Use of plant-derived extracts for stimulating hair growth, and cosmetic or pharmaceutical compositions containing such extracts for the aforementioned use.

Abstract

The present invention relates to the use of at least one plant extract capable of activating GDF11 growth factor (growth differentiation factor 11) for hair growth treatment. The at least one extract is - A high-peptide and sugar-rich extract derived from a culture of plant cells belonging to the species Scabiosa arvensis. - A peptide and sugar-rich extract derived from a culture of somatic embryo-enriched cells of Lotus corniculatus. - Aqueous ethanol extract derived from artichoke heads belonging to the species Quercus scolimus, and / or -A water-soluble extract derived from prickly pear cactus stems, combined with a hydrated ethanol extract derived from coffee beans. Selected from.

Description

TECHNICAL FIELD

[0001] The present invention relates to the use of one or more plant-derived extracts capable of stimulating hair growth by activating growth differentiation factor 11 (GDF11) growth factor.

[0002] These extracts include a combination of an extract derived from cell culture of *Scabiosa arvensis*, an extract derived from somatic embryo-enriched cell culture of *Lotus japonicus*, a hydrous ethanol extract derived from artichoke capitula of *Cynara scolymus*, and a combination of a water-soluble extract derived from *Opuntia* cladodes and a hydrous ethanol extract derived from coffee beans of *Coffea arabica*.

[0003] The present invention also relates to methods for producing these extracts, and to pharmaceutical or cosmetic compositions containing such extracts for stimulating hair growth. BACKGROUND ART

[0004] Hair is a skin appendage distributed on the head of each individual, and performs the functions of protection and thermoregulation.

[0005] Each hair consists of an externally visible portion emerging at the scalp level called the hair shaft, and an internal portion known as the hair follicle. The hair follicle is an invagination of the epidermis that sinks into the skin until it reaches the dermis.

[0006] Each hair follicle is associated with a sebaceous gland responsible for producing sebum, a fatty secretion intended to protect the scalp and hair.

[0007] The lower part of the hair follicle is slightly swollen and is called the hair bulb. It contains various stem cell populations, including a group of mesenchymal-derived, specialized fibroblasts known as dermal papilla cells (DPCs). These cells play a crucial role in maintaining hair follicle vitality, cell proliferation, and hair growth.

[0008] Stem cells within hair follicles are reliably maintained by SOX9, a member of the SOX gene family, which is a transcription factor that binds to DNA. In mice, loss of SOX9 in epidermal tissue leads to visible hair loss associated with the loss of CD34, a stem cell marker (Vidal et al., 2005).

[0009] The lifespan of a single hair is typically 2 to 7 years, and it is estimated that each hair follicle produces approximately 20 hairs throughout its lifespan.

[0010] More specifically, the hair life cycle consists of the following three main phases: • Anagen phase: This is the active growth period for hair. This is the longest period, lasting an average of several years, approximately 2-4 years for men and 3-7 years for women. • Catagen phase: This is a degenerative period in which vital functions decline and hair growth stops. This period lasts for 2-3 weeks. • Telogen phase: This is a resting period in which functional activity completely ceases, but the hair remains within the hair follicle and falls out mainly during washing and combing. This period averages 3-4 months and marks the start of a new cycle.

[0011] Insulin-like growth factor 1 (IGF-1) plays a crucial role in the hair cycle by maintaining the growth phase and delaying the regression phase, effectively promoting hair growth in hair follicles (Weger, 2005).

[0012] Under normal conditions, when a resting hair falls out (called natural shedding), the hair follicle has already started a new cycle, and the new hair is in the growth phase. Each hair follows its own life cycle and does not synchronize with other hairs. This is why hair does not fall out all at once, but rather a few hairs are lost each day.

[0013] Therefore, hair loss is a natural physiological process in which up to 100 hairs can fall out each day. This phenomenon may be more pronounced at certain times of the year, such as September through November, when increased sun exposure during the summer months can lead to a greater increase in hair loss due to the accumulation of free radicals, molecules involved in cellular senescence.

[0014] However, under certain other conditions, an imbalance between the number of hairs in the growth phase and the number of hairs in the degeneration phase can lead to progressive hair loss, a condition known as alopecia or baldness.

[0015] While vegetative head syndrome can affect both men and women, it is far more common and progresses more rapidly in men, with an estimated 50%–80% of men worldwide experiencing it. It generally begins around age 19–20 and resolves by age 32–33.

[0016] Rapid hair thinning manifests as a gradual receding hairline, beginning at the temples in men, or as localized hair loss in specific areas of the scalp. Regardless of the degree of loss, the hair may appear weak or thin.

[0017] Head problems are a complex condition influenced by genetic and hormonal factors, as well as an unbalanced diet of macronutrients and micronutrients, stress, and unhealthy lifestyle habits.

[0018] The most commonly used medications to treat hair loss are minoxidil and finasteride. Minoxidil is a potent vasodilator and antihypertensive drug that promotes cell proliferation in the hair follicles. Finasteride, on the other hand, is a potent inhibitor of the enzyme involved in the production of the hormone dihydrotestosterone (DHT), which is important for the development of major male characteristics but also causes hair follicle atrophy (also known as miniaturization of hair follicles).

[0019] Currently, these are the only two drugs approved by the FDA to treat alopecia; the former is for topical use, and the latter is for oral use. However, their effectiveness may vary from person to person, and they may have serious side effects, so careful consideration is needed regarding the actual benefits.

[0020] In cosmetic medicine, techniques such as hair transplantation, a surgical procedure that densifies the scalp area by transplanting healthy hair follicles harvested from the back and sides of the same individual's head, are also used. However, these are invasive surgical procedures performed under anesthesia, and the transplantation of all hair follicles is not always completely successful.

[0021] Platelet-rich plasma (PRP) is an advanced treatment for alopecia and hair loss. PRP contains growth factors such as platelet-derived growth factor (PDGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), and epidermal growth factor (EGF). These growth factors stimulate hair follicle stem cells, promoting the formation of new hair follicles and, consequently, hair growth.

[0022] In recent years, the growth factor GDF11 (growth differentiation factor 11) has been identified in PRP derived from the blood of young organisms. It is present in higher amounts in the blood of young organisms than in older organisms (Bueno et al., 2016), and its levels decrease with age. Encoded by a specific gdf-11 gene, GDF11 is a member of the "transforming growth factor β" (TGFβ) superfamily, and its regulatory role in embryonic development, particularly in the nervous system and anterior-posterior axis regionalization, was first identified (Williams, 2013; Anderson, 2006).

[0023] Subsequently, it was thought to have a promising rejuvenating role in several organs. In fact, GDF11 has been shown to suppress age-related cardiac hypertrophy (Loffredo et al., 2013), reverse myocardial aging (Sinha et al., 2014), and even reduce some of the adverse effects of functional aging in the brain (Katsimpardiay et al., 2014).

[0024] This remarkable finding was validated by experiments using a "parabiosis" system, in which the blood of young mice containing the factor GDF11 was mixed with the blood of older rats. The two mice were surgically joined, allowing the blood of the young mice to circulate within the older mice, or the older mice received transfusions from the young mice. In the older mice, DNA repair in muscle cells was observed, resulting in healthier and more rejuvenated muscle fibers. Furthermore, muscle grip strength increased, and the mice were able to run longer on a treadmill compared to untreated mice (Loffredo et al., 2013; Sinha et al., 2014).

[0025] The current trend in hair growth-promoting products is shifting towards natural and plant-based products. Plant cell cultures are a valuable source for producing plant extracts containing active ingredients with proven medicinal or cosmetic efficacy. These cultures can be used to produce contaminant-free, sustainable, and standardized products. Furthermore, these production processes can be easily scaled up industrially (Lee et al., 2010).

[0026] Japanese Unexamined Patent Publication No. 05-170627 discloses a hair growth treatment agent containing an extract of prickly pear cactus (*Opuntia ficus indica*). The extract is obtained, for example, by extraction using a solvent at a temperature between room temperature and the boiling point of the solvent. The Examples show extraction using 70% ethanol, water, acetone, or the like at room temperature or under heating.

[0027] U.S. Patent Publication No. 20222226410 discloses a hair growth treatment agent comprising an extract or a fraction of prickly pear cactus (*Opuntia ficus indica*), such as finely ground fruit or a fruit extract thereof.

[0028] WO 2015 / 132755 discloses a functional food composition, cosmetic composition, or pharmaceutical composition based on a combination of plant extracts derived from the flowers or fruits of *Opuntia ficus* and *Oryza sativa* (black rice) for inhibiting 5-alpha reductase. Such preparations are described as being useful for preventing or treating benign prostatic hypertrophy or hyperplasia, androgenetic alopecia, and acne.

[0029] Mintel GNPD Record ID 5430331 "Hair Proteinz Spa Inbuilt Protein Booster", February 2018, XP93134018, discloses a composition comprising argan oil, keratin protein, artichoke extract (*Cynara scolymus*), and ylang-ylang essential oil for strengthening hair and nourishing hair roots.

[0030] International Publication No. 2019 / 028214 discloses a composition comprising one or more of the following extracts: acai (Euterpe oleracea) extract containing cyanidin 3-glycoside and / or cyanidin 3-rutinoside, olive (Olea europaea) extract containing oleuropein, Arabica coffee tree extract, and / or yupei (Tabebuia impetiginosa) extract. This composition further comprises micronutrients including zinc and vitamin D3. The above composition is said to be useful for the non-medical treatment of hair loss and / or for nourishing and rejuvenating hair, skin, and nails.

[0031] Chinese Patent No. 101524426 discloses an aqueous ethanol extract of coffee for stimulating hair growth.

[0032] International Publication No. 2016 / 173867 discloses specific plant extracts, namely peptide / sugar mixtures derived from somatic embryo-enriched cell cultures of Lotus japonicus, and proposes the use of these extracts in cosmetics for combating skin aging and rejuvenating skin tissue.

[0033] In "The Growth Differentiation Factor 11 is involved in skin fibroblast aging and is induced by a preparation of peptides and sugars derived from plant cell cultures" by Tito Annalisa et al., Molecular Biotechnology, Springer US, New York, Vol. 61, No. 3, January 19, 2019, it was disclosed that a specific plant extract, namely a peptide / sugar mixture derived from a somatic embryo-enriched cell culture of Lotus japonicus, can stimulate GDF11 production in skin cells.

[0034] Italian Patent No. 202100020309 discloses an extract of prickly pear cactus (Opuntia ficus indica) and a water-soluble olive extract for use in the prevention and treatment of atopic dermatitis. [Overview of the project] [Problems that the invention aims to solve]

[0035] The fundamental technical problem underlying this invention is to provide a plant-derived extract that is effective in stimulating hair growth and does not have the drawbacks of the aforementioned known products. [Means for solving the problem]

[0036] The inventors have found that, similar to the effects of TGF-β1 growth factor, treatment with recombinant GDF11 protein (rGDF11) can stimulate the gene expression of the stem cell marker SOX9 in human dermal papilla cells, and can also stimulate the expression of insulin-like growth factor IGF-1, which is important for maintaining the hair growth period.

[0037] Furthermore, the inventors discovered that treatment with recombinant GDF11 protein (rGDF11) can stimulate the expression of the proteins noggin and β-catenin in an in vitro model of dermal papilla cell spheroids (three-dimensional (3D) cell aggregates that reproduce the characteristics of hair bulbs).

[0038] Since noggin plays an important role in hair follicle regeneration and growth (Botchkarev, 2001) and β-catenin promotes the induction and prolongation of the hair growth phase (Dong, 2022), this finding is important for identifying products that may be beneficial for hair care.

[0039] Furthermore, the inventors discovered that treating human hair follicle explants with GDF11 can increase the length of the hair shaft and stimulate hair growth.

[0040] In light of these findings, the inventors identified plant extracts that stimulate the expression of GDF11, and consequently, the expression of the genes SOX9 and IGF-1.

[0041] Therefore, in one embodiment, the present invention relates to the use of at least one plant extract as an active ingredient for stimulating hair growth, wherein the extract can activate the GDF11 growth factor, and the at least one plant extract is 1. Extracts containing high levels of peptides and sugars derived from plant cell cultures of Scabiosa arvensis. 2. Extracts containing high levels of peptides and sugars derived from plant cell cultures enriched with somatic cell embryos of Lotus japonicus. 3. A combination of a water-soluble extract derived from the stem segments of prickly pear cactus and an aqueous ethanol extract derived from coffee beans, and / or 4. Hydrated ethanol extract derived from the artichoke flower heads of Cynara scolimus. Selected from.

[0042] In one embodiment of the present invention, the peptide and sugar-rich extract derived from the plant cell culture of Scabiosa arvensis for use according to the present invention is a) A step of homogenizing a plant cell culture of Scabiosa arvensis in physiological saline solution to obtain a homogenate; b) Separating the solid portion of the obtained homogenate from the liquid portion; c) The solid portion is treated with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins and glycosidic bonds, thereby obtaining the peptide and sugar-rich extract. It is obtained by a preparation process that includes the following.

[0043] In one embodiment of the present invention, an extract derived from a somatic cell embryo-enriched plant culture of Lotus japonicus for use according to the present invention is obtained by a preparation process described in European Patent No. 3,288,644 in the name of the present applicant (the contents of which are incorporated herein by reference).

[0044] This process a) A step in which a suspension of Lotus corniculatus plant cell culture is induced to form a somatic cell embryo; b) A step of homogenizing the obtained somatic cell embryos in physiological saline solution to obtain a homogenate; c) Separating the solid portion from the liquid portion of the obtained homogenate; d) A step in which the solid portion is treated with a proteolytic enzyme in an acidic solution to hydrolyze the proteins in the cell wall of the somatic cell embryo and obtain an extract rich in peptides and sugars. Includes.

[0045] In another aspect, the present invention relates to the use of an extract derived from a somatic cell embryo-enriched plant culture of Lotus japonicus for hair growth treatment, wherein the extract is obtained by the above process.

[0046] In one embodiment of the present invention, a water-soluble extract of prickly pear stem segments combined with the aqueous ethanol coffee extract according to the present invention is obtained by a process described in Italian Patent Application No. 102021000020309 in the name of the present applicant (the contents of which are incorporated herein by reference).

[0047] This process i) A step of subjecting pre-washed prickly pear cactus stems to steam treatment; ii) A step of peeling the prickly pear stem segments obtained in step i); iii) Homogenizing the peeled prickly pear stem segments to obtain homogenates; iv) A step of separating the solid portion from the liquid portion of the obtained homogenate, where the liquid portion constitutes the water-soluble extract of the prickly pear cactus. Includes.

[0048] In one embodiment of the present invention, the aqueous ethanol coffee extract combined with the water-soluble extract of prickly pear cactus for use according to the present invention is i) The step of grinding raw (i.e., unroasted) coffee beans to obtain a ground product; ii) Adding low-temperature ethanol, preferably at a temperature of -30° to -10°C, to the pulverized material, and then performing a homogenization treatment to obtain a homogenate in the form of a suspension in which a solid portion exists within a liquid portion; iii) Stirring the suspension at room temperature; iv) Separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the aqueous ethanol extract of coffee beans; v) The step of filtering the aqueous ethanol extract obtained in step iv). It is obtained by a process that includes this.

[0049] In another aspect, the present invention also relates to the use of a water-soluble extract derived from prickly pear stem segments in combination with a water-containing ethanol extract derived from coffee beans for hair growth treatment, wherein these extracts are obtained by the respective processes described above.

[0050] In another embodiment, the present invention also relates to a process for preparing an aqueous ethanol extract of artichoke, i) Step of crushing the artichoke flower heads; ii) Adding low-temperature ethanol, preferably at a temperature of -30° to -10°C, to the pulverized material, and then performing a homogenization treatment to obtain a homogenate; iii) Stirring the suspension at room temperature; iv) Separating the solid portion from the liquid portion of the homogenate, wherein the liquid portion constitutes the aqueous ethanol extract of the artichoke; v) The obtained aqueous ethanol extract is filtered. Includes.

[0051] In another aspect, the present invention further relates to a pharmaceutical or cosmetic composition comprising at least one plant extract as an active ingredient having the effect of activating GDF11 growth factor for the above-mentioned use in hair growth stimulation, and a pharmaceutically and / or cosmetically acceptable vehicle, wherein the at least one plant extract of the pharmaceutical or cosmetic composition is 1. Extracts containing high levels of peptides and sugars derived from plant cell cultures of the species Scabiosa arvensis. 2. Extracts containing high levels of peptides and sugars derived from plant cell cultures enriched with somatic cell embryos of the Lotus japonicus species. 3. Aqueous ethanol extract derived from the artichoke flower heads of the Quercus scolimus species, and / or 4. A combination of a water-soluble extract derived from prickly pear cactus stem segments and a water-containing ethanol extract derived from coffee beans. Selected from.

[0052] In one embodiment, the present invention relates to a pharmaceutical or cosmetic composition comprising a peptide and a sugar-rich extract derived from a Scabiosa arvensis cell culture obtained by the above processes as active ingredients.

[0053] In one embodiment, the present invention relates to a pharmaceutical or cosmetic composition comprising a peptide and a sugar-rich extract derived from a somatic cell embryo-enriched plant culture of Lotus japonicus obtained by the above processes as active ingredients.

[0054] In another aspect, the present invention relates to a pharmaceutical or cosmetic composition comprising an aqueous ethanol extract of artichoke obtained by the above processes as an active ingredient.

[0055] In another aspect, the present invention relates to a composition comprising a water-soluble extract derived from prickly pear stem segments and an aqueous ethanol extract of coffee beans obtained by the above processes as active ingredients for hair growth treatment.

[0056] Herein, the present invention will be described for illustrative and non-limiting purposes, with reference particularly to the figures in the appended drawings, according to its preferred embodiments. [Brief explanation of the drawing]

[0057] [Figure 1] Figure 1 is a bar graph showing the results of the cytotoxicity assay (MTT) of the extracts of the present invention against human keratinocytes. Specifically, the test concentrations were 0.0006% (0.006 mg / ml) for the peptide extract from Scabiosa arvensis cells, 0.0002% (0.002 mg / ml) for the peptide extract from the somatic cell embryo of Lotus japonicus, 0.002% (0.02 mg / ml) for the ethanol extract from artichoke, 0.003% (0.03 mg / ml) for the ethanol extract from coffee, and 0.0005% (0.005 mg / ml) for the water-soluble extract derived from prickly pear stem segments. Furthermore, a mixture containing both the ethanol extract from coffee and the water-soluble extract derived from prickly pear stem segments was also tested. [Figure 2] Figure 2 is a bar graph showing the effects of recombinant GDF11 protein (rGDF11), tested at concentrations of 10 ng / ml and 100 ng / ml, on the expression of the SOX9 gene, which is important for maintaining stem cell characteristics, and the IGF-1 gene, which is important for maintaining the hair growth period, in human dermal papilla cells. The values ​​shown in the graph are expressed as percentages relative to the untreated control sample, which is set to 100%. TGF-β1 growth factor tested at a concentration of 2.5 ng / ml was used as a positive control. Bars represent the standard deviation, and asterisks indicate significant variation. [Figure 3]Figure 3A shows immunofluorescence analysis in an in vitro model of dermal papilla cell spheroids to evaluate the effect of recombinant GDF11 protein tested at a concentration of 10 ng / ml on noggin protein expression. Noggin protein plays a crucial role in hair follicle regeneration and growth. Spheroid cell nuclei are stained blue with 4',6-diamidino-2-phenylindole (DAPI) dye. The target protein is recognized by a primary antibody against the noggin protein epitope and a secondary antibody that binds to the primary antibody, and is labeled with a green fluorescent substance to be detected in green. Figure 3B shows immunofluorescence analysis in an in vitro model of dermal papilla cell spheroids to evaluate the effect of recombinant GDF11 protein tested at a concentration of 10 ng / ml on β-catenin expression. β-catenin plays a crucial role in inducing and maintaining the hair growth phase. Spheroid cell nuclei are stained blue with DAPI dye. The target protein is detected as red, recognized by a primary antibody against β-catenin and a secondary antibody that binds to the primary antibody, and labeled with a red fluorescent substance. [Figure 4] Figure 4 is a bar graph showing the effect of recombinant GDF11 protein tested at a concentration of 100 ng / ml on the elongation of hair shaft length in human hair follicle explants. The y-axis represents the hair shaft length measured at T0 (start of treatment) and 7 days after treatment (T7), and is expressed as a percentage relative to T0, which is set to 100%. [Figure 5] Figure 5 is a bar graph showing the effect of the extract according to the present invention on GDF11 production in human dermal papilla cells (HFDPCs). Measurements were performed using an ELISA assay with a specific antibody against the GDF11 protein. The effects of minoxidil and TGF-β1 factor on GDF11 production were also evaluated. Minoxidil is a drug used to treat male pattern baldness, and TGF-β1 factor is used as a positive control in the assay. Bars represent the standard deviation, and asterisks indicate significant variation. [Figure 6A]Figure 6A is a bar graph showing the effect of the extract according to the present invention on the expression of the SOX9 gene. This gene is important for maintaining the stem cell properties of dermal papilla cells. Furthermore, this graph also shows the effects of TGF-β1 factor and minoxidil. The bars represent the standard deviation, and the asterisks indicate significant variation. [Figure 6B] Figure 6B is a bar graph showing the effect of the extract according to the present invention on the expression of the IGF-1 gene. This gene is important for maintaining the hair growth period of dermal papilla cells. Furthermore, this graph also shows the effects of TGF-β1 factor and minoxidil. The bars represent the standard deviation, and asterisks indicate significant variation. [Figure 7A] Figure 7A is a bar graph showing the effect of the extract according to the present invention on the expression of the GDF-11 gene in human dermal papilla cells under oxidative stress conditions. To induce free radical generation, cells were treated with hydrogen peroxide (H2O2, 100 μM) for 1 hour, grown in culture medium for 7 hours, and then treated again with 100 μM H2O2 for another hour. The cells were then grown overnight in the culture medium. The following day, two more cycles of H2O2 treatment were repeated, with a 7-hour incubation period in the culture medium in between. After the second H2O2 cycle was completed, the cells were treated with the extract according to the present invention for 24 hours. TGF-β1 factor and minoxidil were used as positive controls. The values ​​shown in the graph are expressed as percentages relative to the H2O2 stress sample, which is set to 100%. Bars represent the standard deviation, and asterisks indicate significant variation. [Figure 7B]Figure 7B is a bar graph showing the effect of the extract according to the present invention on the expression of the SOX9 gene in human dermal papilla cells under oxidative stress conditions. To induce free radical formation, cells were treated with hydrogen peroxide (H2O2, 100 μM) for 1 hour, grown in culture medium for 7 hours, and then treated again with 100 μM H2O2 for another hour. The cells were then grown overnight in the culture medium. The following day, the entire treatment cycle was repeated once more, and after completion, the cells were treated with the extract according to the present invention for 24 hours. Recombinant GDF11 protein, TGF-β1 factor, and minoxidil were used as positive controls. The values ​​shown in the graph are expressed as percentages relative to the H2O2 stress sample, which is set to 100%. Bars represent the standard deviation, and asterisks indicate significant variation. [Modes for carrying out the invention]

[0058] The applicant has found that the GDF11 protein is of particular interest in combating hair follicle atrophy and, therefore, can be used to identify extracts or products that can be used in hair care, particularly to stimulate hair growth, by activating the GDF11 protein.

[0059] Similar to TGF-β1 growth factor treatment, recombinant GDF11 (rGDF11) treatment can activate the SOX9 transcription factor in dermal papilla cells, thereby enhancing their vitality. This also enhances the expression of IGF-1 factor in these cells, which is important for extending and maintaining the hair growth period.

[0060] The applicant also observed remarkable effects of exogenous GDF11 treatment on a 3D dermal papilla cell spheroid model (a three-dimensional (3D) cell mass that more effectively reproduces the hair follicle microenvironment) compared to a standard two-dimensional (2D) cell culture.

[0061] The applicant discovered that treating 3D spheroid models with exogenous GDF11 produced remarkable effects on hair follicle activation, regeneration, and growth, as well as on proteins related to hair growth. Specifically, treatment with recombinant GDF11 protein significantly increased the expression of the proteins noggin and β-catenin in these models. Noggin is involved in hair follicle regeneration, and its loss is associated with a decrease in the number of hair follicles and delayed hair follicle growth. On the other hand, β-catenin is involved in the initial stage of the hair life cycle and promotes the induction and duration of the growth phase (anagen).

[0062] Furthermore, the applicant discovered that applying recombinant GDF11 treatment to ex vivo hair follicle explants derived from male patients undergoing hair follicle transplantation using Follicular Unit Extraction (FUE) technology resulted in elongation of the hair shaft. The observed elongation was more pronounced than in untreated control explant samples.

[0063] Based on these findings, the applicant has identified plant extracts for hair treatment that can activate the GDF11 growth factor, meaning that these plant extracts can stimulate the gene expression of this growth factor.

[0064] In this regard, the applicant has found that peptide and sugar-rich extracts derived from Scabiosa arvensis plant cell cultures, as well as extracts derived from Lotus japonicus somatic cell embryo-enriched plant cultures (rich in peptides and sugars), artichoke aqueous alcohol extracts, and combinations of water-soluble extracts derived from prickly pear stem segments and aqueous alcohol extracts derived from coffee beans (e.g., mixtures containing such extracts) are of particular interest for hair treatment and promoting the proliferation of hair follicle cells and the hair growth period.

[0065] These plant extracts can stimulate GDF11 expression in dermal papilla cells to the same or even better effect than the control (TGF-β1 growth factor) used in the assay. Furthermore, they function better than minoxidil (a well-known hair treatment that works by promoting vasodilation of hair follicle capillaries and stimulating cell proliferation, thereby combating hair follicle atrophy (miniaturization)).

[0066] Furthermore, while extracts derived from prickly pear cactus stems and coffee beans individually activate GDF11 production, they show a better (synergistic) effect when used in combination.

[0067] In addition to activating GDF11 expression, the aforementioned extract can also stimulate genes (such as the transcription factors SOX9 and IGF-1) that are activated in dermal papilla cells by recombinant GDF11 treatment.

[0068] Specifically, when an extract containing a water-soluble extract derived from prickly pear cactus stem segments and an aqueous alcohol extract derived from coffee beans was combined, it showed a remarkable effect on both genes compared to treatment with the individual extracts. These extracts were found to be effective in influencing SOX9 gene expression only when tested as a mixture, and unexpectedly, they showed a synergistic effect on IGF-1 expression.

[0069] The applicant discovered that repeated treatment of dermal papilla cells with 100 μM H2O2 (simulating aging) significantly reduced the expression of GDF11 factor. However, by using the extract of the present invention, this condition can be reversed, restoring GDF11 expression to levels similar to those of unstressed cells. The effect of the extract of the present invention is even better than that of positive controls TGF-β1 growth factor and minoxidil.

[0070] Finally, the applicant found that, similar to GDF11 recombinant protein treatment, the extract of the present invention can restore the expression of stem cell marker-related SOX9 transcription factor in dermal papilla cells that mimic the aging process by repeated treatment with 100 μM H2O2.

[0071] Peptides and high-sugar extracts derived from Scabiosa arvensis plant cell cultures are a) A step of homogenizing a Scabiosa arvensis plant cell culture in physiological saline solution to obtain a homogenate; b) Separating the solid portion of the homogenate from the liquid portion; c) It may be obtained by a preparation process comprising the step of treating the solid portion with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins and glycosidic bonds to obtain the peptide and sugar-rich extract.

[0072] Homogenization refers to the crushing of plant materials in a suitable container such as a ceramic mortar with a pre-cooled ceramic pestle, or, in the case of larger quantities, a laboratory or industrial blender or press may be used, along with a metal container having metal blades.

[0073] Preferably, the scabiosa arvensis plant cell culture is obtained by collecting plant tissue from a scabiosa arvensis plant, inducing callus formation from this tissue on a solid substrate, collecting these calluses, and preparing a liquid culture from them.

[0074] Preferably, step b) separating the solid portion of the homogenate from the liquid portion is carried out by centrifugation, sedimentation, or filtration.

[0075] In a preferred embodiment of the present invention, this procedure further includes washing the solid portion separated in step b) with distilled water to remove cytoplasmic component residues, prior to step c) treating the solid portion with a proteolytic enzyme.

[0076] In another preferred embodiment of the present invention, this procedure further includes washing the solid portion separated in step b) with distilled water to remove cytoplasmic component residues before step c) in which the solid portion is treated with a proteolytic enzyme, and then treating the solid portion with a heated EDTA solution, preferably a heated EDTA solution boiled to, for example, about 100°C.

[0077] The EDTA solution may be, for example, an aqueous solution with a concentration of 2 mM EDTA. The time for treating the solid portion of the homogenate with the heated EDTA solution may vary from 10 to 30 minutes, preferably 20 minutes, in order to remove pectin and starch from the cell wall by chelating calcium with EDTA (promoting the binding of pectin and starch in the cell wall).

[0078] The peptide and sugar-rich extract obtained by the above process may be used in its original form, or it may be dried by an established method (e.g., freeze-drying or spray-drying) to produce a powder.

[0079] The present invention also relates to the use of peptides and high-sugar extracts derived from Scabiosa arvensis plant cell cultures for hair growth treatment, wherein the extracts are obtained by the procedure described above.

[0080] Extracts derived from a somatic cell embryo-enriched plant cell culture of Lotus japonicus can be obtained by the preparation method described in European Patent No. 3,288,644 in the name of the present applicant (the contents of which are incorporated herein by reference).

[0081] This delicious, a) A step in which a suspension of Lotus corniculatus plant cell culture is induced to form a somatic cell embryo; b) A step of homogenizing the obtained somatic cell embryos in physiological saline solution to obtain a homogenate; c) Separating the solid portion of the obtained homogenate from the liquid portion; d) The step of treating the solid portion with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins of the somatic cell embryo and obtain an extract rich in peptides and sugars.

[0082] The aforementioned plant cell culture can be obtained by collecting plant tissue from a plant of the genus Lotus, inducing callus formation from this tissue on a solid substrate, collecting these calluses, and preparing a liquid culture from them.

[0083] Preferably, step a) is carried out by adding plant hormones such as thidiazuron (TDZ) and benzylaminopurine (BAP) to these plant cultures.

[0084] The saline solution in step b) is generally a buffer solution such as phosphate-buffered saline (PBS) with a pH of 7.4.

[0085] Preferably, step c) separating the solid portion of the homogenate from the liquid portion is carried out by centrifugation to separate the supernatant (liquid portion) from the pellet (solid portion).

[0086] In a preferred embodiment of the present invention, this procedure further includes washing the solid portion of the homogenate separated in step c) with distilled water to remove cytoplasmic component residues, prior to step d) treating the solid portion with a proteolytic enzyme.

[0087] In another preferred embodiment of the present invention, this procedure further includes washing the solid portion of the homogenate separated in step b) with distilled water to remove cytoplasmic component residues, before step d) in which the solid portion is treated with a proteolytic enzyme, and then treating the solid portion with a heated EDTA solution, preferably a heated EDTA solution boiled to, for example, about 100°C.

[0088] The EDTA solution may be, for example, an aqueous solution with an EDTA concentration of 2 mM. The treatment time for the solid portion of the homogenate with the heated EDTA solution can be between 10 and 30 minutes, preferably 20 minutes, in order to remove pectin and starch from the cell wall through calcium chelation by EDTA (which promotes the binding of pectin and starch in the cell wall).

[0089] The extract obtained by the above procedure may be used in its original form, or it may be dried by a known method (e.g., freeze-drying or spray-drying) to produce a powder.

[0090] The present invention also relates to the use of an extract derived from a somatic cell embryo-enriched plant culture of Lotus japonicus for hair growth treatment, wherein the extract is obtained by the procedure described above.

[0091] The present invention also relates to a method for preparing an aqueous alcohol extract of artichoke, i) Step of crushing the artichoke flower heads; ii) Adding cold ethanol to the pulverized material, and then homogenizing it to obtain a homogenate; iii) Stirring the suspension at room temperature; iv) Separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the aqueous alcohol extract of the artichoke; v) Filtering the obtained aqueous alcohol extract. Regarding methods including

[0092] Preferably, the artichoke heads used belong to the Violet variety of Quercus scholimus.

[0093] Preferably, in step i), the artichoke heads for grinding are first frozen at a temperature of -20°C to -40°C, preferably about -30°C. Furthermore, preferably, before carrying out step i), the artichoke heads, from which the stems and outer leaves have been removed, are washed with water, and then washed with a 3% to 5%, preferably 5%, sodium bicarbonate solution for 20 to 60 minutes, for example, 30 minutes. In the washing step, the weight / volume ratio of the artichoke to the sodium bicarbonate solution may be in the range of 1:3 to 1:5, preferably 1:5. The pre-washing step advantageously ensures that the extract is sterile and uncontaminated and free from bacteria that could metabolize and degrade the active ingredients in the final extract. After washing, the artichoke heads are rinsed with sterile distilled water to remove the bicarbonate residue, and then dried, for example, under a horizontal laminar flow hood, and subjected to step i) of grinding. Before grinding, it is preferable to cool the artichoke head in a freezer to a temperature of -20°C to -40°C, preferably about -30°C.

[0094] Preferably, in step i), the artichokes are ground without solvent while frozen for 3 to 15 minutes, preferably 3 minutes, using a blade homogenizer operating at 1000 to 3000 rpm, preferably 1500 rpm. This yields a more uniform substrate.

[0095] Preferably, in step ii), ethanol is added at a concentration of 70% to 80%, preferably 80%, and in a ratio of 1:1 to 1:2, preferably 1:1 (homogenate weight / solvent volume).

[0096] The ethanol added in step ii) is at a low temperature, i.e., the temperature is -30°C to -10°C, preferably -20°C.

[0097] Preferably, in step ii), ethanol is added to the pulverized material at a temperature of -30°C to -10°C, preferably -20°C.

[0098] Preferably, in step ii), the low-temperature ethanol is added to the pulverized material and two cycles of homogenization are performed. Each cycle of homogenization is performed using a blade homogenizer operating at a speed of 3000 to 5000 rpm, preferably 3800 rpm, for 3 to 15 minutes.

[0099] Advantageously, this allows for better homogenization of the substrate in a small amount of extraction solvent. Furthermore, by using ethanol at the above percentages rather than high-purity ethanol (96%), the extraction yield is optimized, enabling more efficient extraction of polyphenols.

[0100] Preferably, after step ii) and before step iii), an amount of additional ethanol in the above ratio is added to the homogenate in an amount that achieves a final extraction ratio of 1:5 (homogenate weight / solvent volume). For example, after step ii) and before step iii), 80% ethanol may be added to the homogenate in a ratio of 1:4 (homogenate weight / solvent volume) to achieve a final extraction ratio of 1:5.

[0101] Preferably, in step iii), the homogenate is stirred using a mixer at a temperature of 20°C to 30°C, preferably 25°C, for 1 to 4 hours, preferably 2 hours, and then left to stand.

[0102] Preferably, in step iv), separation is carried out by centrifugal separation at a speed of 4000 to 6000 rpm, preferably 5000 rpm, for 5 to 15 minutes, preferably 10 minutes.

[0103] Preferably, in step v), filtration is performed using qualitative filter paper with a pore size of 60 to 68 micrometers, preferably using a vacuum pump.

[0104] The above-mentioned extract may be evaporated using a rotary vaporizer (maximum temperature 25°C) to remove any excess ethanol that may be harmful to cells.

[0105] Next, this may be dried by an established method such as freeze-drying or spray-drying to obtain a powder. Freeze-drying yields a more stable extract and extends its shelf life.

[0106] The water-soluble extract of prickly pear cactus, preferably used in combination with the aqueous ethanol coffee extract for use according to the present invention, is obtained by a preparation process described in Italian Patent Application No. 102021000020309 in the name of the present applicant (the contents of which are incorporated herein by reference).

[0107] This process i) A step of subjecting pre-washed prickly pear cactus stems to steam treatment; ii) A step of peeling the prickly pear cactus stem segments obtained in step i); iii) Homogenizing the peeled prickly pear stem segments to obtain homogenates; iv) a step of separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes a water-soluble extract of prickly pear cactus.

[0108] Preferably, in step i), steam treatment is carried out at a pressure of up to 4.5 bar, preferably 3 to 4.5 bar, for a period of 5 to 30 minutes, preferably 10 to 20 minutes.

[0109] Preferably, in step iii), the peeled prickly pear stem segments are homogenized while frozen, preferably at a temperature of about -30°C.

[0110] Preferably, in step iii), the peeled prickly pear stem segments are subjected to a first homogenization treatment in the absence of a solvent in a rotary blade homogenizer operating at a speed of 2000 rpm to 3500 rpm for 1 to 30 minutes, preferably 3 minutes, and then subjected to a second homogenization treatment in the presence of a solvent in a rotary blade homogenizer operating at a speed of 3500 rpm to 4500 rpm, more preferably 3800 rpm for 5 to 30 minutes, preferably 3 minutes.

[0111] Preferably, in step iii), the solvent is selected from aqueous solution, physiological saline solution, or water, and more preferably, the weight ratio of the homogenate obtained in step iii) to the solvent is in the range of 1:1 to 1:3.

[0112] Preferably, step iv) is carried out by centrifugation or filtration.

[0113] For use according to the present invention, the aqueous ethanol coffee extract, preferably combined with the water-soluble extract of prickly pear cactus, i) A step of grinding green coffee beans to obtain a ground product; ii) Adding low-temperature ethanol at a temperature of -30°C to -10°C, preferably -20°C, to the pulverized material, and then performing a homogenization treatment to obtain a homogenate in the form of a suspension in which a solid portion exists within a liquid portion; iii) Stirring the suspension at room temperature; iv) Separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the aqueous ethanol coffee bean extract; iv) A step of filtering the aqueous ethanol extract obtained in step iv). It is obtained by a process that includes this.

[0114] Preferably, the coffee beans used are of the Arabica coffee plant species, Santos variety, decaffeinated with high-pressure carbon dioxide (CO2), and unroasted.

[0115] Preferably, in step i), the temperature of the coffee beans is -20°C to -40°C, preferably about -40°C.

[0116] Preferably, in step i), grinding is carried out with a rotating blade homogenizer operating at a speed of 3000 to 5000 rpm, preferably 3800 rpm, for 3 to 15 minutes.

[0117] Preferably, in step ii), ethanol is added at a concentration of 96% and in a ratio of 1:1 to 1:3, more preferably 1:2 (homogenate weight / solvent volume).

[0118] Preferably, in step ii), ethanol is added to the pulverized material at a temperature of -20°C to -10°C, preferably -15°C.

[0119] The ethanol added in step ii) is at a low temperature, i.e., the temperature is -30°C to -10°C, preferably -20°C.

[0120] Preferably, in step ii), the homogenization process is carried out using a rotating blade homogenizer operating at a speed of 3000 to 5000 rpm, preferably 4000 rpm, for 3 to 15 minutes.

[0121] Preferably, in step iii), the homogenate is stirred using a mixer at a temperature of 20°C to 30°C, preferably 25°C, for 1 to 4 hours, preferably 2 hours.

[0122] Preferably, in step iv), the separation is carried out by centrifugal separation at a speed of 5000 to 7000 rpm, preferably 6300 rpm, for 5 to 15 minutes, preferably 10 minutes.

[0123] Preferably, in step v), filtration is performed using qualitative filter paper with a porosity of 60 to 68 micrometers, and for microfiltration, a Stericap® PLUS unit (Millipore) with a porosity of 0.22 μm is used.

[0124] The extract may be evaporated using a rotary vaporizer until a suspension with a solid content of 80% ± 4% by weight is obtained.

[0125] The present invention also relates to a process for preparing the above-described aqueous ethanol coffee extract for hair growth treatment, as well as the use of a combination of a water-soluble extract derived from prickly pear stem segments and an aqueous ethanol extract derived from coffee beans, wherein the aforementioned extracts are obtained by the respective processes described above.

[0126] These extracts can be applied simultaneously, separately, or sequentially, especially when applied topically to the scalp.

[0127] When used simultaneously, the above-mentioned extracts may be mixed to form a mixture or composition containing such extracts, which may then be applied topically.

[0128] Preferably, in the combined use of the above-mentioned extracts according to the present invention, the weight ratio of the aqueous ethanol coffee extract to the water-soluble prickly pear stem extract may be in the range of 10:1 to 3:1, and preferably this ratio is 6:1. In particular, these are used in the form of a mixture or composition containing the above-mentioned extracts in the above ratio. Preferably, in the use of a mixture or composition containing the above-mentioned extracts in the above ratio, the concentration of the aqueous ethanol coffee extract must be at least 0.003% by weight of the mixture or composition.

[0129] The present invention further relates to a pharmaceutical or cosmetic composition containing at least one plant extract as an active ingredient that activates GDF11 growth factor for use in the above-mentioned hair growth stimulation, wherein the at least one plant extract in the pharmaceutical or cosmetic composition is 1. Extracts containing high levels of peptides and sugars derived from plant cell cultures of Scabiosa arvensis. 2. Extracts containing high levels of peptides and sugars derived from plant cell cultures enriched with somatic cell embryos of Lotus japonicus. 3. Aqueous ethanol extract derived from the artichoke heads of Quinara scolimus, and / or 4. Selected from a water-soluble extract derived from prickly pear cactus stem segments, combined with a water-containing ethanol extract derived from coffee beans.

[0130] The present invention also relates to a pharmaceutical or cosmetic composition that is rich in peptides and sugars derived from cell cultures of Scabiosa arvensis and contains an extract obtained by each of the above processes as an active ingredient.

[0131] The present invention also relates to a pharmaceutical or cosmetic composition that is rich in peptides and sugars derived from a plant cell culture enriched with somatic cell embryos of Lotus japonicus, and contains an extract obtained by each of the above processes as an active ingredient.

[0132] The present invention further relates to a pharmaceutical or cosmetic composition containing an artichoke-derived aqueous ethanol extract obtained by the above processes as an active ingredient.

[0133] The present invention also relates to a composition containing, as an active ingredient, a combination of a water-soluble extract derived from prickly pear stem segments and an aqueous ethanol extract of coffee beans obtained by the above processes.

[0134] The above-mentioned pharmaceutical or cosmetic composition may be in any topical form, such as a shampoo, serum, conditioner, emulsion, gel, or lotion, for topical application (to the scalp).

[0135] Furthermore, these compositions may include additional components such as carriers, solvents, excitants, and / or cosmetically and / or pharmaceutically acceptable auxiliaries known in the art.

[0136] Preferably, the solvent is a hydrophilic solvent, preferably selected from water and physiological saline solution, or one or more organic solvents suitable for cosmetic and / or pharmaceutical formulations, more preferably selected from alcohols, glycerols, organic acids, amides, amines, aldehydes, or ketones, or a combination thereof if two types of solvents are miscible with each other.

[0137] Carriers that can be used in the above composition include liposomes, preferably multilayer liposomes, cyclodextrins, and silicates.

[0138] Finally, the present invention relates to a pharmaceutically or cosmetic treatment method for stimulating hair growth, comprising topically applying a pharmaceutically or cosmetically effective amount of the above composition, i.e., a composition comprising at least one of the above-mentioned plant extracts, to a scalp requiring treatment.

[0139] As non-limiting examples of the present invention, the following are some examples relating to the preparation of extracts according to the present invention, and experiments in which the biological activity of the GDF11 protein and the extracts of the present invention was confirmed in in vitro cell models, three-dimensional spheroid models, and ex vivo hair follicles. [Examples]

[0140] Example 1: Method for preparing an extract derived from a plant cell culture of Scabiosa arvensis according to the present invention The following are the steps for preparing a peptide and sugar-rich extract derived from a plant cell culture of Scabiosa arvensis according to the present invention.

[0141] Steps for preparing cell cultures Plant callus culture was initiated on solid media from young leaf fragments of Scabiosa arvensis. Specifically, whole leaves of Scabiosa arvensis were sterilized with 70% ethanol (v / v, aqueous solution) for 15 minutes, followed by further sterilization with 1% sodium hypochlorite (v / v, aqueous solution) for another 15 minutes. The leaves were washed three times with water to remove alcohol and hypochlorite, then trimmed to obtain 5mm x 5mm pieces. These pieces were placed on a solid substrate, "Ganborg B5 medium," containing "plant culture agar" (7.5 mg / L), myo-inositol (500 mg / L), and sucrose (30 g / L), and the pH was adjusted to 5.7 using KOH (0.1N). Subsequently, 2,4-dichlorophenoxyacetic acid (1 mg / L), adenine (1 mg / L), and kinetin (0.01 mg / L) were added to the autoclaved culture medium. After incubation in the dark at 20°C for approximately 5 weeks, callus was obtained and then transferred to a liquid culture medium to begin cultivation.

[0142] Growth steps for liquid cultures The callus was collected when it reached a size of approximately 1 cm in diameter (approximately 50 mg in weight) and placed in a flask containing 50 ml of the same medium as above, but without agar. The flask was placed on an orbital shaker in the dark and shaken at a shaking speed of 120 rpm. After about 10 days, the callus began to disintegrate, forming a homogeneous cell culture consisting of single cells or small cell clumps.

[0143] Cell harvesting step When the culture density in a 2L flask reached approximately 150 g / L, the cells were collected from the culture medium using a low-porosity filter (80-100 μm). The cells were then washed with sterile distilled water and frozen at -80°C.

[0144] Preparation of extracts containing peptides and sugars 500 g of frozen cells were mechanically pulverized and then homogenized in PBS (NaCl 136 mM, KCl 2.7 mM, NaH2PO4 12 mM, KH2PO4 1.76 mM, pH 7.4) in a 1:2 ratio (v / w). The resulting homogenate was centrifuged at 8,500 rpm at 4°C for 15 minutes to precipitate insoluble components. The pellet (including cell walls) was then treated with 2 volumes of 2 mM EDTA solution and heated at 100°C for 20 minutes. After cooling, the extract was filtered through a cellulose filter with a pore size of 80-100 μm, washed, and re-filtered to remove residual EDTA. The resulting pellet was then resuspended in 2 volumes of 0.1 N HCl solution and heated for 1 hour (100°C) to hydrolyze the cell wall glycoproteins and dissolve all sugars.

[0145] After heating, the sample was cooled (on ice) and then enzymatically digested using protease at 37°C for 16 hours. Once the enzymatic digestion was complete, the suspension was centrifuged to obtain a clear solution, which was then adjusted to pH 6.5 with 10N NaOH. This constitutes the peptide and sugar-rich extract derived from the cell wall of Scabiosa arvensis.

[0146] Example 2: Method for preparing an extract derived from a culture of somatic embryo-enriched cells derived from Lotus japonicus according to the present invention. Plant callus culture was first initiated on solid medium from young leaves of the Lotus japonicus species, and then somatic cell embryo formation was induced in liquid cell culture. Once the desired density was reached, the somatic cell embryo culture was harvested and processed to obtain extracts. The methods used are as follows:

[0147] a) Preparation of callus: Whole leaves were collected from the Lotus corniculatus plant, sterilized with 70% ethanol (aqueous solution, v / v) for 15 minutes, and then sterilized again with 1% sodium hypochlorite (aqueous solution, v / v) for another 15 minutes. The leaves were washed three times with water to remove alcohol and hypochlorite, and then cut with a sterile blade to a depth of approximately 0.5 cm. 2The leaf fragments were cut into small pieces. All leaf fragments were placed on Gamborg B5 solid medium (containing 7.5 mg / L plant culture agar, 500 mg / L myo-inositol, 30 g / L sucrose, 1 mg / L 2,4-dichlorophenoxyacetic acid, 0.01 mg / L kinetin, 1 mg / L adenine, and pH 5.7). After incubation in the dark at 20°C for about 5 weeks, callus was obtained by proliferation of leaf cells. Every 3-4 weeks, the callus was excised and transferred to fresh culture medium.

[0148] b) Preparation of cell culture: When the callus reaches a diameter of approximately 1 cm (weight approximately 50 mg), remove it and disperse it in a flask containing 50 ml of AB1 liquid culture medium (Gunborg B5 medium containing myo-inositol 500 mg / L, sucrose 30 g / L, 2,4-dichlorophenoxyacetic acid 1 mg / L, kinetin 0.01 mg / L, adenine 1 mg / L, pH 5.7). Place the flask on an orbital shaker in the dark and shake at a shaking speed of 100 rpm. After about 10 days, the callus will decompose and proliferate, forming a cell suspension culture.

[0149] c) Preparation of somatic cell embryos: Plant cell cultures are resuspended in fresh AB2 liquid growth medium (Ganborg B5 medium containing myo-inositol 500 mg / L and sucrose 30 g / L) supplemented with plant hormones (thidiazurone (TDZ) 1 mg / L and benzylaminopurine (BAP) 0.05 mg / L), glutathione 10 mg / L, casein 500 mg / L, 7% ammonium phosphate, and 10% ammonium sulfate, thereby inducing the formation of somatic cell embryos in the cell cultures (cell cultures enriched with somatic cell embryos).

[0150] d) Proliferation of somatic embryonic cell cultures: Somatic embryo induction is performed for 4 weeks, adding fresh culture medium weekly until the differentiation level into somatic embryos reaches 70-90%.

[0151] e) Recovery of somatic embryos: Embryos can be separated from the growth medium by centrifugation at 2000 g, sedimentation, or filtration using a membrane with a pore size of less than 100 microns. The embryos are then frozen at -80°C to maintain their chemical and physical properties.

[0152] f) Homogenization of somatic cell embryos: Frozen somatic cell embryos are mechanically ground (homogenized) in phosphate-buffered saline (PBS) (NaCl 136 mM, KCl 2.7 mM, NaH2PO4 12 mM, KH2PO4 1.76 mM, pH 7.4) in a 1:2 ratio (weight / volume). This step can be carried out in a suitable container such as a pre-cooled ceramic mortar and pestle, or, in larger quantities, a larger container including a metal container may be used, in which case the plant material can be homogenized with metal blades using either a laboratory or industrial blender or press.

[0153] After obtaining a homogenate (lysate) by homogenizing the embryo, the sample is centrifuged, for example, at 4000 rpm and 4°C for about 15 minutes to precipitate insoluble components.

[0154] g) Obtaining an extract containing peptides and sugars from the cell wall: The pellet (containing the cell wall) obtained in step f) is further washed with distilled water to remove the soluble fraction residue and re-filtered. The pellet is weighed and suspended, and heated in 2 volumes (w / v ratio) of 2 mM EDTA solution for 20 minutes. After cooling, the extract is filtered using a filter cloth, then washed and re-filtered to remove residual EDTA. Next, the obtained pellet is heated in 2 volumes of 0.1 N HCl solution under a fume hood for 1 hour to hydrolyze the sugar bonds. After heating, the sample is resuspended and cooled with ice, then enzymatically digested using protease (1 mg of enzyme per 1 ml of suspension) at 37°C for 16 hours. Once the enzymatic digestion is complete, the suspension is centrifuged or re-filtered to obtain a clear solution. This is a hydrophilic extract rich in peptides and sugars derived from the cell wall.

[0155] Example 3: Preparation of water-soluble cactus extract The water-soluble cactus extract according to the present invention was produced by the following method.

[0156] 1.2 kg of cactus stem segments harvested immediately after harvesting were washed under running water to remove any remaining soil. Next, the segments were subjected to high-pressure steam (maximum 4.5 bar) for approximately 15 minutes, then peeled, the thorns were removed, and they were dried on absorbent paper under a horizontal laminar flow hood. The pre-treated segments were then weighed (approximately 1 kg) and transferred to a -30°C freezer.

[0157] Next, the frozen cactus stems were homogenized for 3 minutes at room temperature and 2000 rpm without the use of a solvent. Then, water or physiological saline (phosphate-buffered saline, i.e., PBS) was added in a 1:1 ratio, and the material was subjected to a second homogenization treatment at room temperature and 3800 rpm for 3 minutes.

[0158] The obtained homogenate was centrifuged at 6300 rpm and 4°C for approximately 15 minutes to precipitate the insoluble components. The supernatant obtained by centrifugation was collected and freeze-dried to obtain the water-soluble cactus extract according to the present invention.

[0159] Example 4: Preparation of aqueous ethanol coffee extract The coffee beans used in this invention were obtained from Kimbo Caffe SpA (Naples). These belong to the Arabica coffee plant species, Santos variety, and were caffeinated using high-pressure CO2 extraction. This coffee is characterized by being raw, i.e., unroasted. Roasting is typically carried out at 200°C, which inactivates many of the active molecules present in the coffee.

[0160] Once decaffeinated green coffee beans were obtained, they were transferred to a freezer at -40°C. The aqueous ethanol extract derived from these coffee beans according to the present invention was produced by the following method.

[0161] 500g of frozen coffee beans were subjected to an initial homogenization treatment at room temperature at 3800 rpm for 3 minutes without the use of a solvent. Subsequently, 96% cold ethanol was added in a 1:2 ratio (weight / volume), and two further homogenization cycles were performed at room temperature at 4000 rpm for 3 minutes each.

[0162] The resulting suspension was incubated at 25°C for 2 hours with stirring using a mixer. The resulting suspension was centrifuged at 6300 rpm for 10 minutes, and the supernatant was filtered using a vacuum pump through qualitative filter paper (60-68 micrometers) to remove solid residue. Then, further microfiltration was performed using a Stericap® PLUS filtration unit (porosity 0.22 μm, Millipore) with the vacuum pump again.

[0163] The microfiltered extract was evaporated in a glass flask using a rotary vaporizer (the maximum temperature reached by the sample was 25°C) to obtain the aqueous ethanol coffee extract according to the present invention.

[0164] Example 4a: Preparation of a mixture of aqueous ethanol coffee bean extract and water-soluble cactus stem extract The mixture (composition) according to the present invention was prepared by combining the aqueous ethanol coffee bean extract obtained in Example 4 and the water-soluble cactus stem extract obtained in Example 3 in a ratio of 6:1. In the following test examples, the mixture was used in the following manner.

[0165] Both extracts were individually dissolved while continuously stirring for 5 minutes to obtain 10% aqueous solutions, which were then centrifuged to remove insoluble residue. The resulting supernatant was collected and used in an in vitro assay. In the tests described in the following examples, the supernatants obtained from coffee bean and cactus stem segment extracts were mixed with culture medium and added to cells.

[0166] Example 5: Preparation of aqueous ethanol extract of artichoke Artichoke heads (Cinnamomum scholimus, Violet variety) purchased from a company in Puglia that holds all relevant organic certifications were trimmed of stems and outer leaves, thoroughly washed under running water, and then immersed in a 5% sodium bicarbonate solution (weight / volume ratio 1:5) for 30 minutes. After treatment, the artichokes were rinsed with sterile distilled water to remove bicarbonate residue and dried under a horizontal laminar flow hood. The pre-treated artichoke heads were weighed (approximately 500g) and transferred to a freezer at -30°C.

[0167] The aqueous ethanol extract of artichoke according to the present invention was prepared by the following method.

[0168] 500 g of frozen artichoke heads were subjected to an initial homogenization treatment at room temperature and 1500 rpm for 3 minutes without solvent. Next, 80% cold ethanol was added in a 1:1 ratio (weight / volume), and the heads were subjected to two more homogenization cycles of 3 minutes each at 3800 rpm. After homogenization, 80% ethanol was added in a 1:4 ratio (homogenate weight / solvent volume) to achieve a final extraction ratio of 1:5. The resulting suspension was incubated on a stirrer plate at 25°C for 2 hours with stirring.

[0169] Next, the resulting suspension was centrifuged at 5000 rpm for 10 minutes, and the supernatant was filtered using a vacuum pump through qualitative filter paper (60-68 micrometers) to remove solid residue.

[0170] Next, the extract was evaporated in a glass flask using a rotary vaporizer (the maximum temperature reached by the sample was 25°C) to remove excess ethanol, and then freeze-dried to obtain the aqueous ethanol extract of artichoke according to the present invention.

[0171] Example 6: Cytotoxicity assay Cytotoxicity assays were performed to ensure that the concentrations of the extracts used in the following assays, which are the subject of this invention, are non-toxic to proliferating cells.

[0172] This assay is based on the use of MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], first described by Mosmann in 1983. This is based on the action of the enzyme dehydrogenase in living cell mitochondria, which hydrolyzes the tetrazolium ring (pale yellow) as MTT, forming formazan crystals (dark blue). These crystals are impermeable to the cell membrane and accumulate in the cytoplasm of metabolically active cells. Therefore, the number of viable and healthy cells is directly proportional to the level of formazan produced.

[0173] Initial number per well: 1 × 10 4 Immortalized human keratinocytes (HaCaT cells) were grown in Dulbecco's Modified Eagle Medium (DMEM) (Lonza) supplemented with 10% fetal bovine serum in a 96-well plate for approximately 8 hours. Cells were treated for approximately 48 hours with a mixture containing 0.0006% peptide and high sugar content extract from Scabiosa arvensis, 0.0002% peptide and high sugar content extract from Lotus japonicus, 0.002% aqueous ethanol extract from artichoke, 0.003% aqueous ethanol coffee bean extract, 0.0005% water-soluble cactus stem extract, and 0.003% aqueous ethanol coffee bean extract and 0.0005% water-soluble cactus stem extract. After washing the cells with PBS, they were incubated in 100 μl / well of reaction buffer (pH 7.4 PBS buffer containing 10 mM Hepes, 1.3 mM CaCl2, 1 mM MgSO4, 5 mM glucose, and 0.5 mg / ml MTT colorimetric substrate). After incubation at 37°C in 5% CO2 for 3 hours, 100 μl of solubilization solution (containing 10% Triton-X100 and 0.1N HCl in anhydrous isopropanol) was added to each well. After 16 hours, the colorimetric reaction was measured at 595 nm using a Victor3 plate reader. The MTT results shown in Figure 1 confirmed that no toxicity to cells was observed at the concentration of the test extract.

[0174] Example 7: Analysis of SOX9 and IGF-1 gene expression in human dermal papilla cells Initial number per well: 8 × 10 4 Human dermal papilla cells (HFDPCs) were grown in human hair follicle growth medium (C-26501, Promocell) with appropriate additives in a 6-well plate for 20 hours. The following day, they were treated with fresh medium for 24 hours. Specifically, two concentrations (10 ng / ml and 100 ng / ml) of recombinant GDF11 protein (rGDF11) and 2.5 ng / mL of TGF-β1 were tested. Furthermore, the following extracts were tested in the experiments below: 0.0006% of Scabiosa arvensis extract rich in peptides and sugars, 0.0002% of Lotus japonicus extract rich in peptides and sugars, 0.002% of artichoke extract in aqueous ethanol, 0.003% of coffee bean extract in aqueous ethanol, 0.0005% of water-soluble cactus stem extract, and combinations of the last two extracts. In this experiment, in addition to TGF-β1, 1 μM minoxidil was also tested.

[0175] After processing, cells were washed with PBS and recovered in lysis buffer, then subjected to RNA extraction using the Merck GenElute® Total RNA Purification kit. RNA samples were treated with DNase I (Ambion) at 37°C for 30 minutes to remove genomic DNA contaminants. 2 μl of each sample was loaded onto a 1% agarose gel in the presence of a denaturing loading dye, and quantified using a specific RNA marker (ThermoScientific) as a reference. iBright Analysis Software (ThermoScientific) was used for quantification. 500 ng of total RNA was reverse transcribed using reverse transcriptase (ThermoScientific). Semi-quantitative RT-PCR was performed using a universal 18S primer / competimer pair (Ambion) as an internal standard, with SOX9 in a 4:6 ratio and IGF-1 in a 3:7 ratio.

[0176] PCR products were separated on a 1.5% agarose gel, visualized using an iBright instrument (Thermo Scientific), and analyzed using iBright Analysis Software (Thermo Fisher Scientific).

[0177] The values ​​shown in the graph represent the band intensity ratio of the analyzed gene and the band ratio of the 18S standard, and are therefore related to the actual expression of the target gene. The values ​​were then converted to percentages (%) by setting the value obtained from the untreated control as 100%.

[0178] The primer sequences used for amplification were as follows: • Hs SOX9 forward (for): Sequence ID 1 • Hs SOX9 Reverse (rev): Sequence ID 2 • Hs IGF-1 forward (for): Sequence ID 3 • Hs IGF-1 reverse (rev): Sequence ID 4.

[0179] As shown in Figure 2, treatment of dermal papilla cells with both concentrations of rGDF-11 resulted in an 80% and 66% increase in SOX9 gene expression, and a 63% and 38% increase in IGF-1 gene expression. These increases were comparable to, or greater than, those induced by the positive control TGF-β1.

[0180] Regarding the extracts of the present invention, the results shown in Figure 6A clearly demonstrate that, similar to rGDF11, SOX9 gene expression was enhanced in all extracts. Specifically, SOX9 gene expression increased by approximately 110% in the peptide and high sugar content extract derived from Scabiosa arvensis cells, by 25% in the peptide and high sugar content extract derived from Lotus japonicus embryo cells, and by approximately 80% in the artichoke extract. Notably, the combination of coffee bean extract and cactus stem segment extract showed a 28% increase. In contrast, the two extracts tested individually did not affect SOX9 gene expression.

[0181] Figure 6B shows the effectiveness of treatment with the extracts of the present invention in increasing IGF-1 gene expression. Scabiosa arvensis extract, rich in peptides and sugars, and artichoke extract increased IGF-1 gene expression by approximately 35%, while a combination of peptide and sugar-rich extract derived from Lotus japonicus embryo cells, coffee bean extract, and cactus stem segment extract increased it by approximately 50%. Treatment with individual extracts also showed efficacy, but the combined effect was synergistic and unexpected, exceeding the sum of the effects produced by the two individual extracts.

[0182] Example 8: Analysis of noggin and β-catenin protein expression in an in vitro dermal papilla cell spheroid model In recent years, significant progress has been made in the development of three-dimensional models that mimic the characteristics of tissues. These 3D spheroid-based models are effectively used in screening to identify promising products or compounds because they more accurately reproduce the tissue microenvironment compared to conventional two-dimensional (2D) cell cultures. These models consist of both surface-exposed cells and cells located in the hypoxic center, similar to in vivo conditions.

[0183] When hair papilla cell aggregates are seeded into low-adhesion, round-bottom well plates, individual spheroids are formed that can be used as a hair follicle spheroid model.

[0184] Human dermal papilla cell spheroid formation To create more complex cell lines, we constructed 3D dermal papilla spheroid cultures and evaluated the effects of GDF-11 on the production of proteins related to hair follicle development.

[0185] 3 x 10 3Individual dermal papilla cells (Hair Follicle Dermal Papilla Cells: HFDPCs) were seeded for 48 hours in 50 μl of "Human Hair Follicle Growth Medium" (C-26501, Promocell), supplemented as needed to promote spheroid formation, in a 96-well round-bottom U plate. At the end of the incubation period, microscopic observation revealed that the dermal papilla cell clusters had developed into separate spheroids.

[0186] Immunofluorescence analysis - Analysis of noggin and beta-catenin proteins The obtained spheroids were collected and centrifuged at 1500 rpm for 3 minutes. After washing them twice with PBS buffer, they were fixed with 4% paraformaldehyde for 1 hour. After three further PBS washes, the spheroids were permeabilized with 0.1% Triton-X100 solution in phosphate buffer for 15 minutes. After one more PBS wash, the spheroids were incubated in a "blocking" solution (6% BSA, 5% goat serum, 20 mM MgCl2, and 0.2% Tween) at room temperature for 1 hour. Subsequently, the spheroids were washed with PBS and incubated overnight at 4°C with shaking, together with either a mouse primary antibody against noggin (Noggin Monoclonal Antibody, OTI1C1, Thermo Fisher Scientific) or a mouse primary antibody against β-catenin (Beta-Catenin Monoclonal Antibody, 6F9, Thermo Fisher Scientific). After washing the spheroids three times with PBS, they were incubated with anti-mouse secondary antibodies (Alexa-Fluor-488 for noggin and Alexa-Fluor-588 for β-catenin) at room temperature for 1 hour. The nuclei were stained with 1 μg / ml DAPI (4',6-diamidino-2-phenylindole) in PBS for 10 minutes. Finally, images were acquired using a ZEISS ISM700 confocal microscope and analyzed using ImageJ software.

[0187] As shown in Figure 3, treatment of spheroids with recombinant protein rGDF-11 at a concentration of 10 ng / ml for 24 hours increased the production of both noggin protein, which plays a crucial role in hair follicle regeneration, and β-catenin, which promotes the induction and persistence of the growth phase in the hair growth cycle.

[0188] Example 9: Analysis of hair shaft length in hair follicle explants The effect of GDF11 recombinant protein treatment on hair shaft length was analyzed using hair follicles derived from male patients who underwent hair follicle transplantation using follicular unit extraction (FUE) technology. All patients provided informed consent for the use of their hair follicles for research purposes, and hair follicle units were collected from areas with higher hair follicle density.

[0189] Hair follicles were examined under a light microscope and separated from each other. Only hair follicles exhibiting a more swollen, spherical shape and in the growth phase, which is the active growth period for hair, were selected for testing. These selected hair follicles were then cultured in William E medium (12551032, Gibco-Fisher Scientific) supplemented with 2 mM glutamine, 10 ng / ml hydrocortisone (Merck), 10 μg / ml insulin (Merck), and 1% penicillin and streptomycin (Gibco). After a 24-hour culture period, the hair follicles were treated with 100 ng / ml recombinant GDF11 protein for 7 days. During this period, the culture medium and treatment were changed every other day. After treatment, the hair bulbs were observed under a light microscope, images were acquired, and analyzed using ImageJ software.

[0190] Figure 4 shows that treatment with rGDF11 protein increased hair shaft length by approximately 15% over 7 days, compared to an increase of approximately 8% observed in the untreated case.

[0191] Example 10: Analysis of GDF11 production 8 x 10 per well 3Individual dermal papilla cells (HFDPCs) were grown in 96-well plates using appropriate culture medium and necessary supplements (C-26501, Promocell). The following day, the extracts to be tested were added together with TGF-β1 and minoxidil as positive controls. The test concentrations of the extracts were as follows: 0.0006% peptide and sugar-rich extract from Scabiosa arvensis cells, 0.0002% peptide and sugar-rich extract from Lotus japonicus somatic cell embryos, and 0.002% aqueous ethanol extract from artichoke. As a combination, an aqueous ethanol extract from coffee beans was used at a concentration of 0.003% and a water-soluble extract from fig (Ficus carica) stem segments was used at 0.0005%. Both individual and combined extracts were tested. After 6 hours, the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. Next, the cells were washed three times with washing buffer (PBS 1x, 0.5 mM CaCl2, 1 mM MgCl2, 0.1% Triton) and incubated for 30 minutes with shaking using blocking buffer containing 0.5% non-fat dried milk (NFDM, sc-2334, Santa Cruz Biotechnology Inc). After washing with washing buffer, the cells were incubated with anti-GDF11 primary antibody (Abcam, ab124721) (dilution 1:1000) in washing buffer containing 0.5% NFDM. After shaking for 2 hours, the plate was washed three times with washing buffer and incubated with anti-rabbit peroxidase-labeled secondary antibody (1706515, Biorad) (dilution 1:5000) in washing buffer containing 0.5% NFDM. After incubation for 1 hour, the plate was washed three times with washing buffer. The chemiluminescence reaction was performed using QuantaRed® Enhanced Chemifluorescent HRP Substrate (Thermo Fisher Scientific) according to the manufacturer's instructions. After 15 minutes, the absorbance was measured at 490 nm.

[0192] As shown in Figure 5, GDF-11 production was induced by treatment with the extracts of the present invention. Specifically, the peptide extract derived from Scabiosa arvensis cells, as well as aqueous alcohol extracts derived from artichoke and coffee, increased the induction of GDF-11 production by approximately 50%, while the somatic cell embryo enrichment peptide extract from Lotus japonicus and the water-soluble extract derived from Opuntia ficus indica stem segments increased the induction by approximately 30%. Furthermore, a combination of coffee extract and prickly pear extract showed an increase of approximately 85%, which corresponds to the sum of the effects produced by the individual extracts.

[0193] Example 11: Analysis of GDF11 and SOX9 gene expression in human dermal papilla cells under oxidative stress conditions 8 x 10 per well 4 Individual dermal papilla cells (follicle papilla cells, HFDPCs) were grown in a 6-well plate for 20 hours in "Human Hair Follicle Growth Medium" (C-26501, Promocell) supplemented with appropriate supplements. The following day, the cells were treated with hydrogen peroxide (H2O2, 100 μM) for 1 hour to induce free radical formation, grown in culture medium for 7 hours, and then treated again with 100 μM H2O2 for another hour. Finally, the cells were grown overnight in the culture medium.

[0194] The following day, the cells were subjected to two more H2O2 treatment cycles, interspersed with a 7-hour incubation period in culture medium. After the second H2O2 cycle, the cells were treated with the extract of the present invention for 24 hours.

[0195] After processing, the cells were washed with PBS and then collected in lysis buffer as described in Example 7, followed by RNA extraction, reverse transcription, and RT-PCR reaction. The universal primer pair ratio used for the expression of the GDF-11 and SOX9 genes was 4:6.

[0196] The primer sequences used for amplification were as follows: • Hs SOX9 forward (for): Sequence ID 1 • Hs SOX9 Reverse (rev): Sequence ID 2 • Hs GDF11 forward (for): Sequence ID 5 • Hs GDF11 reverse (rev): Sequence ID 6.

[0197] As shown in Figure 7A, GDF11 expression was reduced by approximately 80% under oxidative stress conditions induced by repeated H2O2 treatment compared to unstressed control cells. However, treatment with the extracts of the present invention reversed this condition, restoring expression to levels similar to those of unstressed control cells. Specifically, treatment with a peptide and high-sugar extract derived from Scabiosa arvensis, and a combination of coffee bean extract and prickly pear stem extract increased GDF11 gene expression by approximately 65%, with a peptide and high-sugar extract derived from Lotus japonicus somatic cell embryo increasing it by approximately 50%, and with artichoke extract increasing it by 80%. The increases shown with positive controls TGF-β1 and minoxidil were equivalent to or less than the effects produced by the extracts of the present invention.

[0198] The results shown in Figure 7B demonstrate an effect on SOX9 gene expression. SOX9 was reduced by approximately 150% compared to unstressed control cells, but treatment with the extract of the present invention increased SOX9 gene expression, offsetting this effect. This effect is similar to that produced by recombinant GDF11. Specifically, similar to rGDF11, treatment with peptides and high-sugar extracts derived from Scabiosa arvensis increased SOX9 gene expression by approximately 65%, with aqueous ethanol extract of artichoke by approximately 50%, with somatic cell embryo extract of Lotus japonicus by approximately 90%, and with a combination of coffee bean extract and prickly pear stem extract by approximately 115%.

[0199] literature

[0200] [ka]

[0201]

change

Claims

1. The use of at least one plant extract capable of activating GDF11 growth factor (growth differentiation factor 11) for hair growth treatment, wherein the at least one extract is - Peptides and sugar-rich extracts derived from plant cell cultures belonging to the species Scabiosa arvensis. - A high-peptide and sugar-rich extract derived from a culture of somatic cell embryo-enriched plant cells belonging to the Lotus species. - Aqueous ethanol extract derived from artichoke heads belonging to the species Quercus scolimus, and / or - A water-soluble extract derived from prickly pear cactus stem segments, combined with a water-containing ethanol extract derived from coffee beans. Select from, use.

2. The peptide and sugar-rich extract derived from the culture of the aforementioned Scabiosa arvensis plant cells, a) A step of homogenizing a culture of Scabiosa arvensis plant cells in physiological saline solution to obtain a homogenate; b) A step of separating the solid portion of the homogenate from the liquid portion; c) The solid portion is treated with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins and glycosidic bonds, thereby obtaining the peptide and sugar-rich extract. The use according to claim 1, obtained by a preparation process including the following.

3. An extract derived from a culture of somatic embryo-enriched plant cells belonging to the aforementioned Lotus species, a) A step in which a culture of suspended Lotus corniculatus plant cells is induced to form a somatic cell embryo; b) A step of homogenizing the obtained somatic cell embryo in physiological saline solution to obtain a homogenate; c) A step of separating the solid portion of the homogenate from the liquid portion; d) A step in which the solid portion is treated with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins of the somatic cell embryo and obtain an extract rich in peptides and sugars. The use according to claim 1, obtained by a preparation process including the following.

4. The water-soluble extract of the aforementioned prickly pear cactus, i) A step of subjecting pre-washed prickly pear cactus stems to steam treatment; ii) A step of peeling the prickly pear stem segments obtained in step i); iii) Homogenizing the peeled prickly pear stem segments to obtain homogenates; iv) A step of separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the water-soluble extract of the prickly pear cactus. The use according to claim 1, obtained by a preparation process including the following.

5. A process for preparing a hydrated ethanol extract of coffee, i) A step of grinding raw coffee beans to obtain a ground product; ii) Adding low-temperature ethanol, preferably at a temperature of -30° to -10°C, to the pulverized material, and then performing a homogenization treatment to obtain a homogenate in the form of a suspension in which one solid portion exists in one liquid portion; iii) Shake the suspension at room temperature; iv) Separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the aqueous ethanol extract of the coffee beans; v) A step of filtering the aqueous ethanol extract obtained in step iv). A process that includes this.

6. Use of a water-soluble extract derived from prickly pear stem nodes in combination with a water-soluble ethanol extract derived from coffee beans for hair growth treatment, wherein the water-soluble extract derived from prickly pear stem nodes is obtained by the process described in claim 4, and the water-soluble extract derived from coffee beans is obtained by the process described in claim 5.

7. A process for preparing an aqueous ethanol extract of artichoke, i) A step of crushing artichoke flower heads, preferably artichoke flower heads belonging to the violet variety of Quercus scolimus; ii) Adding low-temperature ethanol, preferably at a temperature of -30° to -10°C, to the obtained pulverized material, and then performing a homogenization treatment to obtain a homogenate; iii) Shake the suspension at room temperature; iv) Separating the solid portion of the homogenate from the liquid portion, where the liquid portion constitutes the aqueous ethanol extract of the artichoke; v) The step of filtering the obtained aqueous ethanol extract. A process that includes this.

8. The process according to claim 7, wherein, prior to performing step i), the artichoke heads are destemmed and peeled, subjected to a washing step in water, and then subjected to a washing step in a 3% to 5%, preferably 5%, sodium bicarbonate solution for a period of 20 to 60 minutes, preferably 30 minutes, wherein the weight / volume ratio of the artichoke heads to the sodium bicarbonate solution is between 1:3 and 1:5, preferably 1:

5.

9. The process according to claim 7 or 8, wherein the artichoke head is pulverized using a blade homogenizer operating at a speed of 1,000 to 3,000 rpm, preferably 1,500 rpm, for 3 to 15 minutes, preferably 3 minutes, at room temperature and in the absence of a solvent.

10. The process according to any one of claims 7 to 9, wherein in step ii) ethanol is added in a homogenate weight / solvent volume ratio between 1:1 and 1:2, preferably 1:1, in an amount of 70% to 80%, preferably 80%, and / or the pulverized material is subjected to two homogenization cycles of adding the low-temperature ethanol, each homogenization cycle being carried out using a blade homogenizer operating at a speed of 3000 to 5000 rpm, preferably 3800 rpm, for 3 to 15 minutes.

11. Use of an artichoke aqueous ethanol extract for hair growth treatment, wherein the artichoke aqueous ethanol extract is obtained by the process described in claim 10.

12. A pharmaceutical or cosmetic composition comprising at least one plant extract as an active ingredient capable of activating GDF11 growth factor for use in hair growth treatment, wherein the at least one extract is - Peptides and sugar-rich extracts derived from plant cell cultures belonging to the species Scabiosa arvensis. - A high-peptide and sugar-rich extract derived from a culture of somatic cell embryo-enriched plant cells belonging to the Lotus species. - Aqueous ethanol extract derived from artichoke heads belonging to the species Quercus scolimus, and / or A pharmaceutical or cosmetic composition selected from a water-soluble extract derived from a prickly pear cactus stem node combined with a water-containing ethanol extract derived from coffee beans.

13. As an active ingredient, - A peptide and sugar-rich extract obtained by the process described in claim 2, derived from a culture of Scabiosa arvensis cells. - A peptide and sugar-rich extract obtained by the process described in claim 3, derived from a culture of somatic cell embryo-enriched plant cells belonging to the species Lotus japonicus. - A aqueous ethanol extract of artichoke obtained by the process described in any one of claims 7 to 10, and / or - A water-soluble extract derived from a prickly pear cactus stem node obtained by the process described in claim 4, combined with a water-containing ethanol extract derived from coffee beans obtained by the process described in claim 5. A pharmaceutical or cosmetic composition according to claim 12, comprising:

Images