Composition including silybum marianum extract as senotherapeutic agent

Abstract

To provide a senotherapeutic composition that is of natural origin, has reduced undesired side effects, and is obtainable with low effort.SOLUTION: The present invention relates to the use of a composition including or consisting of an extract of flowers of Silybum marianum from which seeds have been removed including less than 0.1 wt.% of silymarin based on the total weight of a dry extract, as a senotherapeutic agent for reducing cellular senescence in a tissue. Furthermore, the present invention relates to a composition including or consisting of an extract of flowers of Silybum marianum from which seeds have been removed as a senotherapeutic agent for use in a method for treating or preventing a disorder associated with cellular senescence in a tissue.SELECTED DRAWING: None

Description

Technical Field

[0001] Field of the Invention The present invention relates to the (cosmetic) use of a composition comprising or consisting of an extract of the flowers of Silybum marianum from which the seeds have been removed, as an agent for treating target senescent cells for reducing cellular senescence in tissues present in the skin, wherein the composition contains less than 0.1% by weight of silymarin based on the total weight of the dry extract. Furthermore, the present invention relates to a composition comprising or consisting of an extract of the flowers of Silybum marianum from which the seeds have been removed, as an agent for treating target senescent cells for use in a method for treating or preventing disorders associated with cellular senescence in tissues present in the skin. Accordingly, the present invention relates to an active ingredient consisting of an extract of the flowers of Silybum marianum from which the seeds have been removed, containing less than 0.1% by weight of silymarin based on the total weight of the dry extract, and its use. In particular, the present invention relates to use as a senotherapeutic active ingredient in the prevention and / or treatment of senescent cells associated with aging or stress in tissues; and / or in the treatment of disorders associated with cellular senescence in tissues; more generally, in the delay and / or prevention of the appearance of signs of tissue aging and tissue deterioration, for an extract of the flowers of Silybum marianum from which the seeds have been removed, containing less than 0.1% by weight of silymarin based on the total weight of the dry extract.

Background Art

[0002] Cellular senescence is a physiological process and is the fate of tumor-suppressive cells characterized by a permanent and irreversible cell cycle arrest and the acquisition of a pro-inflammatory and proteolytic secretome. Aging over time typically correlates with an increase in senescent cells, including skin cells. Indeed, the skin is the first barrier of the human body. It protects the organs from differences in temperature and humidity and from attacks by the external environment. However, excessive chemical and physical stimuli degrade the normal function of the skin and induce its aging.

[0003] Skin aging is driven by a multi - factor process due to both endogenous (e.g., time, genetic factors, hormones) and exogenous (e.g., UV exposure, pollution) factors. Characteristics of endogenous or chronological aging include visible signs such as thin and dry skin, fine wrinkles, reduced elasticity, abnormal pigmentation, hair graying and hair loss.

[0004] Among them, epidermal thinning has been reported to cause a decrease in the proliferation and regeneration ability of basal keratinocytes. In addition to the epidermis, both the dermal - epithelial junction (DEJ) and the dermis also often become thinner. Fibroblasts present in the dermis produce the extracellular matrix (ECM) that provides structural integrity and elasticity to the skin. During aging, the ECM typically undergoes structural changes and degradation, leading to an increase in wrinkles and loss of skin elasticity. In adult tissues, aging is activated upon cell damage as a defense mechanism that contributes not only to the normal physiological process of biological aging in many cell types in response to various stresses but also to the tumor - promoting unrestricted growth signals for tumorigenesis. Cellular aging often promotes tissue remodeling through the following three consecutive processes: stable growth arrest, the senescence - associated secretory phenotype (SASP) that recruits immune cells and modifies the extracellular matrix, and the mobilization of neighboring precursors that reconstruct the tissue.

[0005] Aging markers are described in the process of skin changes associated with aging. Among them, plasminogen activator inhibitor type 1 (PAI - 1), an inhibitor of serine protease, is elevated in skin fibroblasts derived from patients with premature aging syndromes and donors who have aged over time. Furthermore, studies on aging markers have reported that PAI - 1 is a mediator of cell cycle arrest due to its role in inducing aging downstream of the essential cell cycle checkpoint p53.

[0006] Senescent cells exhibit characteristic morphological features, high activity of senescence-associated β-galactosidase (SA-β-gal), increased senescence-associated secretory phenotype (SASP) such as cytokines and matrix metalloproteinases, decreased lamin B1 expression, and translocation of nuclear HMGB1 to the cytoplasm (Wang and Dreesen, 2018: Frontiers in Genetics, Vol.9, Article 247) (Non-Patent Document 1). Furthermore, permanent growth arrest is driven by telomere shortening and cell cycle arrest initiated by enhanced p53 activity and increased p21 and p16INK4A-Rb expression (Lozano-Torres et al., 2019: Nature Reviews in Chemistry, Vol. 3, pages 426-441) (Non-Patent Document 2).

[0007] In addition to the loss of proliferative capacity, senescent cells often lead to enhanced survival and resistance to apoptosis through the activation of various mechanisms called the senescent cell anti-apoptotic pathway (SCAP). These pathways are directly related to many characteristics, among which are growth arrest, metabolic reprogramming, and the intrinsic and extrinsic apoptotic pathways.

[0008] Senescent cells exert pleiotropic effects that can be explained by their ability to secrete numerous inflammatory cytokines (e.g., IL-1α, IL-1β, IL-6, IL-8, (γ-HMGB1)), chemokines (e.g., CXCR2), growth factors (e.g., IGFBP7), and proteases such as matrix metalloproteinases (MMP-1 and MMP-3), known as the senescence-associated secretory phenotype (SASP). Depending on the physiological context, SASP secretion can be either beneficial or harmful, particularly when persistent senescent cells negatively impact the surrounding microenvironment by compromising tissue homeostasis. Indeed, some SASP components, such as IL-6 and IL-8, can enhance senescence-associated growth arrest (SAGA) via autocrine pathways mediated by secreted and membrane-bound IL-1α. However, the same secreted components can act in paracrine signaling to adjacent cells, propagating the senescent phenotype and thus potentially interfering with the regenerative capacity of the surrounding tissue. Furthermore, SASP cytokines promote the infiltration of immune cells, contributing to persistent and chronic inflammation, a hallmark of aging, and age-related dysfunction. In vivo, elevated IL-6 has been detected in nevus melanocytes, while MMPs have been detected in chronologically aged and photoaged skin over time, which are responsible for the degradation of the extracellular matrix (ECM). In senescent cells, HMGB1 translocates from the nucleus to the cytoplasm and extracellular space, promoting the release of SASP factors including IL-1β, IL-6, and MMP-3. SASP secretion is associated with aging and can be decoupled from cell cycle arrest. Indeed, studies in primary human fibroblasts have demonstrated that DNA damage elicits SASP independently of p16INK4A levels. One important actor for SASP transcription is the phosphorylation of the transcription factor NF-kB, which translocates to the nucleus where it binds to the promoters of several SASP genes.

[0009] Senolytics, which are typically understood as agents that can selectively kill senescent cells, were the first potential senotherapy to be successfully tested in preclinical in vivo models. In other words, an agent is considered to have senolytic activity if its activity involves specifically inducing or promoting cell death of senescent cells in tissues, particularly apoptotic cell death. Several senolytic agents have been currently identified, including quercetin that inhibits the PI3K pathway and acts on the Bcl-2 family, p53 / p21, dasatinib that interferes with the dependency receptor EFNB; ABT-763; and navitoclax (also called ABT-263) that targets the Bcl-2 / Bcl-xL proteins. Other senolytic agents are the peptide FOXO4-D-Retro Inverso and 17-DMAG (alvespimycin). Such senolytic agents can selectively kill senescent cells by inducing the apoptosis process but do not kill non-senescent or normal cells. To date, research on agents that selectively kill senescent cells has focused only on single compounds, and little research has been done on mixtures of compounds such as plant extracts.

[0010] Senomorphics are typically understood as agents that suppress markers of senescence or their secretory phenotypes without inducing apoptosis and provide a senomorphic effect by a mechanism different from that of senolytics. In other words, an agent is considered to have senomorphic activity if its activity involves suppressing the senescent phenotype in senescent cells in tissues.

[0011] According to the literature, it has not been described that the above-mentioned senolytic agents can exert senomorphic activity in senescent cells.

[0012] According to the above, preventing the generation or accumulation of senescent cells within an organization seems to be an interesting strategy for preventing or treating aging-related or stress-related tissue deterioration caused by senescent cells. In fact, senescent fibroblasts account for 20 - 60% of all skin cells. However, blocking p16INK4A and p53 or activating telomerase to expand the cell proliferation ability will inevitably lead to an increased cancer risk. Furthermore, MMP-1 and MMP-3, or inflammatory cytokines such as IL-6 and IL-8 secreted from senescent fibroblasts are known as SASP that play important roles in the degradation of ECM that causes skin deterioration. Therefore, a balanced approach that ensures the prevention of the increase in the number of senescent cells and at the same time limits the expansion of cell proliferation ability should be considered to combat aging-related and stress-related cellular senescence within an organization.

[0013] Furthermore, selectively eliminating senescent cells can extend lifespan, improve the healthy period, and may be beneficial for a wide range of disease treatment and prevention (Gonzalez-Gualda et al., 2020, Aging Cell. 2020;19:e13142) (Non-Patent Document 3). These findings provide the validity that suppressing senescent cells is a good target for counteracting the aging process and tissue deterioration. As a result, a reliable approach consists of selectively eliminating senescent cells and / or regulating their functions, especially SASP.

[0014] Some senescent cell targeted treatment agents including senolytic agents and senomorphic agents are known in the art.

[0015] US-A 2019 / 054097 (Patent Document 1) discloses several fairly complex synthetic small molecule senolytic agents that, like ABT-263, selectively kill senescent cells. These compounds also make it possible to interfere with the anti-apoptotic Bcl-2 pathway, which is also involved in cancer. WO2020 / 122392 (Patent Document 2) discloses zotarolimus, a fairly complex immunosuppressant that is a derivative of sirolimus (rapamycin) for use as a senomorphic agent to rejuvenate senescent cells. WO2020 / 132053 (Patent Document 3) discloses dasatinib and quercetin as senescent cell-targeted therapeutic agents for treating obesity-induced neuropsychiatric disorders such as anxiety. These therapeutic agents can have severe undesirable side effects.

[0016] US-A2020 / 121620 (Patent Document 4) teaches senolytic agents for killing senescent cells, focusing on synthetic small molecule drugs typically used for therapeutic applications in other fields. Also, KR-A2020 / 0072924 (Patent Document 5) teaches senolytic agents for killing senescent cells including fibroblasts.

[0017] The drawback of these aforementioned agents and compositions containing such agents is that they focus on specific agents. These are often of synthetic origin, have fairly complex chemical structures, require considerable effort to obtain, and have severe undesirable side effects.

[0018] Therefore, it is desirable to obtain yet another composition having senotherapeutic substances of natural origin with reduced undesirable side effects and obtainable with low effort.

[0019] Nature provides a wide variety of plant-based agents and compositions, including plant extracts.

[0020] The plant Silybum marianum has traditionally been known to be effective for improving liver function, reducing total cholesterol levels, treating diabetes, preventing motion sickness and heart disease. Silybum marianum is sometimes also referred to as (blessed) milk thistle. On the other hand, the name "milk thistle" is ambiguous. It can refer to different species, such as Lactuca serriola, various Sonchus species (e.g., Sonchus asper, Sonchus arvensis, Sonchus oleraceus), as well as Silybum marianum.

[0021] Moreover, it was also considered for changing the appearance of the skin. EP-B1845935 (Patent Document 6) teaches that the compound silymarin or isosilibin can have a number of effects including affecting hair and skin pigmentation. EP-B2658522 (Patent Document 7) teaches the topical administration of a combination of Silybum marianum and Momordica grosvenori extracts that have antioxidant effects and reduce cellular stress factors. US-A2010 / 316743 (Patent Document 8) teaches that, among many other agents, milk thistle extract can be used to promote the inhibition of the formation of advanced glycation end products (AGE). CN-A103961276 (Patent Document 9) teaches milk thistle extract for anti-glycation cosmetic compositions in addition to a number of other plant extracts. CN-B104382833 (Patent Document 10) teaches a plant peptide solution that can contain, inter alia, milk thistle extract as an anti-wrinkle agent and skin improver. JP-A2002 / 034505 (Patent Document 11) teaches a food composition having reduced bitterness and containing polysaccharides and silymarin that can be extracted from milk thistle. US-A2019 / 175677 (Patent Document 12) teaches a Silybum marianum fruit extract containing less than 0.2% silymarin for use in the treatment of acne, seborrhea, seborrheic dermatitis and / or rosacea. WO2014 / 151891 (Patent Document 13) discloses an Nrf2 activator containing a plant extract containing milk thistle extract. This document also mentions a Silybum marianum seed extract that can contain 80% by weight of silymarin as an active ingredient. EP-B0180505 (Patent Document 14) teaches silymarin that can be extracted from milk thistle for obtaining an anti-inflammatory effect. Senotherapeutic uses, particularly developing senolytic and / or senomorphic activities, i.e., targeting senescent cells in tissues, are considered not to have been contemplated. KR-A20200063855 (Patent Document 15) teaches a hydroethanol extract of the flower lacking the seeds of Silybum marianum for promoting melanin production, preventing vitiligo, and for hair whitening and hypopigmentation.However, the above-mentioned senotherapeutic effects have not been disclosed. The hydroethanol extract of the flowers of Silybum marianum containing silybin (the main component of silymarin) has been described to inhibit the formation of glycated proteins in the skin by scavenging free radicals (protection of collagen and elastin) (Seoungwoo Shin et al., Molecules 2015, 20, 3549-3564; doi:10.3390 / molecules20033549) (Non-Patent Document 4).

Prior Art Documents

Patent Documents

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Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Patent Document 9

Patent Document 10

Patent Document 11

Patent Document 12

Patent Document 13

Patent Document 14

Non-Patent Literature

[0023]

Non-Patent Literature 1

Non-Patent Literature 2

Non-Patent Literature 3

Non-Patent Literature 4

Summary of the Invention

Problems to be Solved by the Invention

[0024] There is still an unmet need for senotherapeutic compositions that are of natural origin, have low unwanted side effects, and can be obtained with low effort.

Means for Solving the Problems

[0025] Surprisingly, it has been found that an extract of flowers lacking the seeds of Withania somnifera can be used as an agent for targeting senescent cells. Furthermore, it has surprisingly been found that a composition comprising (or consisting of) at least one extract of flowers lacking the seeds of Withania somnifera as an agent for targeting senescent cells can be used to reduce cellular senescence in tissues and to treat or prevent disorders associated with cellular senescence in tissues present in the skin.

[0026] Accordingly, the present invention relates to the (optionally cosmetic) use of an extract of the flowers of Serenoa repens with seeds removed as an agent for treating senescent cell targets.

[0027] The present invention also relates to an effective amount of an extract of the flowers of Serenoa repens with seeds removed, which contains less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract; or to a composition comprising at least said extract, for the prevention and / or treatment of senescent cells associated with aging or stress in the skin and hair follicles by administering it to the skin or scalp in need thereof; and / or for the treatment of disorders associated with cellular aging in the skin and hair follicles, and relates to a cosmetic skin and scalp care method.

[0028] A first aspect of the present invention relates to the (optionally cosmetic) use of a composition comprising (or consisting of) an extract of the flowers of Serenoa repens with seeds removed, which contains less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, as an agent for treating senescent cell targets to reduce cellular aging in tissues present in the skin.

[0029] Accordingly, the present invention also relates to a method (which is optionally a cosmetic method) for reducing cellular aging in a tissue, which comprises administering to the tissue a sufficient amount of a composition comprising (or consisting of) an extract of the flowers of Serenoa repens with seeds removed, which contains less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, as an agent for treating senescent cell targets.

[0030] Accordingly, the present invention also relates to a method (optionally a cosmetic method) for reducing cell aging in tissues present in the skin of a subject, the method comprising administering, as an agent for targeted treatment of aging cells, to the tissues of the subject, a composition comprising (or consisting of) an extract of the flowers of Silybum marianum from which the seeds have been removed, said composition containing less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract.

[0031] A further aspect of the present invention relates to a composition comprising (or consisting of) an extract of the flowers of Silybum marianum from which the seeds have been removed, said composition containing less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, for use as an agent for targeted treatment of aging cells for use in a method for treating or preventing a disorder associated with cell aging in tissues present in the skin.

[0032] Accordingly, the present invention also relates to a composition comprising an extract of the flowers of Silybum marianum from which the seeds have been removed, said composition containing less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, for use in the prevention and / or treatment of aging cells associated with aging or stress in tissues present in the skin; and / or for use as a senotherapeutic active ingredient in the treatment of disorders associated with cell aging in said tissues.

[0033] Accordingly, the present invention also relates to a method for treating or preventing a disorder associated with cell aging in tissues present in the skin, the method comprising administering to the tissue a sufficient amount of a composition comprising (or consisting of) an extract of the flowers of Silybum marianum from which the seeds have been removed, the composition containing less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, as an agent for targeted treatment of senescent cells in the tissue.

[0034] Accordingly, the present invention also relates to a non-therapeutic method for treating or preventing a disorder associated with cell aging in tissues present in the skin of a subject, the method comprising administering to the tissue in the subject a sufficient amount of a composition comprising (or consisting of) an extract of the flowers of Silybum marianum from which the seeds have been removed as an agent for targeted treatment of senescent cells in the tissue.

[0035] Accordingly, the present invention also relates to a method for treating or preventing a disorder associated with cell aging in a subject, the method comprising administering to the subject a sufficient amount of a composition comprising (or consisting of) an extract of the flowers of Silybum marianum from which the seeds have been removed as an agent for targeted treatment of senescent cells in the subject.

[0036] The present invention also relates to the use of an extract of the flowers of Silybum marianum from which the seeds have been removed, or a composition comprising at least said extract, for preventing and / or treating aging cells associated with aging or stress in tissues, preferably in the skin and hair follicles; and / or for use in cosmetology as a senotherapeutic active ingredient in the treatment of disorders associated with cell aging in tissues, preferably in the skin and hair follicles.

[0037] It has been found that an extract of the flowers of Withania somnifera lacking the seeds of the present invention exhibits new beneficial effects in different tissues as compared to those described in the prior art. In fact, it has been demonstrated that the extract of Withania somnifera from which the seeds have been removed can, firstly, selectively kill senescent cells (e.g., skin and hair follicle cells). Thus, it has been found that the said extract of Withania somnifera has senolytic activity. Secondly, such an extract has been found to regulate the phenotype of senescent cells and to help these cells to remove these harmful cells. Thus, it has been found that the said extract of Withania somnifera has senomorphic activity. This may include the regulation of SASP secretion. It may also involve the proliferative capacity of these senescent cells.

Brief Description of the Drawings

[0038] Figures 1 (A, B, C) show the senolytic activity of Withania somnifera flower extract (SME) in a senescent replication model of human dermal fibroblasts (HDF). Figure 1A shows a comparison of the cell viability of non-senescent and senescent cells untreated or treated with ABT-737 or SME. Figure 1B shows the number of SA-β-Gal positive cells, where senescent cells treated with ABT-737 are compared with senescent cells treated with different concentrations of SME. Figure 1C shows a comparison of the decrease in p21 expression in senescent cells treated with ABT-737 with senescent cells treated with different concentrations of SME. Figures 2 (A, B, C) relate to the senolytic activity of Withania somnifera flower extract (SME) in a senescent replication model of HDF by induction of apoptosis specifically via the caspase-3 / PARP pathway in senescent HDF. Figure 2A shows apoptosis in non-senescent and senescent cells treated with ABT-737 or SME. Figure 2B shows cleaved caspase-3 in non-senescent and senescent cells treated with ABT-737 or SME. Figure 2C shows cleaved poly-ADP-ribose polymerase (PARP) in non-senescent and senescent cells treated with ABT-737 or SME. Figures 3 (A, B, C, D) relate to the senomorphic activity of Serenoa repens flower extract (SME) in a model of HDF senescent replication by inhibition of SASP factor release. Figure 3A shows the expression of p-H2AX in senescent cells treated with ABT-737 or SME. Figure 3B shows the expression of TNF-α in senescent cells treated with ABT-737 or SME. Figure 3C shows the expression of IL-6 in senescent cells treated with ABT-737 or SME. Figure 3D shows the secretion of MMP-1 in senescent cells treated with ABT-737 or SME. Figures 4 (A, B) relate to the senomorphic activity of Serenoa repens flower extract (SME) in a model of HDF senescent replication by restoration of the proliferative capacity of senescent HDF. Figure 4A shows the percentage of cell proliferation in senescent cells treated with ABT-737 or SME. Figure 4B shows the doubling time of cells in senescent cells treated with ABT-737 or SME. Figure 5 relates to the senomorphic activity of Serenoa repens flower extract (SME) in a model of HDF senescent replication by restoration of the type I collagen synthesis ability of senescent HDF. Figure 6 relates to the senolytic activity of Serenoa repens flower extract (SME) in a model of human dermal papilla cell (HFDPC) senescent replication.

[0039] Detailed Description of the Invention The first aspect of the present invention relates to the use, optionally cosmetic, of a composition comprising (or consisting of) an extract of the flower of Serenoa repens with seeds removed, which contains less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, as an agent for treating target senescent cells for reducing cell senescence in tissues present in the skin.

[0040] More generally, the extract according to the invention may be able to delay and / or prevent the appearance of signs of tissue aging and tissue degradation. In a composition consisting of an extract of the flowers of Silybum marianum from which the seeds have been removed, it will be understood that the composition is an extract.

[0041] In particular, the experimental in vitro tests provided in the present application have demonstrated that the extract of the flowers of Silybum marianum without seeds according to the invention achieves senolytic and / or senomorphic activity by targeting senescent cells, particularly skin and hair follicle cells. More advantageously, the said extract of Silybum marianum exerts senolytic and senomorphic activity in the said senescent cells. As a result, the said extract of Silybum marianum according to the invention can be used as an agent that makes it possible to be compatible with a senotherapeutic approach.

[0042] The plant Silybum marianum belongs to the Asteraceae family (white patterned thistle), is a perennial or biennial plant, and its leaves typically have transparent white stripes. Originally, this plant is native from southern Europe to Asia. It is now found throughout the world. The common name "Silybum" is derived from the Greek name by Dioscorides and is widely known as blessed milk thistle because a milky liquid comes out of the split leaves. Silybum marianum is traditionally known to be effective for improving liver function, reducing the total cholesterol level, treating diabetes, preventing motion sickness and heart disease.

[0043] When used through the present invention, the extract of the flowers of Silybum marianum without seeds means the flowers of Silybum marianum from which the seeds have been removed. Preferably, when used herein, the term "flower" essentially refers only to the flowers of Silybum marianum from which the seeds have been removed.

[0044] The extract according to the present invention can be obtained by any means. Optionally, it may be obtained from a commercial supplier, or may be prepared partially or completely from the Serenoa repens plant or one or more parts thereof.

[0045] The extract may be any type of extract. It can be prepared from fresh flowers of Serenoa repens, or from dried flowers of Serenoa repens, or from frozen or lyophilized flowers of Serenoa repens. In either case, the seeds are removed.

[0046] Preferably, the flowers of Serenoa repens can be used in a shredded, mixed or ground form. In a preferred embodiment, the extract is prepared from dried flowers of Serenoa repens, particularly from dried and ground flowers of Serenoa repens.

[0047] As used herein, the term "extract" can be understood in the broadest sense generally understood in the art. An extract can be any substance made by extracting a portion of the raw material (here, the flowers of Serenoa repens with the seeds removed). Typically, extraction is achieved by using a solvent. Thus, in a preferred embodiment, the extract is obtained from a solvent extraction method including cold extraction or hot extraction, ultrasonic extraction, reflux cooling, needle extraction and microwave extraction. In a preferred embodiment, the extract is obtained by performing an ultrasonic extraction method.

[0048] Here, a solvent can be added to the flowers of Serenoa repens and placed together for a time as described herein. In a further step, the solid part is preferably separated by primary extraction. This can be achieved by any means, for example, filtration, sieving, ultrafiltration, cross-flow filtration, centrifugation, precipitation over time, or a combination of two or more of them.

[0049] One or more solvents may be used for extraction. Such solvents may be any solvent suitable for this purpose. Typically, the solvent is liquid under the conditions under which it is used for extraction. The solvent may be selected from the group consisting of organic solvents (e.g., alcohols (e.g., methanol, ethanol, propanol, butanol, pentanol, phenol, glycerol, 1,3-butylene glycol, propanediol, etc.), water (including hot water or subcritical water), aqueous buffers, and combinations of two or more thereof. The organic solvent may be aliphatic or aromatic. Preferably, the solvent may be hydrated.

[0050] The milk thistle flower extract according to the present invention can contain any content of silymarin.

[0051] The component "silymarin" can be understood as generally understood in the art. It can be understood as a mixture of four flavonolignans, namely silybin (also called silybinin), isosilybin, silychristin, and silydianin.

[0052] In a preferred embodiment of the present invention, the extract contains less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract.

[0053] In a preferred embodiment, the milk thistle flower extract is a hydroalcoholic extract or an alcoholic extract. Preferably, the extract is a hydroalcoholic extract.

[0054] Also, the hydroalcoholic or alcoholic extract can be a paste or solid in which the water and alcohol contents are partially or completely removed in one or more further steps after the extraction step. In a preferred embodiment, the hydroalcoholic or alcoholic extract is a dry powder.

[0055] In a preferred embodiment, the extract of the flowers of Seribum marianum is a hydroethanol extract obtained by extraction with an ethanol / water mixture containing 10 - 90% (v / v) ethanol, or containing 25 - 85% (v / v) ethanol, or containing 50 - 82% (v / v) ethanol, or containing 60 - 80% (v / v) ethanol, or containing 65 - 75% (v / v) ethanol, or containing approximately 70% (v / v) ethanol.

[0056] Also, the hydroethanol or ethanol extract can be a paste or solid in which the water and ethanol contents have been partially or completely removed in one or more further steps after the extraction step. In a preferred embodiment, the hydroethanol or ethanol extract is a dry powder.

[0057] The extraction can be carried out at any temperature suitable for this purpose. Preferably, the extraction is carried out at a temperature in the range of 10 - 150°C, or 15 - 100°C, or 20 - 80°C, or 25 - 70°C, or 15 - 25°C, or at approximately 20°C. In a preferred embodiment, the extraction is carried out at ambient temperature (i.e., 15 - 25°C, particularly approximately 20°C).

[0058] In a preferred embodiment, the extraction is carried out at ambient temperature (i.e., 15 - 25°C, particularly approximately 20°C) and ambient pressure (i.e., 980 - 1200 hPa).

[0059] In a preferred embodiment, the extraction is carried out at ambient temperature (i.e., 15 - 25°C, particularly approximately 20°C) and ambient pressure (i.e., 980 - 1200 hPa) using an ethanol / water mixture containing 65 - 75% (v / v) ethanol, particularly approximately 70% (v / v) ethanol.

[0060] In a preferred embodiment, the Schisandra mariana flower extract preferably comprises an extract of Schisandra mariana with seeds removed, and the extraction is carried out using an ethanol / water mixture containing 65 - 75% (v / v) ethanol, particularly about 70% (v / v) ethanol, at ambient temperature (i.e., 15 - 25°C, particularly about 20°C) and ambient pressure (i.e., 980 - 1200 hPa).

[0061] The extraction can be carried out for any period of time suitable for this purpose. For example, the extraction may be carried out for a time in the range of less than 30 minutes, 30 minutes to 1 hour, 1 hour to 2 hours, 90 minutes to 6 hours, 2 hours to 24 hours, 6 hours to 36 hours, 12 hours to 2 days, 1 day to 1 week, 1 week to 1 month, or more than 1 month. In a preferred embodiment, the extraction can be carried out for a time in the range of 6 hours to 18 hours, 20 hours to 2 days, illustratively about 10 hours to 90 hours, 20 hours to 70 hours, or 40 hours to 60 hours (preferably at an ambient temperature of 15 - 25°C). It may be carried out overnight.

[0062] Accordingly, a method for producing an extract of Schisandra mariana according to the present invention includes the following steps: (i) providing a flower lacking Schisandra mariana seeds (optionally dried); (ii) contacting the flower with at least one alcoholic solvent, wherein: (a) the alcohol extraction is carried out at 15 - 60°C, preferably 20 - 50°C, more preferably 25 - 30°C, for 1 hour to 24 hours, preferably 2 hours to 20 hours, most preferably 5 - 15 hours; and / or; (b) the hydroalcoholic ultrasonic extraction is carried out at 15 - 100°C, preferably 15 - 80°C, most preferably 15 - 50°C, for 1 hour to 24 hours, preferably 2 hours to 20 hours, most preferably 5 - 15 hours; (iii) separating the extract from the solid residue of step (ii); and (iv) optionally concentrating the extract by removing at least one of the solvents or a portion thereof.

[0063] The extract according to the invention can be liquid, pasty or solid under standard conditions (room temperature of about 20 °C, standard pressure of about 1013 hPa). In this context, the terms "liquid" and "fluid" can be understood interchangeably. Preferably, the extract is obtained in liquid form first. Then, one or more solvents can optionally be removed to obtain the solid form of the extract. The liquid extract can also be concentrated. Preferably, then, part of one or more solvents is removed. Optionally, a liquid or pasty extract may be obtained. Optionally, the extract may also be diluted with one or more solvents. Optionally, the extract may also be purified by any means (e.g., crystallization, chromatography, etc.). The extract may optionally be referred to as a "tincture" or an "absolute".

[0064] The extract of the flowers of Silybum marianum according to the invention may optionally be obtained from fractions produced by performing ultrafiltration membranes having a certain molecular weight cut-off value, or separation by ion exchange, or various chromatographic methods, or other purification methods including liquid-liquid separation or crystallization or precipitation.

[0065] For example, the extract of the flowers of Silybum marianum according to the invention can be prepared by drying the flowers lacking the seeds of Silybum marianum, grinding it, subsequently extracting it with alcohol or hydroalcohol, and then filtering it under reduced pressure.

[0066] In a preferred embodiment of the invention, the extract comprises or consists of an (preferably hydroalcoholic, particularly hydroethanol) extract of the flowers of Silybum marianum from which the seeds have been removed, and the extract contains less than 0.1% by weight, or less than 0.01% by weight, or less than 0.001% by weight, or 0% by weight of silymarin, based on the total weight of the dry extract.

[0067] In a preferred embodiment, the Schisandra mariana flower extract is a hydroethanol extract obtained by extracting Schisandra mariana flowers with the seeds removed using an ethanol / water mixture containing 60 - 80% (v / v) ethanol (preferably at ambient temperature, for example 15 - 25°C), and the extract contains less than 0.1% by weight of schisandrin, or less than 0.01% by weight of schisandrin, or less than 0.001% by weight of schisandrin, or 0% by weight of schisandrin, based on the total weight of the dry extract.

[0068] As used herein, the term "dry extract" can be understood in the broadest sense as the extract according to the invention as a dry substance, i.e., without solvents. The dry extract may optionally be physically present as a dry substance. However, it will be directly understood that in calculations referring to the percentage of the dry substance, it is not necessary for the dry extract to be present in physical form, and the dry extract may be present in dissolved form. Next, in the calculation, the mass of the solvent is mathematically subtracted from the total mass.

[0069] The dry substance (in physical form) can be obtained by a suitable extraction method and subsequent steps of drying the obtained extract. Drying can be carried out by any method suitable for this purpose known in the art. Drying means removing one or more solvents used for extraction. Removal of one or more solvents can be carried out by any means. For example, it can be achieved by subjecting the extract to a high-temperature drying atmosphere and / or placing the extract on a heating plate to evaporate the solvent. For example, drying can be achieved by evaporation under vacuum and / or elevated temperature (e.g., in a rotary (vacuum) evaporator), or by crystallization of the solid material. Alternatively or additionally, drying can be achieved by atomization or by freeze-drying.

[0070] According to the present invention, the flower extract of Withania somnifera can have any senotherapeutic activity. It can, by any means, reduce cellular senescence in a tissue and / or treat or prevent disorders associated with cellular senescence in the tissue.

[0071] In a preferred embodiment, the flower extract of Withania somnifera has senolytic activity and / or senomorphic activity.

[0072] In a preferred embodiment, the flower extract of Withania somnifera has senolytic activity, including specifically inducing or promoting the cell death of senescent cells in a tissue. In this context, the cell death is preferably apoptotic cell death.

[0073] Preferably, the extract exhibits senolytic activity. In other words, the flower extract of Withania somnifera is important as a senolytic agent or senolytic component.

[0074] When used throughout the present invention, the term "senolytic" can be understood in the broadest sense generally understood in the art. Senolytic activity refers to inducing or promoting the cell death of senescent cells. It can kill senescent cells. It can eliminate senescent cells. The killing can induce or promote apoptosis.

[0075] Preferably, senolytic activity refers to specifically (or: selectively) inducing or promoting the cell death of senescent cells. This can also be considered as the ability to selectively eliminate senescent cells, for example, by inducing apoptosis. It can also be the ability to selectively kill senescent cells found in a tissue with reduced or even no harmful effects on normal resident (i.e., non-senescent) cells of the said tissue. Such senolytic properties can be beneficial, for example, to prevent or attenuate an increase in the number of senescent cells associated with aging and / or stress within a tissue.

[0076] As used herein, the expression "specifically induce or promote cell death of senescent cells" can be understood in the broadest sense as having stronger activity against senescent cells than against corresponding (i.e., comparable) non-senescent cells. In the present specification, the term "specifically" may be selective. The corresponding, and thus comparable, cells are typically of the same tissue type origin and differ essentially only in the degree of senescence. In a preferred embodiment, the induction or promotion of cell death results in more than 1.05-fold, or more than 1.1-fold, or more than 1.2-fold, or more than 1.3-fold, or more than 1.4-fold, or more than 1.5-fold, or more than 2-fold, against senescent cells compared to corresponding (i.e., comparable) non-senescent cells.

[0077] In a preferred embodiment, the flower extract of Withania somnifera has senomorphic activity including suppressing the senescence phenotype in senescent cells in a tissue, restoring the metabolic processes in senescent cells in a tissue, or a combination of two or all of them.

[0078] Preferably, the extract exhibits senomorphic activity. In other words, the flower extract of Withania somnifera is important as a senomorphic agent or a senomorphic component.

[0079] When used throughout the present invention, the term "senomorphic" can be understood in the broadest sense generally understood in the art. Senomorphic activity can include, or be based on, the suppression of the senescent phenotype in senescent cells in a tissue. It can include the ability to inhibit the secretion of senescence-associated secretory phenotype (SASP) factors without killing senescent cells and / or to reverse the growth arrest of senescent cells and / or to restore its ability to maintain the normal metabolic processes of senescent cells, particularly senescent skin and hair follicle cells. Senomorphic activity can include modulating or even reversing the phenotype of senescent cells to that of young cells (or non-senescent cells) by interfering with the induction of senescent cells, targeting senescent cells, reducing or blocking SASP factor secretion, and / or promoting the growth capabilities previously lost by those senescent cells. Senomorphic activity can include suppressing or reducing the secretion of SASP factors by senescent cells and / or restoring the ability to maintain the normal metabolic processes of said senescent cells in order to extend the healthy period and potentially lifespan. Senomorphic activity can be beneficial, for example, for preventing and / or treating cell senescence-related disorders within a tissue, particularly in the skin and hair. The compositions of the present invention can be used to selectively kill senescent cells and / or to inhibit SASP release from senescent cells and / or to regenerate cell proliferation ability and / or to prevent senescence-induced cell deterioration and / or to restore metabolic processes in senescent cells or mixtures thereof.

[0080] As used herein, examples of SASP factors can be selected from the group consisting of tumor necrosis factor alpha (TNF-α), inflammatory cytokines such as IL-1α, IL-1β, IL-6, IL-8; metalloproteinases such as MMP-1 and MMP-3 and γ-H2AX.

[0081] The flower extract of Seribum marianum can optionally also function as a mediator of the immune system that enables (partial) clearance of senescent cells.

[0082] In a preferred embodiment, the extract of the flowers of Seribum marianum has both senolytic activity and senomorphic activity.

[0083] In a preferred embodiment, the extract of the flowers of Seribum marianum has one or more of the following activities in senescent cells in tissue, selected from the group consisting of: (a) inhibiting the release of senescence-associated secretory phenotype (SASP) factors from senescent cells in tissue, in particular, the SASP factors are selected from the group consisting of inflammatory cytokines such as interleukin (IL) selected from the group consisting of γ-H2AX, tumor necrosis factor α, IL-1α, IL-1β, IL-6 and IL-8, and matrix metalloproteinases (MMPs) such as MMP-1 and MMP-3 selected from the group consisting of MMPs; (b) regenerating the cell proliferation ability of senescent cells; (c) preventing senescence-induced cell deterioration of senescent cells; (d) restoring metabolic processes in senescent cells, and (e) inducing or promoting cell death of senescent cells.

[0084] According to a preferred embodiment of the present invention, the extract of the flowers of Seribum marianum or a cosmetic composition containing said extract can be suitable for use in and / or is intended to be used in selectively killing senescent skin and hair follicle cells, and / or inhibiting SASP release from senescent skin and hair follicle cells, and / or regenerating the proliferative capacity of skin and hair follicle cells, and / or preventing senescence-induced skin and hair follicle deterioration, and / or restoring metabolic processes in senescent skin and hair follicle cells or their mixtures.

[0085] In a preferred embodiment, the composition contains 0.0001 to 20% by weight, preferably 0.001 to 15% by weight, more preferably 0.005 to 10% by weight, and particularly 0.01 to 5% by weight of the extract of the flowers of Serenoa repens, based on the total weight of the composition.

[0086] In addition to the extract of the flowers of Serenoa repens, the composition may contain any further additional components. Preferably, such one or more further additional components are suitable for use in cosmetics and / or pharmaceuticals.

[0087] Therefore, the composition according to the present invention may comprise: (A) 0.0001 to 20% by weight, preferably 0.001 to 15% by weight, more preferably 0.005 to 10% by weight, and particularly 0.01 to 5% by weight of the extract of the flowers of Serenoa repens with seeds removed, based on the total weight of the composition; and (B) at least one cosmetically and / or pharmaceutically acceptable carrier. Here, the extract of the flowers of Serenoa repens can be regarded as one (or the only) active ingredient. The term "active ingredient" can be understood in the broadest sense as a component that can exhibit the desired and intended activity alone or, together with one or more carriers that are inactive per se, can exhibit the activity. Preferably, the active ingredient of the present invention is a cosmetic active ingredient.

[0088] As used herein, the terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "cosmetically acceptable carrier", "cosmetically acceptable excipient", "carrier" and "excipient" can be understood interchangeably in the broadest sense as any substance that can support the cosmetic and / or pharmacological acceptability of the composition of the present invention containing the extract of the flowers of Serenoa repens.

[0089] Preferably, neither the extract of the flowers of Serenoa repens according to the present invention nor the composition containing such an extract is toxic when administered to tissues.

[0090] For each of the compositions according to the present invention, techniques for formulation and administration are well known in the art of the present invention.

[0091] The ready-to-use composition is preferably a liquid formulation, particularly a composition suitable for topical administration. The storage form of the composition may also be liquid, but may also be a dry form (e.g., powder, e.g., a powder containing or consisting of the flower extract of Serenoa repens), or a paste or syrup, etc. Optionally, the dry form, paste or syrup may be dissolved or emulsified before being administered to the tissue of interest.

[0092] Cosmetically and / or pharmaceutically acceptable carriers can typically be selected from a list consisting of aqueous buffers, saline, water, alcohol, vegetable oils, mineral oils, polymers, gases or combinations of two or more thereof.

[0093] Optionally, a cosmetic or pharmaceutically acceptable carrier may contain one or more cosmetic or pharmaceutically acceptable additives. In a preferred embodiment, such cosmetic and / or pharmaceutically acceptable additives may be selected from the group consisting of fragrances / aromatics, dyes, pigments, emulsifiers, lubricants, chelating agents, acidity regulators, antibacterial agents, preservatives, antioxidants, and combinations of two or more thereof. For example, a cosmetic and / or pharmaceutically acceptable carrier may optionally contain one or more detergents, triethanolamine, one or more fragrances, one or more foaming agents (e.g., sodium lauryl sulfate (SLS), sodium dodecyl sulfate (SDS)), one or more colorants (e.g., food colorants, pigments), one or more vitamins, one or more salts (e.g., sodium, potassium, calcium, zinc salts), one or more humectants (e.g., sorbitol, glycerol, butylene glycol, propylene glycol, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preservatives (e.g., benzoic acid, methyl paraben), one or more antioxidants, one or more herbal and plant extracts, one or more stabilizers, one or more chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)), one or more polymers (e.g., carboxyvinyl polymer, carboxymethyl cellulose, cellulose, carboxyethyl cellulose), one or more stabilizers, one or more solubilizers, one or more skin softeners, and / or one or more uptake mediators (e.g., polyethyleneimine (PEI), cell-penetrating peptide (CPP), protein transduction domain (PTD), antimicrobial peptide, etc.).

[0094] Furthermore, in addition to the extract of the flowers of Seribum marianum, the composition of the present invention can contain one or more other active ingredients that act synergistically and / or complementarily with the extract of the flowers of Seribum marianum without affecting its activity or its intended use as described above.

[0095] The composition according to the present invention may be a cosmetic or may be included in a cosmetic. Adverse effects on the skin can be reduced or avoided in accordance with safety tests and legal requirements.

[0096] The composition can be for any use. Any route of administration that provides the desired purpose described in the claims is appropriate. Administration can be local or systemic. Preferably, the administration is local. Administration can be topical administration, transdermal administration, oral administration, administration by injection (e.g., intravenous (i.v.), intraarterial (i.a.), intraperitoneal (i.p.), intramuscular (i.m.), and subcutaneous (s.c.) injection), or nasal administration. For example, the composition according to the present invention can be formulated in any suitable form for topical, oral, rectal, transmucosal, nasal, enteral, perenteral, and parenteral administration, together with acceptable carriers and excipients.

[0097] In a preferred embodiment, the composition is a composition for topical use. In a preferred embodiment, the composition is a composition for topical use that is topically administered to a subject having tissue. In other words, the composition according to the present invention can be topically administered. In a preferred embodiment, the composition is a composition for topical use that is topically administered to a part of the skin, such as the face, scalp, or a part of the body, or to the hair. The skin can be any type of skin, including dry skin, normal skin, and oily skin.

[0098] The extract of the flowers of Seribum mariananum can have local and / or systemic effects. In a preferred embodiment, when administered locally and / or topically, it has (mainly or completely) local effects.

[0099] As used in the context of the present invention, the term "subject" can be understood in the broadest sense as any living organism, preferably any animal, more preferably a mammal including a human, particularly a human.

[0100] The composition may be a composition for any use. Preferably, the composition is a cosmetic composition, a pharmaceutical composition, or a nutraceutical composition. In a preferred embodiment, the composition is selected from the group consisting of an emulsion, a gel, an ointment, a toner, a liquid soap, a solid soap, a bath oil, a shower oil, a massage oil, a makeup, a scalp treatment, an aftershave, a shaving product, a deodorant, a shower gel, a shampoo, and combinations of two or more thereof. In a preferred embodiment, the composition is a composition for external use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a surfactant-containing water, an oil, and a spray.

[0101] In an alternative preferred embodiment, the composition is a nutraceutical composition orally administered to a subject having tissue. The extract of the flowers of Silybum marianum may optionally be included in a food, such as a food supplement. Thus, typically, the extract of the flowers of Silybum marianum has a systemic effect. The nutraceutical composition according to the present invention can be formulated into any conventional form suitable for oral administration, such as tablets, capsules, granules, powders, solutions, emulsions and suspensions, together with an acceptable carrier or excipient.

[0102] Cell aging in the context of the present application can be understood in the broadest sense. It can have any reason. In a preferred embodiment, cell aging results from the presence of senescent cells associated with aging, senescent cells associated with stress, or both.

[0103] The tissue from which senescent cells are derived and / or in which senescent cells are located may be any tissue. In a preferred embodiment, the tissue is selected from the group consisting of tissues present in the skin, particularly dermal tissue, epidermal tissue, subcutaneous tissue, hair follicle tissue, and combinations of two or more thereof.

[0104] The intended reduction of cellular senescence can be attempted for any cosmetic and / or therapeutic purpose. In a preferred embodiment of the present invention, the reduction of cellular senescence in a tissue is for improving the appearance or other properties of the skin, hair, or both.

[0105] Senescent cells may be of any origin. In a preferred embodiment, senescent cells are derived from the skin, particularly dermal tissue, epidermal tissue, subcutaneous tissue, hair follicle tissue, and combinations of two or more thereof. In a preferred embodiment of the present invention, senescent cells are derived from tissues selected from the skin, hair follicles, and the body. More generally, the tissue can be composed of cells of mesenchymal origin. Here, senescence can be induced by both endogenous and exogenous factors. Preferably, the tissue can be selected from the skin and / or hair follicles. In a preferred embodiment, senescent skin cells are selected from fibroblasts, melanocytes, keratinocytes, endothelial cells, and adipocytes. In a preferred embodiment, the senescent cells are senescent skin cells selected from fibroblasts, melanocytes, keratinocytes, endothelial cells, and adipocytes.

[0106] In a preferred embodiment of the present invention, reducing cellular senescence in a tissue is for improving the elasticity of the skin, the firmness of the skin, the thickness of the skin, the skin tone, the skin radiance, the skin barrier, the skin moisturization, the rejuvenation of the skin, or two or more of them, or for preventing or ameliorating the sagging of the skin, hyperpigmentation of the skin, hypopigmentation of the skin, loss of elasticity and firmness of the skin, erythema, poor skin barrier function, DNA damage, hair loss, thinning of the hair, hair fragility, grey - hair, or two or more of them.

[0107] Accordingly, a composition containing the flower extract of Seribum marianum can be used to treat, prevent, or improve skin elasticity, skin firmness, skin thickness, skin sagging, skin color, skin radiance, skin barrier, skin moisturization, skin rejuvenation, skin hyperpigmentation, skin hypopigmentation, loss of skin elasticity and firmness, erythema, skin barrier dysfunction, DNA damage, hair loss, hair thinning, hair fragility, white hair, or a mixed state thereof.

[0108] Some fields in which the present application can be used also relate to therapeutic / pharmaceutical uses.

[0109] Accordingly, a further aspect of the present invention also relates to a composition comprising or consisting of an extract of the flower of Seribum marianum with seeds removed, as an agent for treating aging cells, for use in a method for treating or preventing disorders related to cell aging in tissues present in the skin.

[0110] It will be understood that the definitions and preferred embodiments made in the context of the cosmetic uses of the present invention apply, with the necessary modifications, to the compositions for the uses of the present invention.

[0111] In other words, the present application also relates to a method for treating or preventing disorders related to cell aging in tissues present in the skin, wherein the tissue is administered to the tissue in a sufficient amount of a composition comprising or consisting of an extract of the flower of Seribum marianum (as an agent for treating aging cells).

[0112] As used herein, "agent" can be understood in the broadest sense as any component of the composition. It may be a complex component containing different chemical substances, i.e., compounds, or a component consisting of a single component. The extract of the flower of Seribum marianum according to the present invention is preferably a complex component containing many chemical substances. The agent for treating aging cells can also be referred to as a "senotherapeutic component".

[0113] The present invention also relates to a cosmetic composition comprising an extract of a flower lacking seeds of Silybum marianum for use as a senotherapeutic active ingredient in the prevention and / or treatment of aging cells associated with aging or stress in the skin and hair follicles; and / or in the treatment of disorders associated with cellular aging in the skin and hair follicles.

[0114] The present invention also relates to a pharmaceutical composition comprising an extract of a flower of Silybum marianum from which the seeds have been removed for use as a senotherapeutic active ingredient in the treatment of disorders associated with cellular aging in tissues present in the skin; and / or for attenuating aging cells associated with aging in said tissues.

[0115] The present invention also relates to a nutraceutical composition comprising at least an extract of a flower of Silybum marianum from which the seeds have been removed for use as a senotherapeutic active ingredient in the prevention and / or treatment of aging cells associated with aging or stress in the skin and hair; and / or in the treatment of disorders associated with cellular aging in the skin and hair.

[0116] The present invention also relates to a method for treating or preventing a disorder associated with cellular aging in a tissue present in the skin of a subject, wherein the tissue is administered to the tissue of the subject in a sufficient amount of a composition comprising or consisting of an extract of a flower of Silybum marianum from which the seeds have been removed (as an aging cell target treatment agent). The present application also relates to a method for treating or preventing a disorder associated with cellular aging in a subject, wherein a tissue present in the skin is administered to the subject in a sufficient amount of a composition comprising or consisting of an extract of a flower of Silybum marianum from which the seeds have been removed (as an aging cell target treatment agent).

[0117] The present invention also relates to an extract of the flowers of Serenoa repens with seeds removed (as an active ingredient) for use as a senotherapeutic active ingredient in the prevention and / or treatment of aging cells associated with aging or stress in tissues present in the skin; and / or in the treatment of disorders associated with cellular aging in said tissues.

[0118] In view of the above, the present invention also relates to an extract of the flowers of Serenoa repens with seeds removed for use as a senotherapeutic active ingredient in the prevention and / or treatment of aging cells associated with aging or stress in tissues present in the skin; and / or in the treatment of disorders associated with cellular aging in said tissues.

[0119] The disorder to be treated may be any disorder associated with cellular aging. As used herein, the term "disorder" can be understood in the broadest sense as any condition (e.g., a pathological condition) to be treated. It may also be a congenital or acquired disease.

[0120] A subject having a tissue of interest may be affected by a disorder or may have a risk of developing a disorder. As used herein, the term "affected by" can be understood in the broadest sense in the manner that the subject has developed the relevant (pathological) condition. A subject affected by a disorder does not necessarily have medical symptoms and optionally has medical symptoms. The phrases "has a risk" or "has a risk of developing" mean that the subject has a certain risk of having a disorder in the sense of the present invention. In the context of treatment, a subject may also be referred to as a "patient".

[0121] In a preferred embodiment of the present invention, the disorder is selected from the group consisting of (pathological) skin dryness, (pathological) hyperpigmentation, (pathological) hypopigmentation, (pathological) loss of skin elasticity and firmness, erythema, (pathological) hair loss, thinning of hair, hair fragility, gray hair, and tissue fibrosis diseases.

[0122] The disorder may also be a disorder associated with cellular senescence in the tissue. As used herein, the term "disorder associated with cellular senescence in the tissue" may include any physiological disorder in the tissue, including, for example, the skin disorders defined above and tissue fibrosis diseases.

[0123] The disorder may also be a disorder associated with cellular senescence in the skin and hair in the tissue. As used herein, the term "disorder associated with cellular senescence in the skin and hair" can be understood in the broadest sense as a physiological disorder in these tissues, for example, by the accumulation or increased amount of senescent skin and hair follicle cells compared to non-senescent (normal) cells in the tissue.

[0124] The disorder can be induced by both endogenous and exogenous factors, such as time, genetic factors, hormones, light, UV exposure, pollution; more generally external and environmental factors. Examples of the disorder can be wrinkles, fine lines, sagging, dryness, dullness, hyperpigmentation, hypopigmentation, loss of skin elasticity and firmness, erythema, impaired skin barrier function, DNA damage, hair loss, thinning of hair, hair fragility and gray hair.

[0125] Surprisingly, it has been found that extracts containing less than 0.01% by weight of silymarin are particularly beneficial for the above purposes.

[0126] It will be understood that the definitions and preferred embodiments made in the context of the cosmetic use of the present invention and the compositions for the use of the present invention apply, with the necessary modifications, to the compositions of the present invention.

[0127] Throughout this specification and the claims, unless the context requires otherwise, open-ended phrases such as "comprise", "contain", "include", etc., and variations such as "comprises", "contains", "includes", "comprising", "containing", "including", etc., are to be understood to mean including the recited element, component, integer or step, or group of elements, components, integers or steps, but not to exclude any other element.

[0128] The terms "a", "an", and "the" and similar references used in the context of describing the present invention (especially in the context of the claims) are to be construed to cover both the singular and the plural forms unless otherwise indicated herein or clearly contradicted by the context. The description of a range of values herein is merely intended to serve as a shorthand way of referring individually to each separate value within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually and collectively recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contrary to the context. The use of any and all examples, or exemplary language (e.g., "such as", "for example") provided herein is merely intended to better illuminate the invention and is not intended to limit the scope of the invention as otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0129] All components of the present invention will be described in more detail. Although these elements are listed with specific embodiments, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and embodiments described should not be construed as limiting the present invention to only the explicitly described embodiments. This description is to be understood as supporting and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed components. Further, any substitution and combination of all components described in this application should be considered to be disclosed by the description of this application, unless the context indicates otherwise.

[0130] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although the methods and materials described herein are preferred, other methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0131] All documents cited or referenced herein are incorporated by reference and can be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0132] The following examples and drawings are intended to illustrate further embodiments of the present invention without limiting its scope.

Example

[0133] Example 1. Preparation of Silibum marianum Flower Extract The flowers of seed - removed Serenoa repens were dried and pulverized. Subsequently, 100 g of the pulverized Serenoa repens powder was extracted overnight at room temperature by hydro - alcohol extraction using 70% (v / v) ethanol (and 30% (v / v) water). The supernatant was collected by filtration. Ethanol and water were removed from the collected supernatant by rotary vacuum evaporation (Heidolph, Schwabach, Germany). The obtained extract was freeze - dried to obtain Serenoa repens flower extract (SME).

[0134] 2. In vitro comparative test In this test, the senotherapeutic effect of the flower extract (SME) of Serenoa repens was demonstrated. The senotherapeutic effect of the extract of Serenoa repens may be due to its senolytic activity and / or its senomorphic activity in aged skin and hair follicle cells.

[0135] Materials: The reagents were obtained as follows: The agent "ABT - 737", which is generally known as a senolytic agent, was purchased from abcam (MA, USA); 3 - (4,5 - dimethyl - 2 - thiazolyl) - 2,5 - diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Duchefa (Haarlem, Netherlands); Hoechst 33342, that is, the dead - cell apoptosis kit, was purchased from Invitrogen (CA, USA); antibodies and the senescence β - galactosidase staining kit were obtained from CST (MA, USA).

[0136] Induction of senescent cell phenotypes in aged skin Non - aged human dermal fibroblasts (HDF) were obtained from ATCC PCS - 201 - 010 (商標) (VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM; Hyclone, UT, USA) containing 10% (v / v) fetal bovine serum and penicillin / streptomycin (100 IU / 50 μg / mL) at 37 °C in 5% CO₂ in air 2They were cultured in a humidified atmosphere. Non-senescent HDFs from passages 8 - 10 were used in the experiment at a confluence of 80 - 90%. Senescent HDFs were induced by the cumulative population doubling (CPD) corresponding to passage 40. The cells acquired a senescent phenotype after 40 passages.

[0137] Induction of dermal papilla senescent cell phenotypes (HFDPC) Dermal papilla cells were induced by the cumulative population doubling (CPD) corresponding to passage 18. HFDPCs acquired a senescent phenotype after 18 passages.

[0138] Cell viability assay Non-senescent and senescent HDFs were seeded onto 24-well plates respectively. After incubating for 3 days with the treatment of test substances (meaning by SME, ABT-737, etc.), the HDF viability was determined using the MTT assay.

[0139] SA-β-galactosidase staining assay (senescent cell marker) HDFs and HFDPCs were seeded into 6-well plates at a certain cell density. After incubating for 3 days with the treatment of test substances, the cells were stained using a senescence β-galactosidase staining kit (CST, MA, USA). SA-β-gal was used to evaluate the abnormal enzymatic activity observed in senescent cells. The cultures were incubated overnight after adding the staining solution; SA-β-gal in senescent cells stains blue. The proportion of senescent cells was calculated as the number of senescent cells stained blue divided by the total number of cells. The average number of stained cells and total cells was determined.

[0140] Immunofluorescent staining of apoptosis markers and SASP markers HDFs were seeded on 24-well plates. After incubation with the test substances for 3 days, the HDFs were fixed with 4% (w / v) formaldehyde for 15 minutes at room temperature and blocked with 5% bovine serum albumin, 0.3% (w / v) Tween 20 in PBS for 1 hour. The antibodies used, caspase-3, PARP, IL-6, and IL-8 (1:50 dilution; CST, USA) were added to each well and subsequently left at 4 °C overnight. The cells were washed with PBS and incubated with Hoechst33342 (1:10000, Invitrogen, USA) for 10 minutes at room temperature. Subsequently, fluorescence was analyzed by fluorescence microscopy (EVOS; Life Technologies GmbH, Germany).

[0141] Enzyme-linked immunosorbent assay (ELISA) of SASP markers The levels of MMP-1 in the cell supernatants were measured by the Human matrix metalloproteinase 1 (MMP-1) Quantikine ELISA Kit (R&D system Inc., USA) according to the manufacturer's protocol. HDFs were treated with various concentrations of SME 100 and 200 ppm for 3 days, and the cell supernatants were collected after SME treatment.

[0142] Statistical analysis: All data were presented as mean ± standard deviation. The statistical significance of the data was determined by two-way analysis of variance. * p < 0.05, ** p < 0.01 indicated statistical significance.

[0143] Results: The results were obtained by performing three independent assays. Compared with non-senescent cell controls ##p < 0.01 Compared with senescent cell controls * p < 0.05; ** p < 0.01; *** p < 0.001

[0144] To examine whether SMEs have senolytic activity in aged HDFs (Figure 1), cell viability in aged and non-aged HDFs was observed. Aged and non-aged HDFs were treated with SMEs for 3 days. To verify the experimental assay, the effect of a known senolytic chemical compound (ABT-737) was determined by comparing it with the senolytic effect of SMEs (100 and 200 ppm) in aged HDFs (cultured for at least 40 passages) (Figure 1A). Indeed, the 3-day treatment demonstrated a significant specific senolytic effect of SMEs in aged HDFs. Cell viability decreased by 35% in SME-treated aged HDFs, and no change was observed in non-aged HDFs, confirming the specific senolytic activity of SMEs in aged HDFs.

[0145] Furthermore, in Figure 1B, SA-β-Gal activity was examined in this aging model of HDFs treated with ABT-737 as a positive control and SMEs at 50, 100, and 200 ppm. After 3 days of treatment with SMEs, SA-β-Gal staining decreased significantly in a dose-dependent manner, confirming the ability of SMEs to affect the number of aged HDFs. Furthermore, the expression of the aging marker p21 was quantified in non-aged and aged HDFs treated with 100 ppm and 200 ppm of SMEs for 3 days or untreated (Figure 1C). The strong expression of p21 in untreated aged HDFs confirmed the validity of studying this cell cycle inhibitor as an aging marker. The 3-day treatment with SMEs demonstrated a dose-dependent inhibition of p21 expression in aged HDFs, demonstrating the senomorphic activity of SMEs.

[0146] To clarify the mechanism of action of the senolytic effect, apoptotic cells were examined by flow cytometry after annexin V and propidium iodide (PI) staining. Quantification of the percentage of apoptotic HDFs (annexin V-FITC positive) 3 days after treatment was performed (Figure 2A). The assay was verified using known apoptotic compounds. In non-senescent HDFs, 3-day treatment with SME did not change the number of apoptotic HDFs, demonstrating the non-toxicity of SME to non-senescent HDFs. Furthermore, 200 ppm of SME significantly increased the number of apoptotic HDFs in the replicative model senescent HDFs, confirming the senolytic activity of SME shown in Figures 1A and 1B.

[0147] To confirm that the induced apoptosis is specific to senescent HDFs, caspase-3 expression in non-senescent and senescent HDFs was determined. Indeed, caspase-3 plays a central role in the execution phase of cell apoptosis, and its proteolytic cleavage leads to its activation. Inhibition of the cleavage of poly-ADP-ribose polymerase (PARP) (mediated by caspases) promotes cell degradation and serves as a marker for cells undergoing apoptosis. Figures 2B and 2C show the occurrence of cleaved caspase-3 and cleaved PARP, respectively, after 3-day SME treatment at SME concentrations of 100 ppm and 200 ppm. Quantification of the fluorescence intensity analysis of cleaved caspase-3 (Figure 2B) and cleaved PARP (Figure 2C) was obtained. 3-day treatment of senescent HDFs with SME significantly increased the cleavage of caspase-3 and PARP, confirming the specific induction of apoptosis in senescent HDFs by SME and demonstrating the senolytic activity of SME.

[0148] The above data demonstrate that SME enables selective killing of senescent cells (exemplified as human dermal fibroblasts (HDFs)) by inducing them to undergo apoptosis.

[0149] To investigate the senomorphic activity of SME, immunofluorescence intensity measurements of specific SASP markers and SASP regulators were performed. The senomorphic activity of SME in the HDF aging replication model by inhibition of SASP factor release has been reported (Figure 3). Figures 3A, 3B, and 3C show the quantification of immunofluorescence intensity measurements corresponding to γ-H2AX marker, TNF-α, and IL-6 cell expression, respectively. The secretion of MMP-1 is shown in Figure 3D and measured by ELISA assay. The effect of SME on the expression level of the DNA damage response marker γ-H2AX in aged HDFs, which is known to regulate SASP factor release at SME concentrations of 100 ppm and 200 ppm, was investigated (Figure 3A). Aged HDFs have a strong increase in γ-H2AX expression compared to non-aged HDFs. This strong expression was significantly abolished after 3 days of treatment with SME, which induced a dose-dependent decrease in γ-H2AX expression in aged HDFs.

[0150] Furthermore, Figures 3B and 3C show the effect of SME at 100 ppm and 200 ppm on the expression levels of SASP factors exemplified as TNF-α and IL-6, respectively, in aged HDFs. Aged HDFs have a strong increase in TNF-α and IL-6 expression compared to non-aged HDFs. These strong expressions were significantly abolished after 3 days of treatment with SME, which induced a dose-dependent decrease in these SASP factor expressions in aged HDFs.

[0151] Furthermore, Figure 3D shows the MMP-1 secretion levels in the supernatants of cultures of non-aged or aged HDFs treated with 100 ppm and 200 ppm of SME for 3 days or untreated. ABT-737 was used as a positive control. MMP-1 is a well-known SASP factor involved in skin matrix degradation associated with aging. MMP-1 secretion was significantly increased in aged HDFs compared to non-aged HDFs. This strong secretion was significantly abolished after 3 days of treatment with SME, which induced a decrease in MMP-1 secretion in aged HDFs.

[0152] The above results demonstrate the senomorphic activity exerted by SME in senescent cells (exemplified as HDF) by inhibiting SASP secretion.

[0153] To investigate further senomorphic activity of SME, an aging replication model of senescent HDF was used to determine whether SME could restore the proliferative capacity of senescent HDF (Figure 4). For this purpose, evaluation of the ability of SME to stimulate cell proliferation after removal of senescent HDF was made possible by differential staining of senescent and non-senescent HDF. Senescent and non-senescent HDF were mixed at a ratio of 1:9 respectively, treated with SME for 3 days, then the cells were harvested and seeded again. After 3 days of incubation, the cell proliferation rate and population doubling time were measured by cell counting. The results in Figures 4A and 4B were measured by cell counting.

[0154] Figure 4A shows the cell proliferation rates of the mixed senescent and non-senescent HDF after treatment with SME at SME concentrations of 50, 100, and 200 ppm respectively. Indeed, SME significantly increased the proliferation rate at an SME concentration of 200 ppm, demonstrating its ability to improve skin regeneration after removal of senescent HDF. Subsequently, Figure 4B demonstrates the effect of SME on the doubling time of the mixed senescent and non-senescent HDF after 3 days of treatment with SME. Indeed, SME significantly decreased the population doubling time at an SME concentration of 200 ppm, confirming its ability to improve skin regeneration after removal of senescent HDF.

[0155] The above results demonstrate the senomorphic activity exerted by SME in senescent HDF by improving both the proliferation rate and doubling time.

[0156] To demonstrate the senomorphic activity of SME, an aging replication model of HDF was used to determine whether SME could enable the recovery of the ability of aging HDF in the synthesis of type I collagen. For this purpose, an ELISA kit was used to measure the amount of type I collagen in the cell supernatant to evaluate this effect. Figure 5 shows that SME at concentrations of 50, 100, and 200 ppm increased type I collagen synthesis in aging HDF in a dose-dependent manner compared with non-aging HDF. This result confirms that in addition to the ability of SME to selectively kill aging HDF (suggesting senolytic activity), it can also enable the recovery of the ability of aging HDF for SME to synthesize type I collagen (suggesting senomorphic activity). Since stimulation of type I collagen was not observed in aging cells, this effect is considered to be specific to aging cells. Therefore, SME can be used for skin rejuvenation, skin repair of aging skin, and as an anti-wrinkle agent by targeting aging cells.

[0157] Furthermore, SA-β-Gal activity was examined in this aging model of dermal papilla cells treated with ABT-737 as a positive control and SME at concentrations of 50, 100, and 200 ppm (Figure 6). After 3 days of treatment with 200 ppm of SME, SA-β-Gal staining significantly decreased in a dose-dependent manner, confirming the ability of SME to affect the number of dermal papilla cells, and thus, senolytic activity was confirmed. This demonstrates that the above positive effects can be used in various cell types.

[0158] From the above, it was experimentally found that the extract of Withania somnifera (SME) of the present invention can be very well used as an aging cell-targeted treatment agent. In particular, such an extract was found to have senolytic and senomorphic activities. Cell death in aging cells was selectively induced and promoted, particularly by inducing and promoting apoptosis of aging cells. Furthermore, the aging phenotype could be suppressed in surviving aging cells by treatment with the extract of Withania somnifera (SME).

[0159] Therefore, such extracts can be used to reduce cellular senescence in tissues, to improve the appearance or other properties of the skin, hair or both, in particular, to improve skin elasticity, skin firmness, skin thickness, skin color, skin radiance, skin barrier, skin moisturization, skin rejuvenation, or two or more of them, and to prevent or improve skin sagging, skin hyperpigmentation, skin hypopigmentation, loss of skin elasticity and firmness, erythema, skin barrier dysfunction, DNA damage, hair loss, hair thinning, hair fragility, white hair, or two or more of them, and to treat or prevent dryness, hyperpigmentation, hypopigmentation, loss of skin elasticity and firmness, erythema, hair loss, obesity, diabetes, neurodegenerative diseases, osteoarthritis, atherosclerosis, age-related eye diseases and tissue fibrosis diseases.

[0160] 3. Cosmetic formulation examples A cream containing Seribum marianum flower extract (SME) suitable for topical application is prepared according to a conventional method:

[0161]

Table 1

Claims

1. 1. Cosmetic use of a composition comprising or consisting of an extract of flowers of Silybum marianum from which the seeds have been removed, containing less than 0.1% by weight of silymarin, or less than 0.01% by weight of silymarin, or less than 0.001% by weight of silymarin, or 0% by weight of silymarin, based on the total weight of the dry extract, as a senescent cell targeting treatment agent for reducing cellular senescence in tissues present in the skin.

2. 2. The cosmetic use according to claim 1, wherein said extract of Silybum marianum is a hydroalcoholic or alcoholic extract.

3. 3. The cosmetic use according to claim 1 or 2, wherein said extract of Silybum marianum is a hydroalcoholic extract.

4. 4. The cosmetic use according to any one of claims 1 to 3, wherein said extract of Silivum marianum has senolytic activity, comprising specifically inducing or promoting cell death, in particular apoptotic cell death, of senescent cells in said tissue.

5. 5. The cosmetic use according to any one of claims 1 to 4, wherein said extract of Silivum marianum has senomorphic activity inducing the inhibition of the senescent phenotype of senescent cells in said tissue, the restoration of the proliferative capacity of senescent cells in said tissue, the restoration of metabolic processes in senescent cells in said tissue, or a combination of two or all of them.

6. The cosmetic use according to any one of claims 1 to 5, wherein said extract of Silybum marianum has one or more of the following activities against senescent cells in said tissue, selected from the group consisting of: (a) inhibiting the release of senescence-associated secretory phenotype (SASP) factors from senescent cells in a tissue, particularly where the SASP factors are selected from the group consisting of inflammatory cytokines such as γ-H2AX, tumor necrosis factor α, interleukins (IL) selected from the group consisting of IL-1α, IL-1β, IL-6 and IL-8, metalloproteinases such as matrix metalloproteinases (MMPs) selected from the group consisting of MMP-1 and MMP-3; (b) restoring the cell proliferation capacity of senescent cells; (c) preventing senescence-induced cellular deterioration of senescent cells; (d) restoring metabolic processes in senescent cells; and (e) Inducing or promoting cell death of senescent cells.

7. 7. The cosmetic use according to any one of claims 1 to 6, wherein the composition comprises from 0.0001 to 20% by weight, preferably from 0.001 to 15% by weight, more preferably from 0.005 to 10% by weight, in particular from 0.01 to 5% by weight of said extract of Silybum marianum, based on the total weight of the composition.

8. the composition is for topical use, administered topically to a subject having said tissue, in particular the composition is for topical use selected from the group consisting of a solution, a suspension, an emulsion, a cream, a paste, a gel, a lotion, a powder, a soap, a water containing a surfactant, an oil and a spray; or The composition is a nutraceutical composition that is orally administered to a subject having the tissue. Cosmetic use according to any one of claims 1 to 7.

9. Cosmetic use according to any one of claims 1 to 8, wherein cellular senescence results from the presence of age-related senescent cells, stress-related senescent cells, or both.

10. The cosmetic use according to any one of claims 1 to 9, wherein the tissue present on the skin is selected from epidermal tissue, subcutaneous tissue, hair follicle tissue, and combinations of two or more thereof.

11. 11. The cosmetic use according to any one of claims 1 to 10, wherein reducing cellular senescence in said tissue is for improving the appearance or other properties of the skin, hair or both, in particular for improving skin elasticity, skin firmness, skin thickness, skin tone, skin radiance, skin barrier, skin moisturization, skin rejuvenation, or two or more thereof, or for preventing or ameliorating skin sagging, skin hyperpigmentation, skin hypopigmentation, loss of skin elasticity and firmness, erythema, poor skin barrier function, DNA damage, hair loss, hair thinning, hair brittleness, candy hair, or two or more thereof.

12. 1. A composition comprising or consisting of an extract of deseeded Silybum marianaum flowers as a senescent cell targeted treatment for use in a method for treating or preventing disorders associated with cellular senescence in tissues present in the skin.

13. 13. A composition for use according to claim 12, comprising (A) the disorder is selected from the group consisting of dry skin, hyperpigmentation, hypopigmentation, loss of skin elasticity and firmness, erythema, alopecia, hair thinning, hair brittleness, candied hair and tissue fibrosis disorders; (B) at least one extract of Silybum marianum is defined as in any one of claims 1 to 3; (C) the composition is as defined in claim 7 or 8; (D) cellular senescence is defined as in claim 9; (E) the tissue present in the skin is defined as in claim 10; (F) the object is defined as in claim 11; or (G) A combination of any two or more of features (A) to (F). The composition.

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