Diospyros spp. extracts and their use in combating senescence

Abstract

The present invention relates to the use of an extract of aerial parts of Diospyros spp. and / or the use of compositions comprising an extract of aerial parts of Diospyros spp. for combating senescence and for combating skin aging and / or preserving and / or strengthening the immune defenses of the skin and / or preserving and / or strengthening the skin barrier function.

Description

Title of the invention: Extracts of Diospyros spp. and their use in combating senescence TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to the use of an extract of aerial parts of Diospyros spp. and / or the use of compositions comprising an extract of aerial parts of Diospyros spp. for combating senescence and for combating skin aging and / or preserving and / or strengthening the immune defenses of the skin and / or preserving and / or strengthening the skin barrier function. STATE OF THE ART

[0002] The skin is made up of different layers forming a vital barrier for the body against the external environment. This barrier protects the body against external attacks, in particular chemical, mechanical or infectious, and as such a certain number of defense reactions against environmental factors and / or xenobiotics occur at its level.

[0003] The skin is made up of three main parts, the superficial one, the epidermis, the internal part, the dermis and a deeper layer, the hypodermis, which interact.

[0004] The human epidermis is composed of four to five distinct layers (depending on the anatomical site) and four types of cells which are keratinocytes, the vast majority, melanocytes, Langerhans cells and Merkel cells. Each of these cell types contributes through its own functions to the essential role played in the body by the skin, in particular the role of protecting the body from external aggressions. This property is called barrier function.

[0005] Epidermal cells proliferate at its deepest layer, the basal layer, and differentiate during their migration towards the upper layers to successively form the spinous layer consisting of several layers of polyhedral cells arranged on the germinative layers, the granular layer consisting of flattened cells containing distinct cytoplasmic inclusions, the keratohyaline grains, and finally the horny layer (or stratum corneum) which is the most superficial layer of the epidermis. The horny layer is made up of 10 to 30 layers of keratinocytes at the terminal stage of their differentiation, called corneocytes. Corneocytes, the constituent elements of the horny layer, are dead, flat cells containing water and keratin.

[0006] This structure of the epidermal cells participates in the protective function or barrier function of the skin, the stratum corneum, by its structure and composition, constituting the main barrier.

[0007] The skin is not, however, just a physical barrier: it also constitutes an immunological barrier, capable of developing innate and adaptive immune responses, which allow the host to be defended against attacks of various kinds.

[0008] The cutaneous immune system is based on different resident cells with specific and varied functions depending on their location, recruited specialized cellular actors (leukocytes) and soluble mediators (cytokines and chemokines).

[0009] The dermis houses in particular fibroblasts, mast cells, macrophages and dendritic cells, which participate directly or indirectly in the immunology of the skin.

[0010] Within the epidermis, two types of cells play a major role in cutaneous immunity, whether innate or adaptive: • keratinocytes, which represent 90% of the epidermal cell population, which are important in the development of the immune response due to their number and their capacity to secrete cytokines and antimicrobial peptides; • Langerhans cells, which are dendritic cells that act as true sentinels of the cutaneous immune system. They have the ability to detect antigens that cross the stratum corneum and migrate to the lymph nodes to present them to T lymphocytes.

[0011] However, the cutaneous immune system can be disrupted by various intrinsic or extrinsic factors, including aging.

[0012] Fibroblasts, which are the main cells of the dermis, are specialized in the synthesis of elastin and collagen fibers. They ensure the balance between synthesis, maturation and degradation of elastin and collagen fibers. Between the ages of 20 and 80, the population of fibroblasts decreases by half. Thus, over time, this balance will shift towards a degradation of these fibers with the results of a loss of elasticity and tone of the dermis and a flaccidity that no longer opposes the effects of contraction of the underlying muscles, leading to the appearance of wrinkles. Collagen fibers are distributed in all layers of the dermis, they are capable of fixing water and contribute to the hydration of the skin. A decrease in collagen and / or a change in its quality leads to the appearance of deep wrinkles.With aging, elastin fibers, which give elasticity and strength to tissues, become scarce, the skin becomes thinner and wrinkled. The skin must continually face multiple external aggressions that can accelerate the natural aging process. It is particularly sensitive to attacks from free radicals generated both by the normal functioning of our body and by elements. external factors, such as pollution, tobacco, but especially solar radiation. These free radicals are responsible for tissue and cellular changes that lead to skin aging.

[0013] During the aging process, a change in the structure and functions of the skin is observed. This aging is physiological in nature but can also be photo-induced, i.e. due to repeated exposure of the skin to sunlight, particularly ultraviolet light. The main clinical signs of skin aging are the appearance of fine lines and deep wrinkles, which obviously increase with age. The furrows and wrinkles become more pronounced; the skin becomes hollow and loses its firmness; on the surface, the skin loses its luminosity.

[0014] Fibroblasts enter senescence and their proliferation capacity decreases sharply.

[0015] After UV exposure, there is an increase in the expression of certain metalloproteinases in the epidermis and dermis. These enzymes, which alter collagen production in the dermis, also contribute to the appearance of wrinkles. The induction of these enzymes is maintained by repeated exposures. There is a thickening of the dermis due to the accumulation of elastic material. The production of interleukins by the epidermis, following UV irradiation, contributes to the destruction of connective tissue.

[0016] Skin aging is not just an aesthetic phenomenon; other functions are also impacted by this process. Indeed, the loss of immunity as well as the barrier function of the skin are affected by the senescence process.

[0017] Cellular aging leads to a decrease in the ability of cells to proliferate. Apoptosis, or programmed cell death, is a phenomenon responsible for the death of certain cells, occurring physiologically throughout life. But during aging, this phenomenon increases and is responsible for a loss of cells, among other things, at the cutaneous level. If apoptosis intervenes normally in the elimination of damaged cells, it would also intervene abnormally in the elimination of healthy cells during aging, which gradually leads to a cellular rarefaction.

[0018] A senescent cell is a cell that is no longer able to be eliminated or to renew itself, neither completely dead nor completely alive, senescent cells will thus stagnate in our tissues, our immune system becoming less efficient with age to eliminate them. By remaining in our tissues, senescent cells will release bioactive molecules that are toxic to other cells which are still perfectly functional. The more we have of these senescent cells, the more we will contaminate the other cells and the more we will limit the capacity to eliminate and renew them.

[0019] However, it is precisely this accumulation of senescent cells which will generate inflammation, the source of most skin aging problems: the formation of wrinkles, fine lines, brown spots, sagging skin.

[0020] There are different molecular mechanisms that could explain age-associated diseases including mechanisms associated with senescence.

[0021] Cellular senescence corresponds to a process of cessation of cell division after a specific number of cell divisions. When this limit is reached, known as the “Hayflick limit”, cells enter a state of irreversible cell cycle arrest and resistance to apoptosis, with a pro-inflammatory secretion profile and morphological changes, which defines cellular senescence (Kumari and Jat, 2021).

[0022] It has been shown that the senescence process can be induced by several types of cellular stress factors. These stresses include oxidizing chemical agents (H2O2), hyperoxia, and genotoxic agents (irradiation, UV, chemotherapy). In all cases, senescence is a consequence of repeated DNA damage occurring during our lives.

[0023] Senescence is a permanent arrest of the cell cycle that is accompanied by changes in cell morphology and physiology. It involves resistance to apoptosis, and frequently metabolic changes. It is induced by DNA damage.

[0024] In vitro, senescent cells are characterized by a spread morphology which is explained by an abnormal expansion of the endoplasmic reticulum and a modification of the cytoskeleton.

[0025] An excessive accumulation of these senescent cells (not quickly eliminated by our immune system) can accelerate aging at the tissue level. All this leads to a loss of elasticity of the dermis and the appearance of wrinkles.

[0026] Furthermore, the senescent cell is characterized by an increased secretion (called SASP for senescence-associated secretory phenotype) of inflammatory and tissue remodeling factors, such as cytokines, chemokines and proteinases (Fontanilla et al., 2020). This secretion of molecules will lead to the degradation of the dermal matrix and the propagation of senescence. As a result, the skin loses its elasticity and causes the appearance of wrinkles.

[0027] To overcome this senescence process, there are therapies such as the use of senolytics which will modulate the pathways regulating cell death of senescent cells. Senolytics will selectively eliminate senescent cells by causing apoptosis. Senolytics therefore have an effect on slowing down aging but cause a decrease in cells in the skin which is not desirable. There is therefore still a need to find a process which would allow the preservation of senescent cells in order to maintain a constant number of cells in the skin and significantly slow down the aging process.

[0028] Plants of the genus Diospyros are listed, according to botanical classification, within the sub-phylum of Angiosperms in the order of Ericales and in the family of Ebenaceae.

[0029] The genus Diospyros comprises about 500 species, widely distributed in tropical and subtropical regions, with a few species occurring in warm temperate zones both north and south of the equator. They are trees or shrubs, deciduous or evergreen. Terminal buds are absent and the tips of the branches sometimes form a thorn. The leaves are alternate, sometimes finely translucent. The flowers are dioecious or polygamous. The male flowers are arranged in axillary cymes, usually on the basal part of the branches of the current year, they are deciduous shortly after anthesis. The female flowers are usually solitary, axillary. The calyx is usually 3-5(-7)-lobed, sometimes truncate. The corolla is urn-shaped, campanulate or tubular, 3-5(-7)-lobed, deciduous. The fruits are fleshy to leathery berries, usually with a persistent, enlarged calyx.

[0030] The designation Diospyros spp. can designate any species belonging to the genus Diospyros.

[0031] Among the species of Diospyros common in Europe, we can cite Diospyros lotus L. or Lotus Persimmon which is a dioecious shrub or tree which can reach 10 m in height. The leaves are broadly lanceolate, with an entire edge, pubescent above when young, with long hairs underneath. The flowers are more or less sessile in the axils of the leaves, greenish or whitish yellow; the corolla has 4-5 fused lobes; the male flowers are bell-shaped, about 5 mm long; the female flowers are a little larger, more deeply divided, with spreading or reflexed lobes. The fruit is a berry the size of a cherry, yellow or blackish-blue, with a pleasant flavor, edible.

[0032] We can also mention Diospyros kaki Lf or Japanese Persimmon, which is a tree native to China, mainly cultivated for its fruit, the persimmon or persimmon. It is widely cultivated outside China, including in Taiwan, and particularly in Japan, where it is naturalized in certain regions.

[0033] It is a tree that can grow up to 12 m high. The deciduous, entire, elliptical leaves are large, leathery, dark green, shiny above, slightly tomentose below. They turn red in autumn. The flowers bloom on the young shoots of the year. They can be, on the same tree, of three types: hermaphrodite (stamino-pistillate), male (staminate) or female (pistillate). Pollination is done by insects. Individuals are generally monoecious with separate male and female flowers on the same tree, but there are also cultivars Dioecious. When a female flower is not fertilized, the fruit still develops, but does not contain seeds. This species is called a parthenocarp. The fruits are large, globular berries depressed at the top, reminiscent of tomatoes, orange-red in color when ripe. They bear the remains of the persistent calyx at their top. The skin is thin. The flesh is juicy, slightly fibrous, and sweet when ripe. Before maturity, in most cultivars, it has a harsh, astringent taste due to the presence of tannins. The skin is covered with a whitish bloom.

[0034] In traditional Chinese medicine, persimmon leaves have been used as a medicine for the treatment of chronic leg ulcer. Later, they were described to treat cough, hematemesis and to increase salivation and then described as effective against internal hemorrhages, hypertension, cerebral arteriosclerosis and coronary diseases.

[0035] Persimmon leaves have been used for centuries as a raw material for therapeutic drinks. The most widely used preparation is persimmon leaf tea, which is highly valued by the Chinese and Japanese and consumed for reducing blood pressure and eliminating cholesterol.

[0036] Diospyros kaki Lf is increasingly popular in the cosmetic and food industries among others because of its skin lightening effects.

[0037] In the leaves of Diospyros kaki Lf several types of compounds have been highlighted (Xieet al., 2015): flavonoids of the flavonol type such as quercetin, isoquercetin, kaempferol or myricitrin, of the flavonol glucoside type such as rutin, glycosylated derivatives of quercetin or kaempferol, numerous triterpenes such as oleanolic acid, ursolic acid, betulinic acid, pomolic acid, lupeol, α-amyrin, kakisaponins A, B and C or kakidiol, naphthoquinones such as diospyrin, isodiospyrin or neodiospyrin, phytosterols such as campesterol, stigmasterol or [3-sitosterol, coumarins, organic acids, tannins in particular.

[0038] Several pharmacological activities have been highlighted and described in the literature for persimmon leaf extracts:

[0039] - cardiovascular activity (increased coronary blood flow). An excerpt standardized in flavonoids (FLDK-P70) protects rats against cerebral ischemia and could be useful in combating neurodegenerative diseases.

[0040] - other activities described: antiatherosclerosis, antidiabetic, antianaphylactic, he mostatic, anticancer, antimicrobial (on E. coli, B. subtilis, S. aureus, S. mutons, activity due to tannins), anti-tyrosinase (by chrysontemin)

[0041] However, to date, no bibliographic data mentions that an extract of Diospyros spp. can have an activity in the reversion of cellular senescence. Summary of the invention

[0042] The inventors have surprisingly demonstrated that extracts of aerial parts of Diospyros spp. are involved in the process of cellular senescence of the skin and promote the reversion of skin cells, in particular fibroblasts, by reducing the number of senescent skin cells in order to combat the signs of aging, but also against the loss of immunity and a degradation of the skin barrier function.

[0043] Indeed, the inventors have demonstrated that extracts of aerial parts of Diospyros spp. make it possible to return senescent cells to a so-called non-senescent cell state and to resume the cell division cycle.

[0044] To evaluate the change of state from senescent cells to a state of non-senescent cells, the inventors analyzed senescent human dermal fibroblasts to observe the seno-reversion effect and cellular mitochondrial activity.

[0045] The subject of the invention is an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for its use in the fight against senescence, more particularly that of the skin.

[0046] The invention also relates to the use of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for combating senescence, more particularly that of the skin.

[0047] The invention also relates to the use of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for the preparation of a cosmetic or dermatological composition for combating senescence, more particularly that of the skin. The cosmetic or dermatological composition will advantageously comprise an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, with at least one cosmetically or dermatologically acceptable excipient.

[0048] The invention also relates to a method for combating senescence, more particularly that of the skin, comprising the administration, preferably by topical application, to a person in need thereof of an effective quantity of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf.

[0049] The invention also relates to a cosmetic or dermatological composition comprising an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, with at least one cosmetically or dermatologically acceptable excipient (composition according to the invention), in particular for its use in the fight against senescence, more particularly that of the skin.

[0050] The invention also relates to the use of a composition according to the invention for combating senescence, more particularly that of the skin.

[0051] The invention also relates to a method for combating senescence, more particularly that of the skin, comprising the administration, preferably by topical application, to a person in need thereof of an effective amount of a composition according to the invention.

[0052] The invention also relates to an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for its use in the fight against skin aging and / or the preservation and / or strengthening of the skin's immune defenses and / or the preservation and / or strengthening of the skin's barrier function.

[0053] The invention also relates to the use of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function.

[0054] The invention also relates to the use of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, for the preparation of a cosmetic or dermatological composition for combating skin aging and / or preserving and / or strengthening the immune defenses of the skin and / or preserving and / or strengthening the skin barrier function. The cosmetic or dermatological composition will advantageously comprise an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, with at least one cosmetically or dermatologically acceptable excipient.

[0055] The invention also relates to a method for combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function, comprising administering, preferably by topical application, to a person in need thereof an effective amount of an extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf.

[0056] The invention also relates to a composition according to the invention for its use in combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function.

[0057] The invention also relates to the use of a composition according to the invention for combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function.

[0058] The invention also relates to a method for combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function comprising the administration, preferably by topical application, to a person in need thereof. of an effective amount of a composition according to the invention.

[0059] The extract of aerial parts of Diospyros spp., and in particular of Diospyros kaki Lf, or the composition according to the invention is intended more particularly to be applied to aged or mature skin, skin damaged by radiation, in particular UV, in particular by the sun. It is therefore preferably skin having senescent cells.

[0060] “Aged” or “mature” skin will in particular show signs of skin aging, such as wrinkles or fine lines, loss of elasticity, sagging, loss of radiance, pigment spots (also called age spots), etc. This will more particularly be the skin of a person aged 45 or over. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0061] In the present invention, the different species of the genus Diospyros may be abbreviated to Diospyros spp. They include in particular Diospyros kaki Lf, Diospyros lotus L. and Diospyros virginiana L.

[0062] In the present invention, the plant Diospyros kaki Lf may be abbreviated to Diospyros kaki; the plant Diospyros lotus L. may be abbreviated to Diospyros lotus; the plant Diospyros virginiana L. may be abbreviated to Diospyros virginiana.

[0063] By "extract of Diospyros spp." is meant the product of extraction of all or part of a plant of a species belonging to the genus Diospyros, and in particular of the aerial parts thereof. Preferably, it is an extract chosen from an extract of Diospyros kaki Lf, an extract of Diospyros lotus L. and an extract of Diospyros virginiana L.

[0064] By "aerial parts of Diospyros spp." is meant the aerial parts of a tree of the genus Diospyros, namely more particularly the leafy branches (stems and leaves) or the leaves.

[0065] By "extraction product" is meant the product obtained after extraction of a part of the plant, with a solvent, called extraction solvent, that is to say a product consisting of numerous phytochemical compounds of the plant and the extraction solvent, and which can then optionally be obtained in a concentrated or dry form after partial or total evaporation of the extraction solvent. It may be a dry extract. In particular, it is not a hydrolyzate such as an enzymatic hydrolyzate.

[0066] For the purposes of the present invention, the term "dry extract" means an extract free of extraction solvent or support, or containing only trace amounts thereof. significant. Such a dry extract of Diospyros spp. thus contains only material from Diospyros spp.

[0067] By “hydroalcoholic extract” is meant an extract obtained using a hydroalcoholic mixture as extraction solvent.

[0068] By "approximately" is meant in the present invention that the value concerned may be 10% lower or higher, in particular 5%, in particular 1%, than the indicated value. This definition applies in particular when defining the weight content of certain flavonoids, relative to the weight of the dry extract.

[0069] By "polar to non-polar solvent" is meant a solvent chosen from the group consisting in particular of water including subcritical water, water-miscible solvents and more particularly water-miscible alcohols (in particular a C1 to C5 alcohol such as for example ethanol), C3 to C5 glycols, glycerol, acetone, alkyl esters (such as ethyl acetate, or isopropyl acetate), heptane, hexane, limonene, halogenated hydrocarbons (e.g. chloroform, dichloromethane), supercritical CO2, hydrotropic solutions, ionic liquids and mixtures thereof.

[0070] For the purposes of the present invention, the term "moderately polar solvent" means a solvent chosen from the group consisting in particular of C1 to C5 alcohols (in particular ethanol), C3 to C5 glycols (such as propylene glycol, butylene glycol, butanediol, or pentylene glycol), glycerol, acetone, alkyl esters (such as ethyl acetate, or isopropyl acetate), water-miscible solvents mixed with water (such as a hydroalcoholic mixture or an acetone / water mixture, for example), a supercritical CO2 and ethanol mixture, hydrotropic solutions, ionic liquids and a mixture thereof.

[0071] By "hydrotropic solution" is meant a mixture of water and hydrotrope.

[0072] By "hydrotrope" is meant a water-soluble amphiphilic molecule which, at from a certain concentration, can extract hydrophobic compounds and in particular moderately polar compounds by allowing their solubilization in water.

[0073] By "ionic liquid" is meant salts having a melting temperature below 100°C and preferably below room temperature. It may be a natural deep eutectic solvent (NaDES: Natural Deep Eutectic Solvent) which is formed from a mixture in well-defined ratios of hydrogen bond donor compounds and hydrogen bond acceptor compounds.

[0074] By “hydroalcoholic mixture” is meant a mixture of water and one or more water-miscible alcohols, in particular a C1 to C5 alcohol such as, for example, ethanol.

[0075] By “topical application” is meant, within the meaning of the present invention, an app- plication on the skin (including the scalp), and the mucous membranes, preferably the skin.

[0076] By "cosmetically or dermatologically acceptable" is meant, for the purposes of the present invention, that which is useful in the preparation of a cosmetic or dermatological composition, which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for cosmetic or dermatological use, in particular by topical application to the skin.

[0077] For the purposes of the present invention, the term “senescent cells” means cells whose cell cycle has stopped, cell proliferation being irreversibly stopped, and expressing a specific secretory phenotype (SASP).

[0078] For the purposes of the present invention, the term “non-senescent cells” means cells whose cell cycle is still active or reactivated if the cell had entered senescence and which proliferate.

[0079] By "reversion of senescent cells" or "senoreversion" is meant that a senescent cell becomes a non-senescent cell again.

[0080] By “fight” or “fight”, we mean, within the meaning of the present invention, prevent, treat or prevent. Extract of aerial parts of Diospyros spp.

[0081] In the context of the present invention, the extract of aerial parts of Diospyros spp. is obtained from one or more parts of a tree belonging to the genus Diospyros chosen from the aerial parts such as the leafy branches (stem and leaves) or the leaves.

[0082] In the context of the present invention, the extract of aerial parts of Diospyros spp. is advantageously an extract of aerial parts, such as leafy branches (stems and leaves) or leaves, of Diospyros kaki, Diospyros lotus, Diospyros virginiana or a mixture thereof; in particular of Diospyros kaki, Diospyros lotus or a mixture thereof; preferably of Diospyros kaki.

[0083] In a particular embodiment of the invention, the extract is obtained from a culture of Diospyros spp. cells, more particularly from cells of aerial parts of Diospyros spp.

[0084] In another particular embodiment of the invention, the extract is obtained from a culture of Diospyros kaki cells, more particularly from cells of aerial parts of Diospyros kaki.

[0085] According to a particular embodiment of the invention, the extract as defined above is a hydroalcoholic extract, in particular a hydro-ethanolic extract.

[0086] According to a preferred embodiment, the extract according to the invention is obtained or capable of being obtained by an extraction process, in particular as described below. More particularly, the process does not comprise an enzymatic hydrolysis step.

[0087] The aerial parts of the Diospyros spp. plant can be fresh or dried, whole, cut or crushed then subjected to an extraction step.

[0088] A method for preparing an extract according to the invention comprises a step of extracting aerial parts, such as leafy branches (stem and leaves) or leaves, of the plant Diospyros spp. by an extraction solvent which is a polar to apolar solvent, preferably a moderately polar solvent.

[0089] The extraction solvent is advantageously chosen from the group consisting of C1 to C5 alcohols (in particular ethanol), C3 to C5 glycols (such as propylene glycol, butylene glycol, butanediol, or pentylene glycol), glycerol, acetone, alkyl esters (such as ethyl acetate, or isopropyl acetate), water-miscible solvents mixed with water (such as a hydro-alcoholic mixture or an acetone / water mixture for example), a supercritical CO2 and ethanol mixture, hydrotropic solutions, ionic liquids (e.g. NaDES) and a mixture thereof.

[0090] In one embodiment of the invention, the extraction solvent may be chosen from ethyl acetate, isopropyl acetate, a supercritical CO2 and ethanol mixture, a C3 to C5 glycol, a C1 to C5 alcohol, a hydro-alcoholic mixture, a hydrotropic solution, a NaDES and a mixture thereof.

[0091] Advantageously, it will be a hydroalcoholic mixture, in particular a C1 to C5 alcohol / water mixture and preferably an ethanol / water mixture.

[0092] More advantageously, this ethanol / water mixture will be characterized by an ethanol / water proportion of 9.5:0.5 to 1:9 (v / v), in particular of 9.5:0.5 to 1:1 (v / v), preferably of 9:1 to 6:4 (v / v), for example of 8:2 to 6:4 (v / v) or of 9:1 to 7:3 (v / v), in particular about 7:3 (v / v).

[0093] According to a particular embodiment of the invention, the extraction is carried out with stirring in batch or continuous operation, at reflux, at room temperature, or at a temperature between room temperature and reflux, in particular between 50°C and reflux, preferably at reflux. It can be assisted by ultrasound, microwaves, flash detent or extrusion. The extraction can be carried out in particular in a ratio of plant weight (aerial parts) / volume of extraction solvent which can vary from 1 / 3 to 1 / 30, in particular from 1 / 5 to 1 / 15, in particular from 1 / 5 to 1 / 10 (kg / L), for example for a period of 1 minute to 48 hours, in particular from 1 minute to 24 hours, in particular from 1 minute to 10 hours, more particularly from 1 minute to 5 hours, in particular from 15 to 120 minutes. The extraction can be repeated several times, including 1, 2 or 3 times.

[0094] Advantageously, the extraction of Diospyros spp. is carried out conventionally with stirring at a temperature between 50°C and reflux, with a plant weight / extraction solvent volume ratio of 1 / 5 to 1 / 15 (kg / L) for 15 to 120 minutes.

[0095] According to another particular embodiment of the invention, the marc is then separated from the extract by centrifugation or filtration in order to recover a clear liquid phase free of particles. The liquid phase representing the extract can then be more or less concentrated by evaporation of the extraction solvent to obtain a dry extract.

[0096] In another embodiment of the invention, a support may be added during the concentration step so as to obtain an extract containing 1 to 75% dry extract.

[0097] For the purposes of the present invention, the term "support" means a substance capable of solubilizing or dispersing (e.g. by adsorption) within itself the extract according to the invention in a homogeneous manner.

[0098] The support may be maltodextrin, lactose, silica, acacia or arabic gum, glycerin or a glycerin / water mixture, a glycol, a vegetable oil, a hydrotrope, a water / solubilizer mixture (the solubilizer being a compound capable of solubilizing the extract in water) or water / surfactant, or any other cosmetically acceptable support, preferably of bio-sourced origin, such as for example bio-sourced glycols, in particular C3 to C5 (1,2-pentanediol, 1,3-butanediol, 1,3-propanediol, etc.), esterified fatty acids and also hydrotropes such as for example alkyl glycosides (Sepiclear, Apyclean, APXC4, etc.). Advantageously, the extract of Diospyros spp. is placed on a maltodextrin support or on a glycerin / water mixture.

[0099] According to a particular embodiment of the invention, the extract of Diospyros spp. can be decolorized (in particular in order to remove chlorophyll), for example on activated carbon or by concentration / precipitation / filtration. Advantageously, the extract of Diospyros spp. is decolorized on activated carbon.

[0100] According to a particular embodiment of the invention, the extract of Diospyros spp. as described above is characterized by the presence, among others, of flavonoids (genin of the quercetin, myricetin or kaempferol type), glycosylated or not, esterified by a phenolic acid or not and triterpenic acids such as pomolic acid or tormentic acid.

[0101] Thus, the extract may contain, for example, myricitrin (glycosylated myricetin) or astragalin (glycosylated kaempferol).

[0102] The composition of Diospyros spp. extracts can vary qualitatively and quantitatively depending on the species considered, but the majority phytochemical families remain the same: flavonoids and triterpenic acids.

[0103] The extract will advantageously be in a form suitable for topical application. Compositions

[0104] In the context of the present invention, the compositions according to the invention comprise an extract of aerial parts of Diospyros spp. as described above, of preferably an extract of aerial parts, such as leafy twigs (stems and leaves) or leaves, of Diospyros kaki.

[0105] According to one embodiment, the compositions according to the invention contain from 0.01% to 10%, in particular from 0.02% to 5%, in particular from 0.05% to 3%, more particularly from 0.08% to 1%, of extract of aerial parts of Diospyros spp. as described above by weight of dry extract relative to the total weight of the composition.

[0106] Preferably, the compositions according to the invention contain approximately 0.1% of dry extract of aerial parts of Diospyros spp. as described above by weight of dry extract relative to the total weight of the composition.

[0107] More particularly, the compositions according to the invention comprise an extract of aerial parts, such as leafy branches (stems and leaves) or leaves, of Diospyros kaki.

[0108] According to one embodiment, the compositions according to the invention contain from 0.01% to 10%, in particular from 0.02% to 5%, in particular from 0.05% to 3%, more particularly from 0.08% to 1%, of extract of aerial parts, such as leafy branches (stems and leaves) or leaves, of Diospyros kaki by weight of dry extract relative to the total weight of the composition.

[0109] Preferably, the compositions according to the invention contain approximately 0.1% of extract of aerial parts, such as leafy branches (stems and leaves) or leaves, of Diospyros kaki by weight of dry extract relative to the total weight of the composition.

[0110] The compositions according to the invention are advantageously intended for topical application, in particular by application to the skin. They are therefore more particularly in a form suitable for topical application. The composition according to the invention can in particular be applied to sensitive, fragile and / or reactive skin.

[0111] The compositions according to the invention may thus be presented in the forms which are usually known for topical administration, that is to say in particular lotions, milks, emulsions, serums, balms, ointments, masks, creams, dispersions, gels, mousses or sprays.

[0112] They can also be presented in solid form, such as a stick, or be applied to the skin in the form of an aerosol. These compositions may in particular be presented in the form of an oily solution or an emulsion such as an oil-in-water emulsion, a water-in-oil emulsion or a multiple emulsion. Applications

[0113] The extract according to the invention as defined above and the composition according to the invention as defined above are useful in combating senescence, more particularly cellular senescence, in particular that of the skin.

[0114] The extract according to the invention as defined above and the composition according to the invention as defined above are more particularly intended to reduce the number of senescent cells in the skin, in particular the reversion of senescent skin cells.

[0115] The extract according to the invention as defined above and the composition according to the invention as defined above are also intended to promote the proliferation of fibroblasts.

[0116] The extract according to the invention as defined above and the composition according to the invention as defined above are also useful in combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function.

[0117] The fight against skin aging includes in particular the prevention and / or reduction of wrinkles and fine lines, firming of the skin, improvement of skin radiance, prevention and / or reduction of pigment spots, etc.

[0118] The preservation and / or strengthening of the skin's immune defenses includes in particular the preservation and / or strengthening of innate immunity.

[0119] Preserving and / or strengthening the skin barrier function involves, for example, preventing or reducing the alteration of the skin barrier function, maintaining or increasing the thickness of the skin, etc.

[0120] The extract according to the invention or the composition according to the invention is intended more particularly to be applied to aged or mature skin, skin damaged by radiation, in particular UV, in particular by the sun. It is therefore preferably skin with senescent cells.

[0121] “Aged” or “mature” skin will in particular show signs of skin aging, such as wrinkles or fine lines, loss of elasticity, sagging, loss of radiance, pigment spots (also called age spots), etc. This will more particularly concern the skin of a person aged 45 or over.

[0122] The following examples illustrate the invention without limiting its scope. EXAMPLES

[0123] Example 1: 70% reflux ethanol extract of decolorized Diospyroslotus leafy stems

[0124] 20 grams of crushed dry leafy stems of Diospyros lotus are brought into contact with 140 milliliters of 70% (v / v) ethanol and then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel and the marc is rinsed with 50 milliliters of 70% (v / v) ethanol. Then the extract is decolorized by adsorption on activated carbon and filtered again. The solvent is then evaporated to obtain 2.94 grams of a powder with a mass yield of 14.7%. The dry extract obtained contains 5.9% by weight of myricitrin-type flavonoids.

[0125] Example 2: 90% reflux ethanol extract of leafy stems of discolored Diospyros lotus

[0126] 20 grams of crushed dry leafy stems of Diospyros lotus are brought into contact with 140 milliliters of 90% (v / v) ethanol and then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel and the marc is rinsed with 50 milliliters of 90% (v / v) ethanol. Then the extract is decolorized by adsorption on activated carbon and filtered again. The solvent is then evaporated to obtain 2.0 grams of a powder with a mass yield of 10%. The dry extract obtained contains 6.9% by weight of myricitrin-type flavonoids.

[0127] Example 3: 70% ethanol extract with ultrasound of Diospyroskaki leaves

[0128] 9.84 grams of crushed dry leaves of Diospyros kaki are brought into contact with 70 milliliters of 70% (v / v) ethanol in a beaker then extracted under the action of ultrasound (1000 W, 20 kHz) while stirring for 2 minutes. The extract is then filtered under vacuum on Buchner and the marc is rinsed with 30 milliliters of 70% (v / v) ethanol. The solvent is then evaporated so as to obtain 1.34 grams of a powder with a mass yield of 13.6%. The dry extract obtained contains 1.4% by weight of astragalin-type flavonoids.

[0129] Example 4: 70% reflux ethanol extract of Diospyroskaki leaves

[0130] 49.2 grams of crushed dry leaves of Diospyros kaki are brought into contact with 350 milliliters of 70% (v / v) ethanol then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel and the marc is rinsed with 120 milliliters of 70% (v / v) ethanol. The solvent is then evaporated to obtain 7.97 grams of a powder with a mass yield of 16.2%. The dry extract obtained contains 1.2% by weight of astragalin-type flavonoids.

[0131] Example 5: 70% reflux ethanol extract of bleached Diospyros kaki leaves

[0132] 50 grams of crushed dry leaves of Diospyros kaki are brought into contact with 350 milliliters of 70% (v / v) ethanol then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel and the marc is rinsed with 150 milliliters of 70% (v / v) ethanol. Then the extract is decolorized by preconcentration and filtered again. The solvent is then evaporated to obtain 7.93 grams of a powder with a mass yield of 15.9%. The dry extract obtained contains 0.4% by weight of astragalin-type flavonoids.

[0133] Example 6: 70% reflux ethanol extract of leafy stems of discolored Diospyros kaki

[0134] 1626 grams of crushed dry leafy stems of Diospyros kaki are put into contact with 11.5 liters of 70% (v / v) ethanol then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel and the marc is rinsed with 5 liters of 70% (v / v) ethanol. The extract is then decolorized by adsorption on activated carbon and filtered again. The solvent is then evaporated to obtain 149 grams of a powder with a mass yield of 9%. The dry extract obtained contains 0.74% by weight of astragalin-type flavonoids.

[0135] Example 7: Obtaining senescent human dermal fibroblasts

[0136] Senescent human dermal fibroblasts were obtained from primary cultures of human dermal fibroblasts obtained after mammoplasties or abdominoplasties from two donors. Senescence of human dermal fibroblasts was induced by exposure to ionizing radiation (X-ray) according to Bogdanowicz et al (2023). The cells were suspended in phosphate-buffered saline (PBS) and irradiated using an XRAD Smart Irradiator (7.5 Gy; CREFRE, CRCT, Toulouse, France). After exposure to ionizing radiation, cells were seeded in 175 cm2 culture dishes in MEM (minimum essential medium) culture medium (Invitrogen, Villebon sur Yvette, France), supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 50 U / mL penicillin, and 50 mg / mL streptomycin, and cultured for 14 days. Fresh medium was added every other day.Cells are grown in monolayer at 37°C under 5% CO2 in 175 cm2 flasks.

[0137] Example 8: Evaluation of the seno-reversion effect of extracts according to the invention

[0138] At T=0, the seeding of senescent human dermal fibroblasts (obtained in example 7) at 10,000 cells / well of 96-well plate was carried out in culture medium (Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS)).

[0139] 24 hours after seeding, incubation of cells with different concen The extraction of Diospyros spp. extracts (Diospyros kaki and Diospyros lotus are tested) is carried out in culture medium (DMEM + 10% FCS). In parallel, controls (only dimethyl sulfoxide (DMSO) solvent) are carried out without the addition of Diospyros spp. extract.

[0140] After 72 hours of incubation, the medium containing the Diospyros spp. extract is replaced with culture medium (DMEM + 10% FCS).

[0141] 7 days after seeding, the medium is removed and a solution of neutral red (Sigma # N2889) is added to the culture wells. Neutral red is a eurhodin dye that stains the lysosomes of viable cells. Neutral red is indeed a weakly cationic vital dye that accumulates in the lysosomes of living cells (Borenfreund and Puerner, 1985). The absorbance is then measured with a spectrophotometer (Clariostar, 540 nm). The neutral red test is used to measure cell viability and therefore to assess the cytotoxicity of a compound.

[0142] For this, the adherent fibroblasts are first incubated for three hours with the dye, then washed with a washing solution. Then the dye incorporated into the viable cells is extracted using a 1% acetic acid solution for 20 min. Finally, an optical density (OD) reading at 540 nm is carried out using a CLARIOstrar spectrometer (BmgLabtech). In conclusion, the higher the OD at 540 nm, the redder the medium and therefore the more viable cells there are.

[0143] The results obtained are presented in Table 1 below.

[0144] [Tables 1] Compound tested Average in % - extract tested at 100 pg / ml Average in % - extract tested at 300 pg / ml Solvent control (DMSO 0.3%) 100 100 Extract 1 of Diospyros lotus obtained according to example 1 154.67 170.38 Extract 2 of Diospyros lotus obtained according to example 2 131.28 158.74 Extract 3 of Diospyros kaki obtained according to example 3 151.84 213.97 Extract 4 of Diospyros kaki obtained according to example 4 163.82 185.12 Extract 5 of Diospyros kaki obtained according to example 5 131.91 164.00 Extract 6 of Diospyros kaki obtained according to example 6 142.72 150.19

[0145] The control containing only DMSO solvent confirms that the number of cells remains constant and that they do not multiply or proliferate. The result of 100% was expected and thus validates this test.

[0146] For all the extracts tested, a general and significant increase in the number of cells is observed in the presence of an extract of Diospyros spp. independently of the extract of Diospyros lotus or the extract of Diospyros kaki.

[0147] The Diospyros lotus extract obtained according to example 1 induces an increase in the number of cells in a concentration-dependent manner (50% increase at 100pg / mL and 70% increase at 300pg / ml compared to the control treated with DMSO). This concentration-dependent effect is also observed for all the extracts tested above and obtained according to examples 1 to 6.

[0148] The inventors thus demonstrate that these different tested extracts of Diospyros lotus and persimmon allow the reversion of senescent cells into non-senescent cells which leads to a proliferation of fibroblasts.

[0149] Example 9: Evaluation of the seno-reversion effect of a reference antioxidant compound (fisetin)

[0150] The aim of this example is to investigate whether or not an antioxidant compound having anti-aging properties has a reversion activity on senescent cells.

[0151] To do this, a study is carried out on senescent fibroblasts according to example 8, the antioxidant compound being fisetin. Fisetin is a compound of the flavonoid family described as a powerful antioxidant (Khan et al., 2013) and also has senolytic properties in vitro and in vivo (Zhu Y et al., 2017).

[0152] The results obtained in this study are presented in Table 2 below.

[0153] [Tables2] Tested compound Average Control (0.3% DMSO) 97.25 Fisetin at 3.2.108 mol / L 101.59 Fisetin at 3.2.107 mol / L 107.54 Fisetin at 3.2.106 mol / L 110.87 Fisetin at 105 mol / L 103.91

[0154] These results demonstrate that this antioxidant compound does not have senescent cell reversion properties.

[0155] It is interesting to note that a compound with anti-aging efficacy may have no effect in the reversion of senescent cells.

[0156] Example 10: Evaluation of mitochondrial activity / proliferation of extracts according to the invention

[0157] The protocol is similar to that of Example 8 up to 7 days. At 7 days, a solution of WST-1 (Promega) is added for 4 hours. Mitochondrial activity causes the increase in the amount of formazan dye formed which is quantified by measuring the absorbance at 440 nm (Clariostar). The higher the measured value, the greater the metabolic activity.

[0158] The results obtained are presented in Table 3 below.

[0159] [Tables3] Compound Concentration (expressed in pg / ml) Mean ESM Solvent control DMSO 0.3% 100 3.81 Extract 5 of Diospyros kaki obtained according to example 5 50 101.42 1.24 100 105.86 1.89 200 116.09 2.11 300 120.74 1.60 Extract 6 of Diospyros kaki obtained according to example 6 50 101.01 1.36 100 112.06 1.08 200 114.00 2.71 300 114.61 1.28

[0160] SEM = standard error of the mean

[0161] With the control condition, the metabolism of senescent cells is inactive as expected. On the other hand, in the presence of a Diospyros kaki extract, mitochondrial activity increases in a concentration-dependent manner. This test demonstrates that the metabolism of senescent cells is reactivated. These results are in perfect agreement with the results of Example 8.

[0162] The inventors have thus demonstrated that extracts of Diospyros spp. make it possible to reactivate cells which were in senescence. BIBLIOGRAPHICAL REFERENCES

[0163] • Bogdanowicz, P. et al. (2023) Reduction of senescence-associated secretory phenotype and exosome-shuttled miRNAs by Haritaki fruit extract in senescent dermal fibroblasts. Int J Cosmet Sci. 45(4):488-499. • Borenfreund, E. and Puemer, JA (1985) Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24(2-3):119-24. • Fontanilla, P. et al. (2020) Aging: A story of DNA damage, altered nuclear envelope, and inflammation? Medicine / science, vol. 36, 1118-28. • Khan, N. et al. (2013) Fisetin: a dietary antioxidant for health promotion. Antioxid Redox Signal. 19(2):151-62. • Kumari, R. and Jat J. (2021) Mechanisms of Cellular Senescence: Cell Cycle Arrest and Senescence Associated Secretory Phenotype. Forehead. Cell Dev. Biol., vol. 9, item 645593. Zhu, Y. et al. (2017) New agents that target senescent cells: the flavone, fisetin, and the BCL-XL inhibitors, A1331852 and Al 155463. Aging (Albany NY). 9(3):955-963. Xie C., Xie Z., Xu X., Yang D. (2015) Persimmon (Diospyros kaki L.) leaves: A review on traditional uses, phytochemistry and pharmacological properties. Journal of Ethnopharmacology 163, 229-240.

Claims

Claims

1. Extract of aerial parts of Diospyros spp. for its use in combating senescence, in particular cellular senescence, in particular that of the skin.

2. Extract for use according to claim 1, intended to reduce the number of senescent cells in the skin, in particular by reversion of senescent skin cells.

3. Extract for use according to claim 1 to 2, intended to promote the proliferation of fibroblasts.

4. Extract of aerial parts of Diospyros spp. for use in combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin's barrier function.

5. Extract for use according to any one of claims 1 to 4, characterized in that it is chosen from an extract of aerial parts of Diospyros kaki Lf, an extract of aerial parts of Diospyros lotus L., an extract of aerial parts of Diospyros virginiana L. and a mixture thereof.

6. Extract for use according to any one of claims 1 to 5, characterized in that it is obtained by hydroalcoholic extraction, preferably by extraction using an ethanol / water mixture.

7. Cosmetic or dermatological composition comprising an extract of aerial parts of Diospyros spp. and at least one cosmetically or dermatologically acceptable excipient, for use in combating senescence, in particular cellular senescence, in particular that of the skin.

8. Composition for use according to claim 7, intended to reduce the number of senescent cells in the skin, in particular by reversion of senescent skin cells.

9. Composition for use according to claim 7 or 8, intended to promote the proliferation of fibroblasts.

10. Cosmetic or dermatological composition comprising an extract of aerial parts of Diospyros spp. and at least one cosmetically or dermatologically acceptable excipient, for use in combating skin aging and / or preserving and / or strengthening the skin's immune defenses and / or preserving and / or strengthening the skin barrier function.

11. Composition for use according to any one of claims 7 to 10, characterized in that it comprises from 0.01% to 10% of extract of aerial parts of Diospyros spp. by weight of dry extract relative to the total weight of the composition.

12. Composition for use according to any one of claims 7 to 11, characterized in that the extract of aerial parts of Diospyros spp. is chosen from an extract of aerial parts of Diospyros kaki Lf, an extract of aerial parts of Diospyros lotus L., an extract of aerial parts of Diospyros virginiana L. and a mixture thereof.

13. Composition for use according to any one of claims 7 to 12, characterized in that the extract of aerial parts of Diospyros spp. is a hydroalcoholic extract, preferably hydro-ethanolic.

14. Composition for use according to any one of claims 7 to 13, characterized in that it is in a form suitable for topical application.