Use of plant extracts for promoting hair growth and cosmetic or pharmaceutical composition containing such extracts for such 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 the treatment of hair growth, and to a cosmetic or pharmaceutical composition comprising at least one extract as an active ingredient for the treatment of hair growth. The at least one extract is selected from the group consisting of: a peptide and sugar enriched extract derived from a plant cell culture of scabrous raphanus; an extract rich in peptides and sugars derived from a lotus corniculatus plant cell culture rich in somatic embryos; an aqueous ethanol extract derived from artichoke flower heads; and / or a combination of a water soluble extract derived from Opuntia ficus stem nodes and a water ethanol extract derived from coffee beans.

Description

Technical Field

[0001] This invention relates to the use of one or more plant extracts that can promote hair growth by activating GDF11 growth factor (growth differentiation factor 11).

[0002] These extracts include: derived from radish ( Scabiosa arvensis Extracts from cell cultures, derived from *Lotus styrax* (a type of plant) rich in somatic embryos. Lotus japonicus Extracts from cell cultures, artichokes ( Cynara scolymus The aqueous ethanol extract of the flower head, and the water-soluble extract of the stem nodes of the pear cactus and Arabica coffee ( Coffea arabica A combination of aqueous ethanol extracts of soybeans.

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

[0004] Hair is a skin appendage distributed on an individual's head, serving protective and thermoregulatory functions.

[0005] Each hair consists of an externally visible part, the hair shaft, which extends from the scalp; and an internal part called the hair follicle. The hair follicle is a recessed structure in the epidermis that embeds itself into the skin and extends deep into the dermis.

[0006] Each hair follicle is connected to a sebaceous gland, which is responsible for secreting sebum, a fatty secretion that protects the scalp and hair.

[0007] The slightly enlarged area at the base of the hair follicle is called the hair bulb. It contains various populations of stem cells, including a special type of fibroblast derived from mesenchyme, known as dermal papilla cells (DPCs). These cells play a crucial role in maintaining hair follicle vitality, cell proliferation, and hair growth.

[0008] The maintenance of stem cells in hair follicle bulbs depends on a transcriptional regulator that can bind to DNA, namely SOX9, a member of the SOX gene family: In mice, the absence of SOX9 in epidermal tissue leads to a significant reduction in hair, accompanied by the absence of the dryness marker CD34 (Vidal et al., 2005).

[0009] The normal lifespan of a single hair is 2 to 7 years, and it is estimated that each hair follicle can produce about 20 hairs during its lifespan.

[0010] More specifically, the hair life cycle consists of three main stages: • Growth phase: The period of active hair growth, which is also the longest phase, lasting an average of several years, about 2-4 years for men and about 3-7 years for women.

[0011] • Regression phase: This is the regression phase, during which hair's life activities weaken and growth stops, lasting 2-3 weeks.

[0012] • Telogen effluvium: This is the resting phase, where hair function completely ceases but remains attached to the follicle, primarily falling out during shampooing and combing. This phase lasts an average of 3-4 months, marking the beginning of a new cycle.

[0013] Insulin-like growth factor-1 (IGF-1) plays a key role in the hair cycle by maintaining the anagen phase and delaying the catagen phase, effectively promoting hair growth in hair follicles (Weger, 2005).

[0014] Normally, when hair in the resting phase falls out (called the exogenous phase), the hair follicle has already started a new cycle, and new hair is in the growth phase. Each hair follows an independent life cycle, and they are not synchronized with each other. Therefore, hair does not fall out at the same time, but rather in small amounts each day.

[0015] Hair loss is a natural physiological process, with up to a hundred hairs falling out every day. This phenomenon is more pronounced in certain seasons, such as from September to November, when increased sunlight in summer leads to the accumulation of free radicals (molecules that cause cell aging), which in turn exacerbates hair loss.

[0016] However, under other specific conditions, an imbalance between the number of hairs in the growth phase and the number of hairs in the telogen phase can lead to progressive hair loss, a condition known as baldness or alopecia.

[0017] Baldness can affect both men and women, but it is more common and develops more rapidly in men: it is estimated that 50% to 80% of men worldwide will experience baldness. This usually begins around age 19-20 and stabilizes by age 32-33.

[0018] Rapid thinning of hair manifests as a receding hairline, which in men typically begins at the temples or appears as localized hair loss in specific areas of the scalp. Regardless of the degree of hair loss, the hair may appear fragile or thinner.

[0019] Baldness is a complex condition influenced by genetics, hormones, imbalances in macronutrients and micronutrients, stress, and unhealthy lifestyle habits.

[0020] The most commonly used medications for treating hair loss are minoxidil and finasteride. Minoxidil is a potent antihypertensive vasodilator that promotes cell proliferation in hair follicles. Finasteride, on the other hand, is a potent enzyme inhibitor responsible for synthesizing dihydrotestosterone (DHT), a key hormone in the development of male primary sexual characteristics that can also cause hair follicle atrophy (also known as hair follicle miniaturization).

[0021] Currently, these two drugs are the only ones approved by the U.S. Food and Drug Administration (FDA) for the treatment of hair loss; the former is a topical preparation, and the latter is an oral preparation. However, their efficacy varies from person to person and may cause significant side effects, requiring careful weighing of actual benefits against risks.

[0022] Cosmetic medicine also employs techniques such as autologous hair transplantation, a surgical procedure that uses healthy hair follicles from the patient's own Hippocrates crown to thicken bald areas. However, this type of surgery requires anesthesia, is invasive, and not all transplanted hair follicles will survive.

[0023] Platelet-rich plasma (PRP) is an advanced treatment for alopecia and hair loss. PRP is rich in various 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 factors can stimulate hair follicle stem cells, promoting the formation of new hair follicles and thus achieving hair regrowth.

[0024] Recently, a growth factor, GDF11 (growth differentiation factor 11), was discovered in PRP derived from young blood. The level of this factor in the blood of young organisms is significantly higher than that in older organisms (Bueno et al., 2016), and its level gradually decreases with age. GDF11 is encoded by the specific gdf-11 gene and belongs to the "transforming growth factor β" (TGFβ) superfamily. It was initially discovered for its regulatory role in embryonic development, particularly its crucial role in nervous system development and differentiation of anterior and posterior regions (Williams, 2013; Anderson 2006).

[0025] Later, it was discovered that it has potential rejuvenating regulatory effects in multiple organs. In fact, studies have shown that GDF11 can reduce age-related cardiac hypertrophy (Loffredo et al., 2013), reverse muscle aging (Sinha et al., 2014), and even alleviate some of the harmful effects of functional aging of the brain (Katsimpardiay et al., 2014).

[0026] This significant discovery was validated experimentally in a "heterologous symbiosis" system, where blood from young mice containing the GDF11 factor was mixed with blood from older mice. The two mice were surgically connected, allowing the blood from the young mouse to circulate within the older mouse, or the older mouse to receive a blood transfusion from the young mouse. In the older mice, DNA repair in muscle cells was observed, resulting in healthier and younger muscle fibers. Furthermore, muscle grip strength was enhanced, and compared to untreated mice, the experimental mice ran for a significantly longer time on a treadmill (Loffredo et al., 2013; Sinha et al., 2014).

[0027] Current trends in hair growth products are shifting towards natural and plant-based products. Plant cell cultures are a valuable source of plant extracts containing active ingredients with proven medicinal or cosmetic efficacy. Products made from these cultures are contaminant-free, sustainable, and standardized. Furthermore, their production processes are easily scaled up industrially (Lee et al., 2010).

[0028] JH05170627A discloses a pear-bearing cactus ( Opuntia ficus indica A hair growth treatment agent derived from an extract. This extract can be obtained, for example, by extraction with a solvent at a temperature between room temperature and the solvent's boiling point. In the embodiments, solvents such as 70% ethanol, water, or acetone are used, and extraction is carried out at room temperature or under heating conditions.

[0029] US 20222226410 A discloses a pear-bearing cactus ( Opuntia ficus indica Hair growth treatments containing extracts or portions thereof (such as finely ground fruit or fruit extracts).

[0030] WO 2015132755 A1 discloses a nutritional supplement, cosmetic, or pharmaceutical composition based on a compound of plant extracts derived from the flowers or fruits of cacti and rice (black rice) for inhibiting 5-α reductase. Such preparations are allegedly used to prevent or treat benign prostatic hyperplasia, androgenetic alopecia, and acne.

[0031] Mintel GNPD record ID 5430331, "Hair Protein Care Built-in Protein Enhancer" (February 2018, XP93134018), indicates its ingredients include argan oil, keratin, and artichoke (…). Cynara scolymus Extracts and ylang-ylang essential oil are used to strengthen hair strands and nourish hair roots.

[0032] WO 2019028214 A1 discloses a composition comprising one or more of the following extracts: acai berry extract containing cyanidin-3-glycoside and / or cyanidin-3-rutin, olive extract containing oleuropein, Arabica coffee extract, and / or Tabebuia purpurea extract. The composition also contains micronutrients containing zinc and vitamin D3. The above composition is claimed to be used for non-pharmacological treatment of hair loss and for nourishing and restoring hair, skin, and nails.

[0033] CN101524426 discloses a coffee aqueous ethanol extract for promoting hair growth.

[0034] WO 2016173867 discloses a specific plant extract, namely a peptide / glycan mixture derived from a culture of Gallus gallus domesticus cells rich in somatic embryos, and proposes its use in cosmetics for anti-skin aging and skin tissue regeneration.

[0035] In their paper "Growth differentiation factor 11 participates in the aging process of skin fibroblasts and is induced by peptide and sugar preparations derived from plant cell cultures," published in the journal Molecular Biotechnology (Springer US, New York, Vol. 61, No. 3, January 19, 2019), Tito Annalisa et al. disclosed that specific plant extracts (i.e., peptide / sugar mixtures derived from Lophatherum gracile cell cultures rich in somatic embryos) can stimulate skin cells to produce GDF11.

[0036] IT 2021 0002 0309 revealed the pear cactus ( Opuntia ficus indica Extracts and water-soluble olive extracts are used for the prevention and treatment of atopic dermatitis.

[0037] The technical problem to be solved by the present invention is to provide a plant extract that can effectively promote hair growth and does not have the defects of the above-mentioned prior art products. Summary of the Invention

[0038] The inventors of this invention have discovered that treatment with recombinant GDF11 protein (rGDF11) has an effect similar to TGF-β1 growth factor, which can promote the gene expression of the stem cell marker SOX9 in human dermal papilla cells and the expression of insulin-like growth factor IGF-1, which is crucial for maintaining the hair growth phase.

[0039] Furthermore, the inventors discovered that treatment with recombinant GDF11 protein (rGDF11) in an in vitro dermal papillary sphere (a three-dimensional (3D) cell aggregate) model that replicates the characteristics of hair follicle bulbs can stimulate the expression of Noggin and β-catenin proteins.

[0040] This finding is significant for identifying products with hair care benefits: Noggin protein plays a key role in hair follicle regeneration and growth (Botchkarev, 2001), while β-catenin promotes the induction and persistence of the anagen phase (Dong, 2022).

[0041] In addition, the inventors discovered that treating human hair follicle explants with GDF11 can stimulate hair growth by extending the hair shaft length.

[0042] Based on these findings, the inventors identified plant extracts that could promote the expression of GDF11, and subsequently SOX9 and IGF-1.

[0043] Therefore, one aspect of the present invention relates to using at least one plant extract as an active ingredient to stimulate hair growth, wherein the extract can activate GDF11 growth factor, wherein The at least one plant extract is selected from: 1. An extract rich in peptides and sugars derived from cell cultures of radish plant; 2. An extract rich in peptides and sugars derived from a culture of Gallus gallus domesticus cells rich in somatic embryos; 3. A combination of a water-soluble extract of the stem node of the pomegranate cactus and an ethanolic extract of coffee beans; and / or 4. Aqueous ethanol extract of artichoke flower heads.

[0044] In one embodiment of the present invention, the peptide- and sugar-rich extract derived from *Radix Adenophorae* cell cultures used in the present invention is obtained by the following preparation method: a) Homogenize the cell culture of *Radix acutissima* in a saline solution to obtain a homogenate; b) Separate the solid portion from the liquid portion of the resulting homogenate; c) Treat the solid fraction in an acidic solution with a proteolytic enzyme to hydrolyze cell wall proteins and glycosidic bonds, thereby obtaining an extract rich in peptides and sugars.

[0045] In one embodiment of the present invention, the extract derived from a culture of *Lotus Root* rich in somatic embryos used in the present invention can be obtained by the preparation method described in the applicant’s European patent EP 3288644, the contents of which are incorporated herein by reference.

[0046] The method includes the following steps: a) Inducing the formation of somatic embryos in suspension cultured Lophatherum sp. plant cells; b) Homogenize the obtained somatic embryos in a saline solution to obtain a homogenate; c) Separate the solid portion from the liquid portion of the resulting homogenate; d) Treat the solid fraction with proteolytic enzymes in an acidic solution to hydrolyze the cell wall proteins of the somatic embryo, thereby obtaining an extract rich in peptides and sugars.

[0047] Another aspect of the invention relates to the use of an extract derived from a culture of *Lotus styrax* rich in somatic embryos for hair growth treatment, the extract being prepared by the method described above.

[0048] In one embodiment of the present invention, the water-soluble extract of the prickly pear cactus stem node used in combination with the aqueous ethanol coffee extract according to the present invention is obtained by the method described in Italian Patent Application No. 102021000020309 under the name of the applicant, the contents of which are incorporated herein by reference.

[0049] The method includes the following steps: i) Steam treatment of pre-cleaned pear cactus stem sections; ii) Peel the pear cactus stem segments obtained in step i); iii) Homogenize the peeled pear cactus stem segments to obtain a homogenate; iv) Separate the solid portion of the resulting homogenate from the liquid portion, wherein the liquid portion constitutes a water-soluble extract of the prickly pear cactus.

[0050] In one embodiment of the present invention, a coffee aqueous ethanol extract, used in combination with a water-soluble pear cactus extract according to the present invention, is obtained by a preparation method comprising the following steps: i) Grind green coffee beans (i.e., unroasted coffee beans) to obtain the ground product; ii) Add cold ethanol at -30°C to -10°C (preferably -20°C) to the milled product, and then homogenize to obtain a slurry in the form of a suspension of solids in liquids; iii) Stir the suspension at room temperature; iv) Separate the solid portion from the liquid portion of the homogenate, the liquid portion constituting an aqueous ethanol extract of coffee beans; v) Filter the aqueous ethanol extract obtained in step iv).

[0051] Another aspect of the present invention relates to the use of a combination of a water-soluble extract of the stem node of the prickly pear cactus and an ethanolic extract of coffee beans in the treatment of hair growth, wherein the extracts are obtained by their respective methods as described above.

[0052] In another aspect, the present invention also relates to a method for preparing an artichoke aqueous ethanol extract, comprising the following steps: i) Grind the artichoke heads; ii) Add cold ethanol at -30° to -10°C (preferably -20°C) to the ground product, and then homogenize to obtain a slurry; iii) Stir the suspension at room temperature; iv) Separate the solid portion from the liquid portion of the homogenate, the liquid portion constituting the artichoke aqueous ethanol extract; v) The aqueous ethanol extract obtained by filtration.

[0053] On the other hand, the present invention relates to a pharmaceutical composition or cosmetic composition comprising at least one plant extract capable of activating GDF11 growth factor as an active ingredient (for the aforementioned use in promoting hair growth), and a pharmaceutically and / or cosmetically acceptable carrier, wherein the at least one plant extract of the composition is selected from: 1. An extract rich in peptides and sugars derived from cell cultures of radish plant; 2. An extract rich in peptides and sugars derived from a culture of *Lotus Root* plant cells rich in somatic embryos; 3. An aqueous ethanol extract of artichoke flower heads; and / or 4. A combination of a water-soluble extract derived from the stem nodes of the pear cactus and an aqueous ethanol extract derived from coffee beans.

[0054] In one aspect, the present invention relates to a pharmaceutical composition or cosmetic composition comprising an extract rich in peptides and sugars as an active ingredient, the extract being derived from a radish cell culture obtained by the corresponding method described above.

[0055] In one aspect, the present invention relates to a pharmaceutical or cosmetic composition comprising, as an active ingredient, a peptide- and sugar-rich extract derived from a Gallus gallus domesticus cell culture rich in somatic embryos obtained by the corresponding methods described above.

[0056] In another aspect, the present invention relates to a pharmaceutical composition or cosmetic composition comprising an artichoke aqueous ethanol extract as an active ingredient obtained by the corresponding methods described above.

[0057] In another aspect, the present invention relates to a composition comprising a water-soluble extract of a prickly pear cactus stem node and an aqueous ethanol extract of coffee beans as active ingredients for the treatment of hair growth, both obtained by the corresponding methods described above.

[0058] The present invention will now be described in conjunction with the accompanying drawings, according to preferred embodiments thereof. This description is exemplary and not restrictive. Attached Figure Description

[0059] Figure 1This is a bar chart showing the results of the MTT assay (method to detect the cytotoxicity of the extracts of this invention against human keratinocytes). Specifically, the following extracts were tested: 0.0006% (0.006 mg / ml) of *Radix Adenophorae* cell peptide extract, 0.0002% (0.002 mg / ml) of *Radix Adenophorae* somatic cell embryo peptide extract, 0.002% (0.02 mg / ml) of *Artichoke* ethanol extract, 0.003% (0.03 mg / ml) of coffee ethanol extract, and 0.0005% (0.005 mg / ml) of water-soluble extract from *Opuntia ficus-indica* stem nodes; in addition, a mixture containing both coffee ethanol extract and water-soluble extract from *Opuntia ficus-indica* stem nodes was also tested.

[0060] Figure 2 A bar chart is shown illustrating the effects of recombinant GDF11 protein (rGDF11) at concentrations of 10 ng / ml and 100 ng / ml on the expression of SOX9 and IGF-1 genes in human dermal papillary cells. These two genes are crucial for maintaining stem cell properties and the hair growth cycle, respectively. Values ​​reported in the chart are expressed as a percentage relative to untreated control samples, with values ​​for untreated control samples set at 100%. TGF-β1 growth factor at a concentration of 2.5 ng / ml served as a positive control. Error bars represent standard deviation, and asterisks indicate significant differences.

[0061] Figure 3 Table A presents the results of immunofluorescence analysis performed in an in vitro dermal papillary spheroid model, aiming to evaluate the effect of recombinant GDF11 protein at a concentration of 10 ng / ml on Noggin protein expression. Noggin protein plays a crucial role in hair follicle regeneration and growth. The spheroid cell nuclei were stained blue with DAPI (4',6-diamidinyl-2-phenylindole) dye, while the target protein appeared green. This protein was recognized by a primary antibody targeting the Noggin protein epitope and a secondary antibody bound to the primary antibody, which was labeled with a green fluorescent group.

[0062] Figure 3 Table B demonstrates an immunofluorescence analysis performed in an in vitro dermal papillary spheroid model to assess the effect of recombinant GDF11 protein at a concentration of 10 ng / ml on β-catenin expression. β-catenin plays a crucial role in the induction and maintenance of hair growth. DAPI dye was used to stain the spheroid cell nuclei blue, while the target protein appeared red. This protein is recognized by a primary antibody targeting β-catenin and a secondary antibody binding to the primary antibody, which is labeled with a red fluorescent pheromone.

[0063] Figure 4A bar graph is presented to report the effect of 100 ng / ml recombinant GDF11 protein on hair shaft length elongation in human hair follicle explants. The Y-axis represents the hair shaft length measurements at T0 (at the start of treatment) and 7 days after treatment (T7), expressed as a percentage relative to T0, where T0 is set to 100%.

[0064] Figure 5 This report presents a bar chart illustrating the effect of the extract of this invention on GDF11 expression in human dermal papillary cells (HFDPC). The measurement was performed using an ELISA assay with a specific antibody targeting the GDF11 protein. The effects of minoxidil and TGF-β1 on GDF11 production were also evaluated: minoxidil is a treatment for androgenetic alopecia, and TGF-β1 served as a positive control. Error bars represent standard deviation, with asterisks indicating significant differences.

[0065] Figure 6A This is a bar chart reporting the effect of the extract of this invention on SOX9 gene expression. This gene is crucial for maintaining the stemness of dermal papillary cells. The chart also includes the effects of TGF-β1 and minoxidil. Error bars represent standard deviations, and asterisks indicate significant differences.

[0066] Figure 6B This is a bar chart reporting the effect of the extract of this invention on IGF-1 gene expression. This gene is crucial for maintaining the hair growth phase in dermal papilla cells. The chart also includes the effects of TGF-β1 and minoxidil. Error bars represent standard deviation, and asterisks indicate significant differences.

[0067] Figure 7A A bar graph is presented to report the effect of the extract according to the present invention on GDF-11 gene expression in human dermal papillary cells under oxidative stress. To induce free radical generation, cells were treated with hydrogen peroxide (H2O2, 100 µM) for 1 hour, then grown in culture medium for 7 hours, followed by a second treatment with 100 µM H2O2 for 1 hour. Cells were then grown overnight in culture medium. This treatment process was repeated the following day for two cycles of H2O2 treatment, interspersed with 7 hours of culture medium incubation. After the second round of H2O2 treatment, cells were treated with the extract of the present invention for 24 hours. TGF-β1 and minoxidil were used as positive controls. The values ​​reported in the figure are expressed as a percentage relative to the H2O2-stressed sample, with the H2O2-stressed sample value set at 100%. Error bars represent standard deviation, and asterisks indicate significant differences.

[0068] Figure 7BThis is a bar chart reporting the effect of the extract of the present invention on SOX9 gene expression in human dermal papillary cells under oxidative stress. To induce free radical formation, cells were treated with hydrogen peroxide (H2O2, 100 µM) for 1 hour, then grown in culture medium for 7 hours, followed by treatment with 100 µM H2O2 for 1 hour. Cells were then grown overnight in culture medium. The complete treatment cycle was repeated the following day, followed by treatment with the extract of the present invention for 24 hours. Recombinant GDF11 protein, TGF-β1 factor, and minoxidil were used as positive controls. Values ​​reported in the figure are expressed as a percentage relative to the H2O2-stressed sample, with the H2O2-stressed sample value set at 100%. Error bars represent standard deviation, and asterisks indicate significant differences. Detailed Implementation

[0069] The applicant discovered that the GDF11 protein is particularly useful in combating hair follicle atrophy, and therefore can be used to identify extracts or products. By activating it, it can be applied in the field of hair care, especially to promote hair growth.

[0070] Treatment with recombinant GDF11 (rGDF11), similar to treatment with TGF-β1 growth factor, activates and enhances the activity of the SOX9 transcription factor in dermal papilla cells. It also increases the expression level of IGF-1 in these cells, which is crucial for the prolongation and maintenance of the hair growth phase.

[0071] The applicant also observed that exogenous GDF11 treatment had a surprising effect on the three-dimensional dermal papillary sphere model, which, compared to standard two-dimensional cell culture, more effectively replicated the hair follicle microenvironment.

[0072] The applicant discovered that treatment of a 3D spherical model with exogenous GDF11 had a surprising effect on proteins related to hair follicle activation, regeneration, and growth, as well as hair regrowth. Specifically, treatment with recombinant GDF11 significantly increased the expression levels of Noggin and β-catenin proteins in the model. Noggin is involved in hair follicle regeneration, and its deficiency is associated with reduced hair follicle numbers and delayed growth; β-catenin is involved in the initial stage of the hair life cycle, promoting the induction and persistence of the growth phase (anagen phase).

[0073] Furthermore, the applicant discovered that treating in vitro hair follicle explants from male patients undergoing follicle unit extraction (FUE) hair follicle transplantation with recombinant GDF11 resulted in hair shaft elongation. The observed elongation was more significant than that observed in untreated control explant samples.

[0074] Based on the above findings, the applicant has identified plant extracts that can activate GDF11 growth factor for hair treatment, which means that they can effectively stimulate the gene expression of this growth factor.

[0075] Against this backdrop, the applicant's extracts derived from plant cell cultures of *Radix Achyranthes bidentata* rich in peptides and sugars; extracts derived from plant cell cultures rich in somatic embryos of *Lobelia chinensis* rich in peptides and sugars; an ethanolic water extract of *Artichoke*; and a combination of a water-soluble extract of *Pyracantha fortuneana* stem nodes and an ethanolic water extract of coffee beans (e.g., a mixture of the above extracts) have significant effects on hair care, promoting hair follicle cell proliferation, and prolonging the hair growth phase.

[0076] These plant extracts effectively stimulated the expression of GDF11 in dermal papilla cells, with effects comparable to or even better than the experimental control group (TGF-β1 growth factor). Furthermore, they outperformed the well-known hair treatment agent minoxidil, which combats hair follicle atrophy (or miniaturization) by promoting follicular capillary dilation and stimulating cell proliferation.

[0077] In addition, although the stem and node extracts of prickly pear cactus and coffee bean extracts can activate GDF11 production on their own, they show a better (synergistic) effect when used together.

[0078] In addition to activating GDF11 expression, the above extracts can also stimulate genes activated in dermal papillary cells when treated with recombinant GDF11, such as transcription factors SOX9 and IGF-1.

[0079] Specifically, a mixture of water-soluble extracts from the stem nodes of the pomegranate cactus and an aqueous ethanol extract from coffee beans showed surprising effects on the two genes analyzed compared to the single extracts: it was found that these extracts only effectively affected SOX9 gene expression when tested together as a mixture, rather than when tested individually, and unexpectedly showed a synergistic effect on IGF-1 expression.

[0080] The applicant discovered that repeatedly exposing dermal papillary cells to oxidative stress (simulating aging) with 100 µM H2O2 significantly reduced GDF11 expression. However, the extract of this invention reversed this state, restoring GDF11 expression to levels similar to those in unstressed cells. The extract of this invention is even more effective than the positive controls TGF-β1 growth factor and minoxidil.

[0081] Finally, the applicant discovered that, similar to recombinant GDF11 protein therapy, the extract of the present invention can restore the expression of SOX9 transcription factor associated with stem cell markers in dermal papillary cells, wherein repeated treatment with 100 µM H2O2 can mimic the aging process.

[0082] An extract rich in peptides and sugars can be obtained from radish plant cell cultures by a preparation method including the following steps: a) Homogenize the cell culture of radish plant in a saline solution to obtain a homogenate; b) Separate the solid portion from the liquid portion of the resulting homogenate; c) Treat the solid fraction with proteolytic enzymes in an acidic solution to hydrolyze cell wall proteins and glycosidic bonds, thereby obtaining an extract rich in peptides and sugars.

[0083] Homogenization refers to the process of pulverizing plant material in a suitable container, such as using a pre-cooled ceramic mortar and pestle, or for larger volumes, using a metal container with metal blades and a laboratory or industrial mixer or press.

[0084] Preferably, the radish plant cell culture is obtained through the following steps: obtaining plant tissue from the radish plant, inducing the tissue to form callus on a solid culture medium, harvesting the callus and establishing a liquid culture therefrom.

[0085] Preferably, step b) separates the solid and liquid portions of the homogenate by centrifugation, sedimentation, or filtration.

[0086] In a preferred embodiment of the present invention, the method further includes: washing the solid portion separated in step b) with distilled water before treating the solid portion with proteolytic enzyme in step c) to remove residual cytoplasmic components.

[0087] In another preferred embodiment of the invention, the method further includes: washing the solid portion separated in step b) with distilled water to remove residual cytoplasmic components before treating the solid portion with proteolytic enzymes in step c), and then treating the solid portion with heated EDTA solution, preferably heated to boiling (e.g., about 100°C).

[0088] The EDTA solution can be, for example, an aqueous solution with an EDTA concentration of 2 mM. The time for treating the homogenized solid portion with the heated EDTA solution can be 10 to 30 minutes (preferably 20 minutes). Pectin and starch are removed from the cell wall by the calcium chelation effect of EDTA (which promotes the binding of pectin and starch in the cell wall).

[0089] The peptide- and sugar-rich extracts obtained by the above methods can be used in their current form or dried to produce powder using known methods (e.g., freeze-drying or spray-drying).

[0090] The present invention also relates to the use of a peptide- and sugar-rich extract derived from a radish plant cell culture for hair growth treatment, wherein the extract is obtained using the method described above.

[0091] The extract derived from plant cultures rich in Gallus gallus domesticus somatic cell embryos can be obtained by the preparation method described in the European patent EP 3288644 under the name of the applicant, the contents of which are incorporated herein by reference.

[0092] The method includes the following steps: a) Inducing the formation of somatic embryos in suspension cultured Lophatherum sp. plant cells; b) Homogenize the obtained somatic embryos in a saline solution to obtain a homogenate; c) Separate the solid portion from the liquid portion of the resulting homogenate; d) Treat the solid fraction with proteolytic enzymes in an acidic solution to hydrolyze the cell wall proteins of the somatic embryo, thereby obtaining an extract rich in peptides and sugars.

[0093] Plant cell cultures can be obtained by harvesting plant tissue from Lophatherum gracile, inducing the tissue to form callus on a solid substrate, collecting the callus, and establishing a liquid culture from it.

[0094] Preferably, step a) is carried out by adding plant hormones, such as thiabendazole (TDZ) and benzylaminopurine (BAP), to the plant culture.

[0095] The saline solution in step b) is usually a buffer solution, such as phosphate-buffered saline (PBS) with a pH of 7.4.

[0096] Preferably, step c) separates the solid and liquid portions of the resulting homogenate by centrifugation, thereby resulting in the separation of the supernatant (liquid portion) and the precipitate (solid portion).

[0097] In a preferred embodiment of the present invention, before step d) treating the solid portion with a proteolytic enzyme, the homogenized solid portion separated in step c) is further washed with distilled water to remove residual cytoplasmic components.

[0098] In another preferred embodiment of the invention, the method further includes washing the homogenized solid portion separated in step b) with distilled water to remove residual cytoplasmic components before treating the solid portion with proteolytic enzymes in step d), and then treating the solid portion with heated EDTA solution (preferably boiled, for example, at about 100°C).

[0099] The EDTA solution can be, for example, an aqueous solution with an EDTA concentration of 2 mM. The homogenized solid portion can be treated with the heated EDTA solution for 10 to 30 minutes, preferably 20 minutes, to remove pectin and starch from the cell wall through the calcium chelation of EDTA (which promotes the binding of pectin and starch in the cell wall).

[0100] The extract obtained by the method can be used in its current form or dried using known methods (e.g., freeze-drying or spray-drying) to produce a powder.

[0101] The present invention also relates to the use of an extract derived from a Lotus japonicus plant cell culture rich in somatic embryos in the treatment of hair growth, the extract being obtained by the above method.

[0102] The present invention also relates to a method for preparing a water-ethanol extract of artichoke, comprising the following steps: i) Grinding artichoke flower heads; ii) Adding cold ethanol to the grinding product and then homogenizing to obtain a homogenate; iii) Stirring the suspension at room temperature; iv) Separating the solid part from the liquid part in the homogenate, wherein the liquid part constitutes the water-ethanol extract of artichoke; v) Filtering the obtained water-ethanol extract.

[0103] Preferably, the artichoke flower heads used are from the violet variety of artichoke.

[0104] Preferably, in step i), the artichoke flower heads to be ground are first frozen at a temperature of -20°C to -40°C (preferably about -30°C). In addition, preferably, before performing step i), the artichoke flower heads from which the stalks and outer leaves have been removed are first washed with water and then soaked in a 3% to 5% (preferably 5% concentration) sodium bicarbonate solution for 20 to 60 minutes (e.g., 30 minutes). In the washing step, the weight-to-volume ratio of artichoke to the sodium bicarbonate solution can be 1:3 to 1:5, preferably 1:5. This pre-washing step can ensure that the extract is sterile, free of contamination, and free of bacteria that may metabolize and degrade the active ingredients in the final extract. After washing, the artichoke flower heads are rinsed with sterile distilled water to remove bicarbonate residues, then dried (e.g., dried under a horizontal laminar flow hood), and subjected to the grinding in step i). Preferably, the artichoke flower heads are placed in a freezer and cooled to -20°C to -40°C (preferably about -30°C) before grinding.

[0105] Preferably, in step i), the grinding is carried out using a blade homogenizer while the artichoke is still frozen and without using a solvent. The rotation speed is 1000 - 3000 revolutions per minute (preferably 1500 revolutions per minute), and the grinding time is 3 - 15 minutes (preferably 3 minutes), so as to obtain a more uniform matrix.

[0106] Preferably, in step ii), the ethanol addition concentration is 70% to 80% (preferably 80%), and the ratio is between 1∶1 and 1∶2, preferably the ratio is 1∶1 (homogenate weight / solvent volume).

[0107] The ethanol added in step ii) is cold ethanol, i.e., at a temperature of -30°C to -10°C, preferably -20°C.

[0108] Preferably, in step ii), when ethanol is added to the milled product, the temperature of the milled product is -30°C to -10°C, preferably -20°C.

[0109] Preferably, in step ii), the ground product undergoes two homogenization cycles with the addition of cold ethanol. Each homogenization cycle is performed using a blade homogenizer at a speed of 3000-5000 rpm, preferably 3800 rpm, for a duration of 3 to 15 minutes.

[0110] Advantageously, this allows for better homogenization of the matrix with a smaller extraction volume; furthermore, using the aforementioned percentage of ethanol instead of pure ethanol (96%) optimizes the extraction rate and allows for more efficient extraction of polyphenols.

[0111] Preferably, after step ii) and before step iii), additional ethanol of the above concentration is added to the homogenate to achieve a final extraction ratio of 1:5 (homogeneous weight / solvent volume). For example, after step ii) and before step iii), 80% ethanol can be added to the homogenate at a ratio of 1:4 (homogeneous weight / solvent volume) to achieve a final extraction ratio of 1:5.

[0112] Preferably, in step iii), the slurry is stirred with a stirrer at a temperature of 20°C to 30°C (preferably 25°C) for 1 to 4 hours (preferably 2 hours).

[0113] Preferably, in step iv), the separation is carried out by centrifugation at a speed of 4000-6000 rpm (preferably 5000 rpm) for 5-15 minutes (preferably 10 minutes).

[0114] Preferably, in step v), the filtration is performed using qualitative filter paper with a pore size of 60-68 micrometers, preferably with the aid of a vacuum pump.

[0115] The aforementioned extracts can be concentrated using a rotary evaporator (maximum temperature 25°C) to remove excess ethanol that is harmful to cells.

[0116] The extract can then be dried using mature methods such as freeze-drying or spray-drying to form a powder. Freeze-drying yields a more stable extract, thus extending its shelf life.

[0117] According to the present invention, the water-soluble pear cactus extract, preferably used in combination with the aqueous ethanol coffee extract, is obtained by the preparation method described in Italian Patent Application No. 102021000020309 under the name of the applicant, the contents of which are incorporated herein by reference.

[0118] The method includes the following steps: i) Steam treatment of the pre-cleaned pear cactus palm leaves; ii) Peel the pear cactus stem segments obtained in step i); iii) Homogenize the peeled pear cactus stem segments to obtain a homogenate; iv) Separate the solid portion from the liquid portion of the resulting homogenate, wherein the liquid portion constitutes a water-soluble pear cactus extract.

[0119] Preferably, the steam treatment in step i) is carried out at a pressure not exceeding 4.5 bar, preferably 3 bar to 4.5 bar, for a duration of 5 minutes to 30 minutes, preferably 10 minutes to 20 minutes.

[0120] Preferably, in step iii), the peeled pear cactus stem segments are homogenized under frozen conditions, preferably at a temperature of about -30°C.

[0121] Preferably, in step iii), the peeled pear cactus stem section is homogenized for the first time in a rotary blade homogenizer without solvent, the rotary blade homogenizer running at a speed of 2000 rpm to 3500 rpm for 1 to 30 minutes, preferably 3 minutes, and then homogenized for the second time in the presence of solvent, the rotary blade homogenizer running at a speed of 3500 rpm to 4500 rpm (more preferably 3800 rpm) for 5 to 30 minutes, preferably 3 minutes.

[0122] Preferably, the solvent in step iii) is selected from aqueous solution, salt solution or water; more preferably, the weight ratio of the homogenate obtained in step iii) to the solvent is 1:1 to 1:3.

[0123] Preferably, step iv) is performed by centrifugation or filtration.

[0124] According to the use of the present invention, the aqueous ethanol coffee extract, preferably used in combination with the water-soluble pear cactus extract, is obtained by a method comprising the following steps: i) Grind green coffee beans to obtain the ground product; ii) Add cold ethanol at -30°C to -10°C (preferably -20°C) to the ground product, followed by homogenization to obtain a slurry in which the solid portion is suspended in the liquid portion. iii) Stir the suspension at room temperature. iv) Separate the solid portion of the homogenate from the liquid portion, which constitutes an aqueous ethanol coffee bean extract. iv) Filtration step iv) to obtain the aqueous ethanol extract.

[0125] It is preferred to use Arabica species ( Coffea arabica Santos coffee beans are decaffeinated using high-pressure carbon dioxide (CO2) and are unroasted.

[0126] Preferably, the coffee bean temperature in step i) is between -20°C and -40°C, more preferably about -40°C.

[0127] Preferably, the grinding in step i) is carried out in a rotary blade homogenizer with a speed of 3000-5000 rpm, preferably 3800 rpm, for a duration of 3 to 15 minutes.

[0128] Preferably, in step ii), 96% ethanol is added in a ratio of 1:1 to 1:3, more preferably in a ratio of 1:2 (slurry weight / solvent volume).

[0129] Preferably, in step ii), ethanol is added to the grinding product at a temperature of -20°C to -10°C (preferably -15°C).

[0130] The ethanol added in step ii) is cold ethanol, that is, its temperature is between -30°C and -10°C, preferably -20°C.

[0131] Preferably, in step ii), the homogenization operation is carried out in a rotary blade homogenizer at a speed of 3000-5000 rpm, preferably 4000 rpm, for a duration of 3 to 15 minutes.

[0132] Preferably, in step iii), the homogenate is stirred with a stirrer at a temperature of 20°C to 30°C, preferably 25°C, for 1 to 4 hours, preferably 2 hours.

[0133] Preferably, in step iv), the separation is carried out by centrifugation at a speed of 5000-7000 rpm (preferably 6300 rpm) for a duration of 5 to 15 minutes (preferably 10 minutes).

[0134] Preferably, in step v), qualitative filter paper with a pore size of 60-68 micrometers is used for filtration, while microfiltration is performed using a Stericap™ PLUS device (Millipore) with a pore size of 0.22 micrometers.

[0135] The aforementioned extract can be concentrated using a rotary evaporator until a suspension with a dry matter percentage of 80% ± 4% (by weight) is obtained.

[0136] The present invention also relates to a method for preparing the above-mentioned aqueous ethanol coffee extract, and the use of combining a water-soluble extract of prickly pear cactus stem nodes with an aqueous ethanol coffee bean extract in hair growth treatment, wherein the aforementioned extracts are obtained by their respective methods described above.

[0137] These extracts, especially when applied topically to the scalp, can be used simultaneously, individually, or sequentially.

[0138] When used simultaneously, the above-mentioned extracts can be mixed to form a mixture or composition containing such extracts, and then applied topically.

[0139] Preferably, when the extracts are used in combination according to the present invention, the weight ratio of the aqueous ethanol coffee extract to the water-soluble prickly pear cactus stem node extract ranges from 10:1 to 3:1, more preferably 6:1. Specifically, it is used in the form of a mixture or composition containing the extracts in the above proportions. Preferably, when using a mixture or composition containing the extracts in the above proportions, the concentration of the aqueous ethanol coffee extract should be at least 0.003% of the total weight of the mixture or composition.

[0140] This invention also relates to a pharmaceutical or cosmetic composition containing at least one plant extract as an active ingredient, which activates GDF11 growth factor to achieve the aforementioned hair growth promotion purpose, wherein the at least one plant extract in the composition is selected from: 1. An extract rich in peptides and sugars derived from cell cultures of radish plant; 2. An extract rich in peptides and sugars derived from a culture of *Lotus Root* plant cells rich in somatic embryos; 3. An aqueous ethanol extract of artichoke flower heads; and / or 4. A combination of a water-soluble extract derived from the stem nodes of the pear cactus and an ethanolic extract derived from coffee beans.

[0141] The present invention also relates to a pharmaceutical composition or cosmetic composition comprising an extract rich in peptides and sugars as an active ingredient, the extract being derived from a radish cell culture obtained by the corresponding method described above.

[0142] The present invention relates to a pharmaceutical composition or cosmetic composition comprising, as an active ingredient, an extract rich in peptides and sugars, derived from a Gallus gallus domesticus cell culture rich in somatic embryos obtained by the corresponding methods described above.

[0143] This invention relates to a pharmaceutical composition or cosmetic composition comprising an artichoke aqueous ethanol extract as an active ingredient, obtained by the corresponding methods described above.

[0144] The present invention also relates to a composition comprising a water-soluble extract derived from the stem nodes of the pomegranate cactus and an aqueous ethanol extract of coffee beans as active ingredients, both obtained by the methods described above.

[0145] The above-mentioned pharmaceutical compositions or cosmetic preparations may be in any topical dosage form, such as shampoos, serums, conditioners, lotions, gels or washes that can be applied topically (to the scalp) for external use on the scalp.

[0146] In addition, these compositions may include additional components known in the art, such as carriers, solvents, excipients and / or cosmetic and / or pharmaceutically acceptable excipients.

[0147] Preferably, the solvent is a hydrophilic solvent, preferably selected from water and aqueous salt solutions, or one or more organic solvents compatible with cosmetic and / or pharmaceutical preparations, more preferably selected from alcohols, glycerol, organic acids, amides, amines, aldehydes or ketones, or a combination of two solvents (if they are miscible).

[0148] Carriers that can be used in the above compositions include liposomes (preferably multilayered liposomes), cyclodextrins, and silicates.

[0149] Finally, the present invention relates to a pharmaceutical or cosmetic treatment method for promoting hair growth, comprising applying a pharmaceutically or cosmetically effective amount of the above-described composition (i.e., containing at least one of the above-described plant extracts) topically to the scalp in need of treatment.

[0150] The following are some examples of the preparation of the extract of the present invention, and experimental examples demonstrating the bioactivity of GDF11 protein and the extract of the present invention in in vitro cell models, three-dimensional spherical models and in vitro hair follicles. These examples are not intended to limit the present invention.

[0151] Example Example 1: Preparation method of extract derived from cell culture of radish plant according to the present invention.

[0152] The following are the steps of a method for preparing an extract rich in peptides and sugars derived from a radish plant cell culture according to the present invention.

[0153] Steps for preparing cell cultures Plant callus cultures were established on solid culture media using leaves of *Radix Adenophorae* seedlings as the starting material. Specifically, whole leaves of *Radix Adenophorae* were sterilized with a 70% ethanol aqueous solution for 15 minutes, followed by a 1% sodium hypochlorite aqueous solution for 15 minutes. After washing three times with water to remove alcohol and hypochlorite, the leaves were cut into 5mm × 5mm fragments and placed on a solid substrate, "B5 Gamborg medium." This substrate contained "plant agar" (7.5 mg / L), inositol (500 mg / L), sucrose (30 g / L), and the pH was adjusted to 5.7 with potassium hydroxide (0.1 N). Then, 2,4-dichlorophenoxyacetic acid (1 mg / L), adenine (1 mg / L), and kinetin (0.01 mg / L) were added to the autoclaved medium. Callus tissue was obtained after incubation in the dark at 20°C for approximately 5 weeks, and then transferred to liquid culture medium to obtain cultures.

[0154] Growth steps of liquid culture When the callus reaches approximately 1 cm in diameter (weighing approximately 50 mg), remove it and place it in a flask containing 50 ml of the same culture medium (without agar). Place the flask on a shaker in the dark and shake at 120 rpm. After approximately 10 days, the callus begins to decompose and form a homogeneous cell culture consisting of single cells or small cell aggregates.

[0155] Cell harvesting steps When the culture reached a density of approximately 150 g / L in a 2 L flask, the cells were collected through a low-pore filter (80–100 μm) to remove the culture medium. The cells were then washed with sterile distilled water and stored frozen at -80 °C.

[0156] Preparation of peptide and sugar extracts 500 g of frozen cells were mechanically disrupted and homogenized in PBS (NaCl 136 mM, KCl 2.7 mM, NaH2PO4 12 mM, KH2PO4 1.76 mM, pH 7.4) (1:2 volume / weight ratio). The resulting homogenate was centrifuged at 8500 rpm for 15 minutes at 4 °C to precipitate the insoluble components. The precipitate (containing cell walls) was treated with twice the volume of 2 mM EDTA solution and heated at 100 °C for 20 minutes. After cooling, the extract was filtered through a cellulose membrane with a pore size of 80-100 μm, washed, and filtered again to remove residual EDTA. The resulting precipitate was then resuspended in twice the volume of 0.1 N hydrochloric acid solution and boiled at 100 °C for 1 hour to hydrolyze and dissolve all sugars in the cell wall glycoproteins.

[0157] After boiling, the sample was cooled on ice and then enzymatically hydrolyzed with protease at 37°C for 16 hours. After hydrolysis, the suspension was centrifuged to obtain a clear solution, and the pH was adjusted to 6.5 with 10 N NaOH. This yielded an extract rich in peptides and sugars derived from the cell walls of *Radish arugula*.

[0158] Example 2: Preparation method of extract derived from *Lotus Root* cell culture rich in somatic embryos according to the present invention Using leaves from *Lotus Root* seedlings as raw material, plant callus cultures were first established on solid culture media, followed by induction of somatic embryos from liquid cell cultures. Once the desired density was reached, the somatic embryo cultures were collected and processed to prepare extracts.

[0159] The method used is as follows: a) Callus preparation: Intact leaves from *Lotus Root* plants were collected and disinfected with 70% ethanol aqueous solution for 15 minutes, followed by 1% sodium hypochlorite aqueous solution for 15 minutes. After rinsing three times with water to remove ethanol and sodium hypochlorite, the leaves were cut into fragments of approximately 0.5 cm² using a sterile blade. All leaf fragments were placed on solid B5 Gamborg medium, the composition of which was: plant agar 7.5 mg / L, 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. After incubation at 20°C in the dark for approximately 5 weeks, leaf cells proliferated to form callus. Every 3-4 weeks, the callus was excised and transferred to fresh culture medium.

[0160] b) Preparation of cell culture: When the callus tissue reaches a diameter of approximately 1 cm (weighing approximately 50 mg), it is picked and dispersed in a flask containing 50 mL of AB1 liquid medium (B5 Gamborg medium contains: 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). The flask is placed on a shaker in the dark and shaken at 100 rpm. After approximately 10 days, the callus tissue decomposes and proliferates to form a cell suspension culture.

[0161] c) Preparation of somatic embryos: Plant cell cultures were resuspended in fresh AB2 liquid growth medium (B5 Gamborg medium containing 500 mg / L inositol and 30 g / L sucrose) and plant hormones (1 mg / L thidiazuron (TDZ), 0.05 mg / L benzylaminopurine (BAP), 10 mg / L glutathione, 500 mg / L casein, 7% ammonium phosphate and 10% ammonium sulfate) to induce the formation of somatic embryos (cell cultures rich in somatic embryos) in the cell cultures.

[0162] d) Growth of somatic embryonic cell cultures: Somatic embryo induction continued for 4 weeks, with fresh culture medium added weekly until the differentiation level of somatic embryos reached 70-90%.

[0163] e) Collection of somatic embryos: Somatic embryos can be separated from the culture medium by centrifugation at 2000g, sedimentation, or filtration using a membrane with a pore size of less than 100 micrometers. The embryos are then frozen at -80°C to preserve their chemical and physical properties.

[0164] f) Homogenization of somatic embryos: Frozen somatic embryos were mechanically homogenized in phosphate-buffered saline (PBS) (NaCl 136 mM, KCl 2.7 mM, NaH2PO4 12 mM, KH2PO4 1.76 mM, pH 7.4) at a ratio of 1:2 (weight / volume). This step can be performed in a suitable container such as a pre-cooled ceramic mortar; for larger samples, a metal container with a metal blade can be used, and homogenization can be completed using a laboratory or industrial mixer or press.

[0165] After obtaining homogenized lysate from somatic embryos, the sample is centrifuged (e.g., at 4°C and 4000 rpm for about 15 minutes) to precipitate insoluble components.

[0166] g) Obtaining peptide and sugar extracts from the cell wall: The precipitate (containing the cell wall) obtained in step f) was further washed with distilled water to remove any soluble residues and filtered again. The precipitate was weighed, suspended, and boiled for 20 minutes with 2 mM EDTA solution (w / v). After cooling, the extract was filtered through a filter cloth, washed, and filtered again to remove residual EDTA. The resulting precipitate was then boiled for 1 hour with 2 mM 0.1 N hydrochloric acid solution in a fume hood to hydrolyze the sugar bonds. After boiling, the sample was resuspended, cooled on ice, and then enzymatically hydrolyzed at 37°C for 16 hours with a protease (1 mg of enzyme per mL of suspension). After hydrolysis, the suspension was centrifuged or filtered to obtain a clear solution: thus yielding a hydrophilic extract rich in peptides and sugars from the cell wall.

[0167] Example 3: Preparation of water-soluble cactus extract The method for preparing the water-soluble cactus extract according to the present invention is as follows: Take 1.2 kg of freshly harvested cactus stem segments and rinse them with running water to remove soil residue. Then, place the stem segments in a high-pressure steam environment (pressure up to 4.5 bar) for about 15 minutes. After peeling and removing the thorns, place them under a horizontal laminar flow hood and dry them with absorbent paper. Weigh the pre-treated stem segments (about 1 kg) and transfer them to a -30℃ freezer for storage.

[0168] Subsequently, the frozen cactus stem segments were homogenized for the first time at 2000 rpm for 3 minutes at room temperature without adding any solvent. After that, a second homogenization treatment was carried out at 3800 rpm for 3 minutes at room temperature, and an aqueous solution or salt solution (phosphate buffered solution, i.e., PBS) was added at a 1:1 ratio.

[0169] The resulting homogenate was centrifuged at 6300 rpm for about 15 minutes at 4°C to precipitate the insoluble components. The supernatant obtained by centrifugation was collected and lyophilized to obtain the water-soluble cactus extract of this invention.

[0170] Example 4: Preparation of water-ethanol coffee extract The coffee beans used in this invention were obtained from Kimbo Caffè SpA (Naples) and belong to the Arabica variety. Coffea arabica The Santos variety is decaffeinated using a high-pressure carbon dioxide process. Furthermore, this coffee is characterized by being made from green beans, meaning it has not been roasted. Roasting is typically done at 200°C, a process that deactivates many of the active molecules present in coffee.

[0171] Upon receiving decaffeinated green coffee beans, they are immediately transferred to a freezer at -40°C. According to the present invention, an aqueous ethanol extract of coffee beans is prepared using the following method: Take 500g of frozen coffee beans and perform an initial homogenization process at 3800 rpm for 3 minutes at room temperature (without adding solvent). Then perform two additional homogenization processes at 4000 rpm (3 minutes each) at room temperature, and add cold 96% ethanol at a ratio of 1:2 (weight / volume).

[0172] The resulting suspension was placed in a stirrer and incubated at 25°C for 2 hours. The resulting suspension was centrifuged at 6300 rpm for 10 minutes, and the supernatant was filtered using a vacuum pump. Residual solids were removed by filtration using 60-68 micron qualitative filter paper. Subsequently, microfiltration was performed again using a Stericap™ PLUS filtration device (0.22 μm porosity, Millipore) with a vacuum pump.

[0173] The microfiltered extract was placed in a glass flask and concentrated using a rotary evaporator (the maximum temperature of the sample did not exceed 25°C) to obtain the aqueous ethanol coffee extract of the present invention.

[0174] Example 4a: Preparation of a mixture of aqueous ethanol coffee bean extract and water-soluble cactus stem node extract The preparation method of the mixture (composition) of the present invention is as follows: The aqueous ethanol coffee bean extract obtained according to Example 4 and the water-soluble cactus stem and node extract obtained according to Example 3 are mixed in a 6:1 ratio. This mixture is used in subsequent test examples as follows: Both extracts were dissolved in water at a concentration of 10%, stirred for 5 minutes, and then centrifuged to remove insoluble residues. The resulting supernatant was collected for in vitro testing. The supernatants of the coffee bean extract and the cactus stem node extract were mixed in a culture medium for addition to the cells to be treated in the tests described in subsequent examples.

[0175] Example 5: Preparation of Artichoke Extract in Aqueous Ethanol artichoke ( Cynara scolymus The artichoke heads (violet variety) were purchased from a company in the Puglia region with all relevant organic certifications. After removing the stems and outer skin, they were thoroughly washed with running water and then soaked in a 5% sodium bicarbonate solution (weight / volume ratio 1:5) for 30 minutes. Following treatment, the artichoke heads were rinsed with sterile distilled water to remove bicarbonate residue and then dried under a horizontal laminar flow hood. The pre-treated artichoke heads were weighed (approximately 500 grams) and transferred to a -30°C freezer.

[0176] The hydroethanol artichoke extract according to the present invention is prepared by the following method: Take 500g of still-frozen artichoke heads and homogenize them initially at 1500 rpm for 3 minutes at room temperature (without adding solvent). Then add cold 80% ethanol (weight / volume ratio 1:1) and homogenize twice at 3800 rpm for 3 minutes each time. After homogenization, add 80% ethanol again at a ratio of 1:4 (homogeneous material weight / solvent volume), resulting in a final extraction ratio of 1:5. Place the resulting suspension on a shaker and incubate at 25°C with shaking for 2 hours.

[0177] The resulting suspension was then centrifuged at 5000 rpm for 10 minutes, and the supernatant was filtered through qualitative filter paper (60-68 microns) using a vacuum pump to remove residual solids.

[0178] The obtained extract was placed in a glass flask and excess ethanol was removed by evaporation using a rotary evaporator (maximum sample temperature 25°C). Subsequently, it was freeze-dried to obtain the hydroethanol artichoke extract of the present invention.

[0179] Example 6: Cytotoxicity Detection To ensure that the concentration of the extract involved in this invention is not toxic to the growing cells in subsequent tests, cytotoxicity testing was performed.

[0180] This assay is based on MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide], a method first described by Mosmann in 1983. The principle is that mitochondrial dehydrogenases in surviving cells hydrolyze the MTT tetrazolium ring (pale yellow) to form formazan crystals (dark blue). These crystals cannot penetrate the cell membrane and accumulate in the cytoplasm of metabolically active cells. Therefore, the number of surviving healthy cells is positively correlated with the level of formazan products.

[0181] The initial quantity is 1×10 per hole. 4 HaCaT cells (immortalized human epidermal cells) were cultured in 96-well plates in DMEM (Dulbecco's Modified Eagle Medium) (Lonza) containing 10% fetal bovine serum for approximately 8 hours. Cells were treated with 0.0006% peptide- and sugar-rich extract from *Radix Araliae*, 0.0002% peptide- and sugar-rich extract from *Radix Salviae*, 0.002% aqueous ethanol extract from *Artichoke*, 0.003% aqueous ethanol extract from coffee beans, 0.0005% water-soluble cactus stem node extract, and a mixture containing 0.003% aqueous ethanol extract from coffee beans and 0.0005% water-soluble cactus stem node extract for approximately 48 hours. Cells were then washed with PBS and incubated with 100 µl / well of reaction buffer containing 10 mM Hepes, 1.3 mM CaCl2, 1 mM MgSO4, 5 mM glucose, and 0.5 mg / ml MTT colorimetric substrate (in PBS buffer, pH 7.4). After incubation at 37°C and 5% CO2 for 3 hours, 100 μl of dissolving solution (anhydrous isopropanol solution containing 10% Triton-X100 and 0.1 N HCl) was added to each well. After 16 hours, the colorimetric reaction was measured at 595 nm using a Victor3 plate reader. The MTT results are as follows: Figure 1 As shown, the concentration of the measured extract did not produce any toxicity to the cells.

[0182] Example 7: Analysis of SOX9 and IGF-1 gene expression in human dermal papillary cells Human dermal papillary cells (HFDPC) were introduced at an initial number of 8 × 10⁻⁶. 4Samples were cultured per well in 6-well plates for 20 hours in Human Hair Follicle Growth Medium (C-26501, Promocell) (with appropriate additives). The next day, the treated samples were added to fresh medium and cultured for 24 hours. Two concentrations (10 ng / ml and 100 ng / ml) of recombinant GDF-11 protein (rGDF11) and 2.5 ng / mL of TGF-β1 were specifically tested. Subsequent experiments also tested the following extracts: 0.0006% peptide- and sugar-rich radish extract, 0.0002% peptide- and sugar-rich spider lily root extract, 0.002% aqueous ethanol artichoke extract, 0.003% aqueous ethanol coffee bean extract, 0.0005% water-soluble cactus stem node extract, and a combination of the last two extracts. In addition to TGF-β1, 1 µM minoxidil was also tested in this experiment.

[0183] After treatment, cells were washed with PBS and then collected in lysis buffer for 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. Quantification was performed using IBright analysis software (ThermoScientific). 500 ng of total RNA was reverse transcribed using reverse transcriptase (ThermoScientific). Semi-quantitative RT-PCR was performed using a universal 18S primer / competitive primer pair (Ambion) as an internal standard, with a SOX9 ratio of 4:6 and an IGF-1 ratio of 3:7.

[0184] PCR products were separated by 1.5% agarose gel electrophoresis, visualized using an iBright instrument (ThermoScientific), and analyzed using iBright Analysis software (Thermo Fisher Scientific).

[0185] The reported values ​​in the figure represent the ratio of the intensity of the analyzed gene band to the intensity of the 18S standard band, thus obtaining a value related to the actual expression of the target gene. The values ​​are then converted to percentages (%), with the values ​​obtained from the untreated control group as 100% baseline.

[0186] The primer sequences used for amplification are as follows: • Hs SOX9 forward primer (for): SEQ ID NO 1; • Hs SOX9 reverse primer (rev): SEQ ID NO 2; • Hs IGF-1 forward primer (for): SEQ ID NO 3; • Hs IGF-1 reverse primer (rev): SEQ ID NO 4.

[0187] Figure 2 The results showed that treatment of dermal papillary cells with two concentrations of rGDF-11 increased SOX9 gene expression by 80% and 66%, respectively, and IGF-1 gene expression by 63% and 38%, respectively; these increases were greater than or equal to the increases produced by the positive control TGF-β1.

[0188] For the extract of the present invention, Figure 6A The results show that all extracts, like rGDF11, promoted SOX9 gene expression. Specifically: the peptide- and sugar-rich extract from radish increased SOX9 gene expression by approximately 110%; the peptide- and sugar-rich extract from bird's eye root somatic cell embryos increased it by 25%; and the artichoke extract increased it by approximately 80%. Notably, the combination of coffee bean extract and cactus stem node extract increased it by 28%, while neither of the two extracts tested individually had any effect on SOX9 gene expression.

[0189] Figure 6B The efficacy of the extract treatments of this invention on IGF-1 gene expression was demonstrated. A combination of peptide- and sugar-rich radish extract and artichoke extract increased IGF-1 gene expression by approximately 35%, while a combination of peptide- and sugar-rich extracts derived from Lophatherum gracile somatic embryos, coffee bean extract, and cactus stem node extract resulted in an increase of approximately 50%. Single extract treatments were equally effective, but the combined effects were synergistic and unexpected, significantly stronger than the sum of the effects of the two single extracts.

[0190] Example 8: Expression analysis of Noggin and β-catenin proteins in an in vitro dermal papillary spheroid model In recent years, significant progress has been made in the development of three-dimensional models that simulate tissue characteristics. Compared to traditional two-dimensional (2D) cell culture, these three-dimensional, spherical-based models can more accurately reproduce the tissue microenvironment and have therefore been successfully used for screening to identify potential products or compounds. They contain cells with exposed surfaces and cells located in a hypoxic core, similar to the in vivo environment.

[0191] When dermal papillary cell aggregates are seeded onto low-adhesion round-bottomed plates, they can form independent spheres, which can be used as a hair follicle sphere model.

[0192] Formation of human dermal papillary cell spheroids To construct a more complex cellular system, a 3D dermal papillary cell spheroid culture was established to evaluate the effect of GDF-11 on the production of hair follicle development-related proteins.

[0193] 3×10 3 One dermal papilla cell (follicular dermal papilla cell, HFDPC) was seeded in a 96-well round-bottom U-shaped plate and cultured in 50 µl of "human hair follicle growth medium" (C-26501, Promocell) for 48 hours, with supplements added as needed to promote spheroid formation. After incubation, the aggregates of dermal papilla cells had formed different spheroids, which could be observed under a microscope.

[0194] Immunofluorescence analysis – Noggin and β-catenin protein analysis Collect the obtained spheres and centrifuge at 1500 rpm for 3 minutes. Wash twice with PBS buffer and fix with 4% paraformaldehyde solution for 1 hour. After washing three times with PBS buffer, permeate with phosphate buffer containing 0.1% Triton-X100 for 15 minutes. After washing again with PBS, incubate the spheres in blocking solution (containing 6% BSA, 5% goat serum, 20 mM MgCl2 and 0.2% Tween) at room temperature for 1 hour. After washing with PBS, incubate the spheres overnight at 4°C with shaking, adding either anti-Noggin mouse primary antibody (Noggin monoclonal antibody, OTI1C1, Thermo Fisher Scientific) or anti-β-catenin mouse primary antibody (Beta-Catenin monoclonal antibody, 6F9, Thermo Fisher Scientific) during incubation. After washing three times with PBS, cells were incubated with anti-mouse secondary antibodies for 1 hour at room temperature: Alexa-Fluor-488 anti-mouse secondary antibody was used for Noggin, and Alexa-Fluor-588 anti-mouse secondary antibody was used for β-catenin. Cell nuclei were stained with 1 µg / ml DAPI (4',6-diamidinyl-2-phenylindole) in PBS for 10 minutes. Finally, images were acquired using a Zeiss ISM700 confocal microscope and analyzed using ImageJ software.

[0195] like Figure 3 As shown, treatment of spheres with 10 ng / ml of recombinant rGDF-11 protein for 24 hours increased the production of Noggin protein, which plays a key role in hair follicle regeneration, and β-catenin, which promotes the induction and persistence of the growth phase in the hair growth cycle.

[0196] Example 9: Hair shaft length analysis in hair follicle explants Hair follicles from male patients who underwent follicle transplantation using follicle unit extraction (FUE) were used to analyze the effect of recombinant GDF11 protein treatment on hair shaft length. All patients gave informed consent for the use of hair follicles for study purposes, and follicle units were collected from areas with high follicle density.

[0197] Hair follicles were isolated after observation under an optical microscope, and only those in the anagen phase (the active growth phase of hair, characterized by a more enlarged follicular bulb) were selected for study. The selected follicles were then cultured in E-Williams medium (12551032, Gibco-Fisher Scientific) supplemented with 2 mM glutamine, 10 ng / ml hydrocortisone (Merck), 10 µg / ml insulin (Merck), and 1% penicillin-streptomycin (Gibco). After 24 hours of culture, the follicles were treated with recombinant GDF11 protein at a concentration of 100 ng / ml for 7 consecutive days. The medium was changed and treatment repeated every other day during this period. After treatment, the follicular bulbs were observed under an optical microscope, and images were acquired and analyzed using ImageJ software.

[0198] Figure 4 The results showed that after treatment with rGDF11 protein, hair shaft length increased by about 15% within 7 days, while the untreated group only increased by about 8%.

[0199] Example 10: GDF11 Generation Analysis 8×10 per hole 3Dermal papilla cells (follicular dermal papilla cells, HFDPC) were grown in 96-well plates using suitable culture medium and necessary supplements (C-26501, Promocell). The next day, the test extracts were added, along with TGF-β1 and minoxidil as positive controls. Extract concentrations were as follows: 0.0006% for *Radix Adenophorae* cell extract (rich in peptides and sugars), 0.0002% for *Radix Pithoniae* somatic cell embryo extract (rich in peptides and sugars), and 0.002% for *Artichoke* aqueous ethanol extract. Combined extracts consisted of 0.003% coffee bean aqueous ethanol extract and 0.0005% cactus stem node water-soluble extract. All single and combined extracts were tested. After 6 hours, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 minutes. The cells were then washed three times with wash buffer (PBS 1x, 0.5 mM CaCl2, 1 mM MgCl2, 0.1% Triton) and incubated with shaking in blocking buffer containing 0.5% skim milk powder (NFDM, sc-2334, Santa Cruz Biotechnology Inc) for 30 minutes. After washing with wash buffer, the cells were incubated with anti-GDF11 primary antibody (Abcam, ab124721) (1:1000 dilution) in wash buffer containing 0.5% NFDM. After shaking for 2 hours, the plates were washed three times with wash buffer and incubated with peroxidase-conjugated anti-rabbit secondary antibody (1706515, Biorad) (1:5000 dilution) in wash buffer containing 0.5% NFDM. After 1 hour of incubation, the plates were washed three times with wash buffer. Chemiluminescence reaction was performed using QuantaRed™ enhanced chemiluminescence HRP substrate (Thermo Fisher Scientific) according to the manufacturer's instructions. After 15 minutes, the absorbance at 490 nm was measured.

[0200] like Figure 5 As shown, the extract treatment of the present invention can induce the production of GDF-11: Specifically, peptide extracts from radish cells and water-ethanol extracts of artichoke and coffee increase GDF-11 production by about 50%, peptide extracts rich in somatic embryos of Lophatherum gracile and water-soluble extracts of Pyracantha fortuneana stem nodes increase GDF-11 by about 30%, and the combination of coffee extract and Pyracantha fortuneana extract increases GDF-11 by about 85%, which is equivalent to the sum of the effects of the two extracts acting alone.

[0201] Example 11: Analysis of GDF11 and SOX9 gene expression in human dermal papillary cells under oxidative stress 8×10 per hole 4One well of dermal papilla cells (hair follicle dermal papilla cells, HFDPC) was cultured in human hair follicle growth medium (C-26501, Promocell) with appropriate supplementation in 6-well plates for 20 hours. The next day, the cells were treated with hydrogen peroxide (H2O2, 100 µM) for 1 hour to induce free radical formation, followed by growth in medium for 7 hours, and then treated with 100 µM H2O2 for 1 hour. Finally, the cells were grown overnight in medium.

[0202] The treatment process was repeated the following day, with two more H2O2 cycles, interspersed with 7-hour incubation in culture medium. After the second H2O2 cycle, the cells were treated with the extract of this invention for 24 hours.

[0203] After treatment, the cells were washed with PBS and collected in lysis buffer for RNA extraction, followed by reverse transcription and RT-PCR reactions as described in Example 7. The universal primer pair ratio for detecting GDF-11 and SOX9 gene expression was 4:6.

[0204] The primer sequences used for amplification are as follows: • Hs SOX9 forward primer (for): SEQ ID NO 1 • Hs SOX9 reverse primer (rev): SEQ ID NO 2 • Hs GDF11 forward primer (for): SEQ ID NO 5 • Hs GDF11 reverse primer (rev): SEQ ID NO 6 Figure 7A The results shown indicate that under oxidative stress induced by repeated H2O2 treatment, GDF11 expression was reduced by approximately 80% compared to unstressed control cells. However, treatment with the extracts of this invention reversed this condition, restoring expression levels to levels comparable to those of unstressed control cells. Specifically, combined treatment with a peptide- and sugar-rich radish extract, along with coffee bean extract and prickly pear cactus stem node extract, increased GDF11 gene expression by approximately 65%; treatment with a peptide- and sugar-rich lobe rhizome cell extract increased GDF11 gene expression by approximately 50%; and artichoke extract increased GDF11 gene expression by approximately 80%. Positive controls TGF-β1 and minoxidil showed the same or lower increases in efficacy as the extracts of this invention.

[0205] Figure 7BThe results revealed the effect on SOX9 gene expression: compared with unstressed control cells, SOX9 expression decreased by approximately 150%, while treatment with the extracts of this invention offset this effect by increasing SOX9 gene expression, with effects similar to recombinant GDF11. Specifically, treatment with a radish extract rich in peptides and sugars, similar to rGDF11, increased SOX9 gene expression by approximately 65%; water-ethanol artichoke extract increased SOX9 gene expression by approximately 50%; Lophatherum gracile somatic embryo extract increased SOX9 gene expression by approximately 90%; and the combination of coffee bean extract and Pyrus pyrifolia stem node extract increased SOX9 gene expression by approximately 115%.

[0206] References 1. Valerie PI Vidal, Marie-Christine Chaboissier, Susanne Lützkendorf, George Cotsarelis, Pleasantine Mill, Chi-Chung Hui, Nicolas Ortonne, Jean-Paul Ortonne, Andreas Schedl (2005). Sox9 Is Essential for Outer Root Sheath Differentiation and the Formation of the Hair Stem Cell Compartment, Current Biology, Vol. 15, No. 15, pp. 1340-1351.

[0207] 2. Weger N and Schlake T (2005). IGF-1 signaling controls the hair growth cycle and the differentiation of hair shafts. Journal of Investigative Dermatology, Vol. 125, No. 5, pp. 873-882.

[0208] 3. JL Bueno, M. Ynigo, C. de Miguel, 1 RM Gonzalo-Daganzo, A. Richart, C. Vilches, C. Regidor, JA Garcıa-Marco, E. Flores-Ballester & JRCabrera (2016). Growth differentiation factor 11 (GDF11) – a promising anti-aging factor – is highly concentrated in platelets. Vox Sanguinis, Nov; 111(4):434-436.

[0209] 4. Williams G, Zentar MP, Gajendra S, Sonego M, Doherty P, Lalli G. Transcriptional basis for the inhibition of neural stem cell proliferation and migration by the TGFβ-family member GDF11 (2013). PLOS One, 8(11): e78478.

[0210] 5. Anderson O, Reissmann E, Ibanez CF (2006). Growth differentiation factor 11 signals through the transforming growth factor-β receptor ALK5 to regionalize the anterior–posterior axis. EMBO reports, 7, 831-837.

[0211] 6. Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Yalamanchi P, Sinha M, Dall'Osso C, Khong D, Shadrach JL, Miller CM, Singer BS, Stewart A, Psychogios N, Gerszten RE, Hartigan AJ, Kim MJ, Serwold T, Wagers AJ, Lee RT1 (2013). Growth differentiation factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell, 153(4):828–839.

[0212] 7. Sinha M, Jang YC, Oh J, Khong D, Wu EY, Manohar R, Miller C, Regalado SG, Loffredo FS, Pancoast JR, Hirshman MF, Lebowitz J, Shadrach JL, Cerletti M, Kin MJ, Serwold T, Goodyear LJ, Rosner B, Lee RT, Wagers AJ (2014). Restoring systemic GDF11 levels reverses age-related dysfunction in mouse skeletal muscle. Science, 344 (6184):649-652.

[0213] 8. Katsimpardiay L, Litterman NK, Schein PA, Miller CM, Loffredo FS, Wojtkiewicz GR, Chen JW, Lee RT, Wagers AJ, Rubin LL (2014). Vascular and neurogenic rejuvenating of theaging mouse brain by young systemic factors, Science, 344 (6184): 630-634.

[0214] 9. Lee E, Jin Y, Park J, Yoo Y, Hong S, Amir Z, Yan Z, Kwon E, Elfisk A, Tomlinson S, Halbritter F, Waibel T, Yun B, Loake G (2010). Cultured cambial meristematic cells as a source of plant natural products. Nature Biotechnology, 28(11):1213-1217.

[0215] 10. Botchkarev VA, Botchkareva NV, Nakamura M, Huber O, Funa K, Lauster R, Paus R, Gilchrest BA (2001). Noggin is required for induction of the hair follicle growth phase in postnatal skin. FASEB Journal, 15(12):2205-14.

[0216] 11. Dong WS (2022). The Molecular Mechanism of Natural Products Activating Wnt / β-Catenin Signaling Pathway for Improving Hair Loss, Life, 12(11), 1856.

Claims

1. Use of at least one plant extract capable of activating GDF11 growth factor (growth differentiation factor 11) for hair growth treatment, wherein said at least one extract is selected from: - An extract rich in peptides and sugars derived from cell cultures of radish plant; - An extract rich in peptides and sugars derived from a culture of *Lotus Root* plant cells rich in somatic embryos; - An aqueous ethanol extract derived from artichoke flower heads; and / or - A combination of a water-soluble extract derived from the stem nodes of the pear cactus and an aqueous ethanol extract derived from coffee beans.

2. The use according to claim 1, wherein the peptide- and sugar-rich extract derived from *Radix Adenophorae* cell cultures is obtained by a preparation method comprising the following steps: a) Homogenize the cell culture of radish plant in a saline solution to obtain a homogenate; b) Separate the solid portion from the liquid portion of the resulting homogenate; c) Treat the solid fraction in an acidic solution with a proteolytic enzyme to hydrolyze cell wall proteins and glycosidic bonds, thereby obtaining an extract rich in peptides and sugars.

3. The use according to claim 1, wherein the extract derived from a *Lotus Root* plant cell culture rich in somatic embryos is obtained by a preparation method comprising the following steps: a) Inducing the formation of somatic embryos in suspension cultured Lophatherum sp. plant cells; b) The obtained somatic embryos are homogenized in a saline solution to obtain a homogenate; c) Separate the solid portion from the liquid portion of the homogenate; d) Treat the solid fraction with a proteolytic enzyme in an acidic solution to hydrolyze the cell wall proteins of the somatic embryo, thereby obtaining an extract rich in peptides and sugars.

4. The use according to claim 1, wherein the water-soluble extract derived from the stem node of the pomegranate cactus is obtained by a preparation method comprising the following steps: i) Steam treatment of pre-cleaned pear cactus stem sections; ii) Peel the pear cactus stem segments obtained in step i); iii) Homogenize the peeled pear cactus stem segments to obtain a homogenate; iv) Separate the solid portion of the homogenate from the liquid portion, wherein the liquid portion constitutes a water-soluble extract of prickly pear cactus.

5. A method for preparing an aqueous ethanol extract of coffee, comprising the following steps: i) Grind green coffee beans to obtain the ground product. ii) Add cold ethanol at -30°C to -10°C, preferably -20°C, to the milled product, followed by homogenization to obtain a slurry in the form of a suspension of solids in a liquid fraction. iii) Shake the suspension at room temperature. iv) Separating the solid portion from the liquid portion in the homogenate, the liquid portion constituting the coffee bean aqueous ethanol extract. v) Filter the aqueous ethanol extract obtained in step iv).

6. The use of a combination of a water-soluble extract derived from the stem nodes of the prickly pear cactus and an aqueous ethanol extract derived from coffee beans for hair growth treatment, wherein the water-soluble extract derived from the stem nodes of the prickly pear cactus... The aqueous ethanol extract derived from coffee beans is prepared according to claim 4. Prepared according to the method of claim 5.

7. A method for preparing an artichoke aqueous ethanol extract, comprising the following steps: i) Grind artichoke heads, preferably, the artichoke heads are from the violet variety of artichoke. ii) Add cold ethanol at a temperature of -30°C to -10°C, preferably -20°C, to the obtained ground product, followed by homogenization to obtain a homogenate. iii) Shake the suspension at room temperature. iv) Separate the solid portion from the liquid portion of the homogenate, the liquid portion constituting the artichoke aqueous ethanol extract. v) The aqueous ethanol extract obtained by filtration.

8. The method according to claim 7, wherein before performing step i), the artichoke heads are stripped of their stems and outer skin, washed in water, and then washed in a 3% to 5% sodium bicarbonate solution, preferably 5%, for 20 to 60 minutes, preferably 30 minutes, wherein the weight-to-volume ratio of the artichoke heads to the sodium bicarbonate solution is 1:3 to 1:5, preferably 1:

5.

9. The method according to claim 7 or 8, wherein the artichoke heads are ground for 3 to 15 minutes, preferably 3 minutes, by means of a blade homogenizer operating at 1000-3000 rpm, preferably 1500 rpm, at room temperature and in the absence of solvent.

10. The method according to any one of claims 7 to 9, wherein in step ii), 70% to 80%, preferably 80%, of ethanol is added at a homogenate weight / solvent volume ratio of 1:1 to 1:2, preferably 1:1; and / or wherein the milled product is subjected to two homogenization cycles with the addition of the above-mentioned cold ethanol, each homogenization cycle being performed for 3 to 15 minutes by means of a blade homogenizer operating at a speed of 3000-5000 rpm, preferably 3800 rpm.

11. Use of an artichoke aqueous ethanol extract for hair growth treatment, wherein the artichoke aqueous ethanol extract is prepared by the method of claim 10.

12. A pharmaceutical composition or cosmetic composition comprising at least one plant extract as an active ingredient, said plant extract being capable of activating GDF11 growth factor (growth differentiation factor 11) for hair growth treatment, wherein said at least one plant extract is selected from: - An extract rich in peptides and sugars derived from cell cultures of radish plant; - An extract rich in peptides and sugars derived from a culture of *Lotus Root* plant cells rich in somatic embryos; - An aqueous ethanol extract derived from artichoke flower heads; and / or - A combination of a water-soluble extract derived from the stem nodes of the pear cactus and an aqueous ethanol extract derived from coffee beans.

13. The pharmaceutical composition or cosmetic composition according to claim 12, comprising, as an active ingredient: - An extract rich in peptides and sugars derived from a cell culture of *Radix Achyranthes bidentata* obtained by the method according to claim 2; - An extract rich in peptides and sugars derived from a somatic embryo-rich Lophatherum gracile plant cell culture obtained by the method according to claim 3; - Artichoke aqueous ethanol extract obtained by the method according to any one of claims 7-10, and / or - A combination of the water-soluble extract derived from the stem nodes of the pear cactus prepared by the method according to claim 4, and the aqueous ethanol extract derived from coffee beans prepared by the method according to claim 5.

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