.01% and 0.2% by weight of the Pale Iris dedifferentiated cell extract composition.

5. Cosmetic composition according to any one of the preceding claims, comprising at least one other cosmetically acceptable agent.

6. A cosmetic composition according to the preceding claim, wherein said at least one other cosmetically acceptable agent is selected from soothing agents, restructuring agents, regenerating agents, revitalizing agents, sunscreens, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizing agents, thickening and gelling agents, smoothing agents, agents that enhance the firmness, elasticity and / or barrier effect of the skin, antioxidants, opacifiers, thermal waters, mattifying agents, chemical or mineral filters, trace elements, stabilizing agents, foaming agents, perfumes, ionic or non-ionic emulsifiers, fillers, sequestering agents and chelating agents, perfumes, filters, essential oils, coloring materials, pigments, hydrophilic or lipophilic active ingredients,Lipid vesicles encapsulating one or more active ingredients and / or preservatives.

7. A cosmetic composition according to any one of the preceding claims, said composition being formulated as a cream, ointment, balm, mask, milk, lotion, serum, bi-phase serum comprising an oily phase and an aqueous phase, spray, paste, etc. foam, aerosol, stick, shampoo, conditioner, patches, oil-in-water or water-in-oil or multiple emulsions, aqueous or oily gel, anhydrous liquid, paste or solid product, and / or oil dispersion in an aqueous phase using spherules, these spherules being polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

8. A method for anti-aging cosmetic skincare on a healthy subject, comprising a step of applying to the skin of said subject a cosmetic composition according to any one of claims 1 to 7

9. Cosmetic use in healthy subjects of a cosmetic composition according to any one of claims 1 to 7, to inhibit the expression of MMP-2 and MMP-9 in epidermal keratinocytes.

10. Cosmetic use in healthy subjects of a cosmetic composition according to any one of claims 1 to 6, for the anti-aging care of the skin.

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