WO2025046002A1 - Use of plant-derived extracts for hair growth stimulation and cosmetic or pharmaceutical compositions containing such extracts for said use - Google Patents

Abstract

The present invention concerns the use of at least one plant extract capable of activating the GDF11 (Growth Differentiation Factor 11) growth factor and a cosmetic or pharmaceutical composition comprising at least one extract as an active ingredient for treating hair growth. The at least one extract is chosen from the following: • an extract rich in peptides and sugars derived from Scabiosa arvensis cell cultures; • an extract rich in peptides and sugars derived from Lotus japonicus cell cultures enriched in somatic embryos; • a hydroethanolic extract from artichoke flower heads belonging to the species Cynara scolymus; and/or a water-soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans.

Description

Title: Use of plant-derived extracts for hair growth stimulation and cosmetic or pharmaceutical compositions containing such extracts for said use

DESCRIPTION

Field of the invention

The present invention relates to the use of one or more plant-derived extracts that can stimulate hair growth by activating the GDF11 growth factor (Growth Differentiation Factor 11).

These extracts include an extract derived from cell cultures of Scabiosa arvensis, an extract derived from somatic embryo-enriched cell cultures of Lotus japonicus, a hydroethanolic extract from artichoke flower heads of Cynara scolymus, and a combination of a water-soluble extract from prickly pear pads and a hydroethanolic extract from coffee beans of Coffea arabica.

The present invention also relates to methods for producing these extracts and to pharmaceutical or cosmetic compositions containing such extracts for stimulating hair growth.

Prior Art

Hair are skin appendages distributed on the head of each individual, performing functions of protection and thermoregulation.

Each hair consists of an external visible portion called the shaft, which emerges at the scalp level, and an internal part known as the hair follicle. The hair follicle is an invagination of the epidermis that sinks into the skin until it reaches the dermis.

Each hair follicle is associated with a sebaceous gland responsible for the production of sebum, a fatty secretion that has the purpose of protecting the scalp and hair.

The lower part of the follicle, which is slightly enlarged, is called the hair bulb. It contains various populations of stem cells, including a group of specialized fibroblastic cells of mesenchymal origin known as dermal papilla cells (DPC). These cells play a crucial role in maintaining follicle vitality, cell proliferation, and hair growth.

The maintenance of stem cells in the hair bulb is ensured by a transcriptional regulator that binds DNA, a member of the SOX gene family, SOX9: in mice, a loss of SOX9 in epidermal tissue leads to a visible loss of hair associated with a loss of a sternness marker, CD34 (Vidal et al., 2005).

A single hair has a normal life of 2 to 7 years and it is estimated that each follicle produces about 20 hairs throughout its life.

More specifically, the hair life cycle consists of three main phases:

• ANAGEN phase: this is the active growth phase during which the hair grows. It is the longest phase and can last an average of several years, approximately 2-4 years in men and 3-7 years in women.

• CATAGEN phase: this is the involution phase during which vital functions decrease and the hair stops growing. This phase lasts 2-3 weeks.

• TELOGEN phase: this is the resting phase during which functional activities cease completely, but the hair remains in the follicle and mainly falls out during washing and combing. This phase averages 3-4 months and marks the beginning of a new cycle.

The insulin-like growth factor, IGF- 1 (Insulin Growth Factor 1) plays a crucial role in the hair cycle by maintaining the anagen phase and delaying the catagen phase, effectively promoting hair growth in the follicle (Weger, 2005).

Under normal conditions, when a hair in the telogen phase falls out (called exogen), the follicle has already begun a new cycle with a new hair in the anagen phase. Each hair follows its life cycle and is not synchronized with others. This is why hair do not fall out all at once, but a few strands are lost daily.

Hair loss is thus a natural physiological process that can result in the shedding of up to a hundred hairs daily. This phenomenon may be more noticeable at certain times of the year, such as from September to November, when increased sun exposure during the summer can lead to greater hair loss due to the accumulation of free radicals, molecules responsible for cellular aging.

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

Baldness can affect both men and women but is much more common and aggressive in males: it is estimated that between 50% and 80% of men worldwide experience baldness. It generally starts around the age of 19- 20 and settles by the age of 32-33.

The rapid hair thinning appears as a gradual receding of the hairline, starting at the temples in men, or localized loss in certain areas of the scalp. Regardless of the extent of the loss, the hair may appear weak or thinner.

Baldness is a complex condition influenced by genetic and hormonal factors, as well as an unbalanced diet in macronutrients, micronutrients, stress, and an unhealthy lifestyle.

To address hair loss, the most used drugs are Minoxidil and Finasteride. Minoxidil is a potent antihypertensive vasodilator that promotes cell proliferation in the hair bulb. On the other hand, Finasteride is a potent inhibitor of the enzyme responsible for the production of the hormone dihydrotestosterone (DHT), which is crucial for the development of primary male characteristics but also leads to the atrophy of hair follicles (also known as follicle miniaturization)."

Currently, these are the only two drugs approved by the FDA for treating alopecia, the first one is for topical use, and the second one is for oral use. However, their effectiveness may vary from person to person, and they can have significant side effects, which need to be carefully weighed against their actual benefits.

Aesthetic medicine also uses techniques such as hair autotransplantation, a surgical procedure that thickens bald areas by implanting healthy bulbs taken from the same individual's Hippocratic crown. However, these are invasive surgical procedures performed under anesthesia and they do not always result in the complete success of all transplanted bulbs.

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 follicle stem cells, promoting the formation of new follicles and thus hair growth.

Recently, a growth factor, GDF11 (Growth Differentiation Factor 11), has been identified in PRP derived from young blood. It is present in the blood of young organisms in higher amounts than in that of older organisms (Bueno et al., 2016), its amount decreases with advancing age. GDF1 1, encoded by a specific gdf- 11 gene, is a member of the superfamily of "Transforming Growth Factor |3" (TGF[3) and was initially identified for its regulatory role in embryonic development, in particular of the nervous system and anteroposterior regionalization (Williams, 2013; Anderson 2006). Later, it has been attributed a potential rejuvenating role in several organs. In fact, it was shown that GDF11 was able to reduce heart enlargement due to age (Loffredo et al, 2013), to revert muscle aging (Sinha et al, 2014) and even reduce some deleterious effects due to functional aging of the brain (Katsimpardiay et al., 2014).

This remarkable discovery has been validated by experiments in which the blood of a young mouse, containing the factor GDF11, was mixed with one belonging to an old rat, using a "parabiotic" system. The two mice were surgically joined to allow the young mouse's blood to circulate in the old mouse's body, or the old mice received blood transfusions from young mice: in the old mouse was observed that the DNA of muscle cells was repaired and the muscle fibers became healthier and younger. Moreover, the muscle grip strength increased and the mouse was able to run longer on the tapis roulant compared to the untreated mice (Loffredo et al, 2013; Sinha et al, 2014).

The current trend in hair growth-promoting products is moving towards natural and plant-based products. Plant cell cultures represent a valuable source for creating plant extracts that contain active ingredients with proven pharmaceutical or cosmetic effectiveness. These cultures produce products that are free of contaminants, sustainable, and standardized. Additionally, their production can be easily scaled up industrially (Lee et aL, 2010).

JH05 170627 A discloses a hair growth treatment comprising extract of prickly pear (Opuntia ficus indica). The extract is obtained e.g. 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, or water, or acetone etc. at room temperature or with warming.

US 20222226410 A discloses a hair growth treatment comprising extracts or parts of prickly pear {Opuntia ficus indica), such as finely ground fruit or fruit extract. WO 2015132755 Al discloses nutraceutical, cosmetic or pharmaceutical compositions based on a combination of vegetal extracts from flowers or fruits of Opuntia ficus and Oryza sativa (Black rice) for inhibition of the 5-alpha reductase. Such preparation are said to be useful in prevention or treatment of benign prostatic hypertrophy or hyperplasia, of androgenic alopecia and acne..

Mintel GNPD record ID 5430331 “Hair Proteinz Spa Inbuilt Protein Booster”, Feb. 2018, XP93134018 shows a composition comprising argan oil, keratin protein, artichoke extract (Cynara scolymus) and ylang ylang essential oil for strengthening hair and nourishing roots.

WO 2019028214 Al discloses compositions comprising one or more of the following extracts: an Euterpe oleracea extract comprising cyanidin 3-glycoside and/or cyanidin 3-rutinoside, an Olea europaea extract comprising oleuropein, a Coffea arabica extract, and/or a Tabebuia impetiginosa extract. The compositions further comprise a micronutrient comprising zinc and vitamin D3. The above compositions are said to be useful for non-medicinal treatment of hair loss and/or nourishment and rejuvenation of hair, skin and nails.

CN 101524426 discloses hydroethanolic extracts of coffee for stimulating hair growth.

WO 2016173867 discloses certain plant extracts, namely a peptide/ sugar mixture derived from somatic embryo-enriched cell cultures of Lotus japonicus, and proposes the use of the extracts in cosmetics against skin aging and for skin tissue rejuvenation.

Tito Annalisa et al. “The Growth Differentiation Factor 11 is involved in skin fibroblast ageing and is induced by a preparation of peptides and sugars derived from plant cell cultures”, Molecular Biotechnology, Springer US, New York, vol. 61 no. 3, January 19, 2019 discloses that certain plant extracts, namely a peptide/ sugar mixture derived from somatic embryo-enriched cell cultures of Lotus japonicus, were able to stimulate the production of GDF11 in skin cells.

IT 2021 0002 0309 discloses extracts of prickly pear (Opuntia ficus indica) and water-soluble olive extract for use in prevention and treatment of atopic dermatitis.

The technical problem underlying the present invention is to provide plant-derived extracts effective in stimulating hair growth without the disadvantages of the aforementioned known art products.

Summary of the Invention

The authors of the present invention have found that treatment with recombinant GDF11 protein (rGDFl l), similar to the effects of TGF-p i growth factor, can stimulate the gene expression of the stem cell marker SOX9 in human dermal papilla cells as well as stimulate the expression of insulin-like growth factor IGF- 1, which is essential for maintaining the hair growth phase.

Furthermore, the authors discovered that treatment with the recombinant protein GDF11 (rGDFl l) can stimulate the expression of the proteins Noggin and P-catenin in an in vitro model of dermal papilla spheroids - three-dimensional (3D) cell aggregates that replicate the characteristics of the hair bulb.

The findings are important for identifying products that could benefit hair care as noggin plays a crucial role in hair follicle regeneration and growth (Botchkarev, 2001), while [3-catenin promotes the induction and duration of the anagen growth phase of the hair (Dong, 2022).

Moreover, the authors discovered that treating human hair follicle explants with GDF11 can stimulate hair growth by increasing the length of the hair shaft.

In light of these discoveries, the authors identified plant extracts capable of stimulating the expression of GDF11 and, in turn, the genes SOX9 and IGF- 1.

Thus, in an aspect thereof, the present invention relates to the use of at least one plant extract as an active ingredient to stimulate hair growth, wherein said extract can activate the GDF11 growth factor, wherein said at least one plant extract is selected from:

1. An extract rich in peptides and sugars derived from plant cell cultures of Scabiosa arvensis;

2. An extract rich in peptides and sugars derived from plant cell cultures enriched in somatic embryos of Lotus japonicus;

3. A combination of a water-soluble extract derived from prickly pear pads and a hydroethanolic extract derived from coffee beans; and / or

4. A hydroethanolic extract from artichoke flower heads of Cynara scolymus.

In one embodiment of the invention, the extract rich in peptides and sugars from plant cell cultures of Scabiosa arvensis for use according to the invention is obtained by a preparation process comprising the steps: a) homogenizing plant cell cultures of Scabiosa arvensis in an aqueous saline solution, obtaining a homogenate; b) separating the solid part from the liquid part of the obtained homogenate; c) treating said solid part with proteolytic enzymes in an acidic solution to hydrolyze the proteins of the cell walls and the glucosidic bonds, obtaining the extract rich in peptides and sugars.

In one embodiment of the invention, the extract from plant cultures enriched in somatic embryos of the species Lotus japonicus for use according to the invention is obtained by the preparation process described in European patent EP 3288644 in the name of the Applicant, the content of which is incorporated herein by reference.

This process comprises the following steps: a) inducing plant cell cultures of Lotus japonicus in suspension to form somatic embryos; b) homogenizing the obtained somatic embryos in an aqueous saline solution, obtaining a homogenate; c) separating the solid part from the liquid part of the obtained homogenate; d) treating the solid part with proteolytic enzymes in an acidic solution to hydrolyze the proteins of the cell walls of the somatic embryos, obtaining an extract rich in peptides and sugars.

In another aspect, the present invention relates to the use of an extract from plant cultures enriched in somatic embryos of Lotus japonicus for the treatment of hair growth, wherein the extract is obtained by the above-described process.

In one embodiment of the invention, the water-soluble extract of prickly pear pads, used in combination with the hydroethanolic coffee extract according to the present invention, is obtained through the process described in Italian patent application No. 102021000020309 in the name of the Applicant, the content of which is incorporated herein by reference.

This process comprises the following steps: i) subjecting previously cleaned prickly pear pads to steam treatment; ii) peeling the prickly pear pads obtained from step i); iii) homogenizing the peeled prickly pear pads, obtaining a homogenate; iv) separating the solid part from the liquid part of the obtained homogenate, the liquid part constituting the water-soluble extract of prickly pear.

In one embodiment of the present invention, the hydroethanolic coffee extract used in combination with the water-soluble prickly pear extract for use according to the invention is obtained by a process comprising the following steps: i) grinding green (i.e. not toasted) coffee beans, obtaining a ground product; ii) adding cold ethanol having a temperature between -30° and - 10° C, preferably -20° C, to the ground product, and proceeding with subsequent homogenization to obtain a homogenate in the form of a suspension of a solid part in a liquid part; iii) agitating the suspension at room temperature; iv) separating the solid part of the homogenate from the liquid part, which constitutes the hydroethanolic extract of coffee beans; v) filtering the obtained hydroethanolic extract in step iv) .

In another aspect, the present invention also relates to the use of a water- soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans for the treatment of hair growth, wherein the extracts are obtained by their respective processes described above.

In another aspect, the present invention also relates to a process for the preparation of a hydroethanolic extract of artichoke comprising the following steps: i) grinding artichoke flower heads; ii) adding cold ethanol having a temperature between -30° and - 10° C, preferably -20° C, to the ground product, and proceeding with subsequent homogenization, obtaining a homogenate; iii) agitating the suspension at room temperature; iv) separating the solid part from the liquid part of the homogenate, which constitutes the hydroethanolic extract of artichoke; v) filtering the obtained hydroethanolic extract.

In another aspect, the present invention further relates to a pharmaceutical or cosmetic composition comprising at least one plant extract as an active ingredient capable of activating the GDF11 growth factor for the use described above in stimulating hair growth, and a pharmaceutically and/or cosmetically acceptable vehicle, wherein the at least one plant extract of the pharmaceutical or cosmetic composition is selected from:

1. An extract rich in peptides and sugars derived from plant cell cultures of the species Scabiosa arvensis;

2. An extract rich in peptides and sugars derived from plant cell cultures enriched in somatic embryos of the species Lotus japonicus;

3. A hydroethanolic extract from artichoke flower heads of the species Cynara scolymus; and / or

4. A combination of a water-soluble extract derived from prickly pear pads and a hydroethanolic extract derived from coffee beans.

In one aspect, the present invention relates to a pharmaceutical or cosmetic composition comprising as an active ingredient an extract rich in peptides and sugars derived from Scabiosa arvensis cell cultures obtained by the respective process indicated above.

In one aspect, the present invention relates to a pharmaceutical or cosmetic composition comprising as an active ingredient an extract rich in peptides and sugars derived from Lotus japonicus cell cultures enriched in somatic embryos obtained by the respective process indicated above.

In another aspect, the present invention relates to a pharmaceutical or cosmetic composition comprising as an active ingredient a hydroethanolic extract of artichoke obtained by the respective process indicated above.

In another aspect, the present invention relates to a composition comprising, as active ingredients for the treatment of hair growth, a water-soluble extract derived from prickly pear pads and a hydroethanolic extract of coffee beans, obtained by the respective processes described above.

The present invention will be now described for illustrative and nonlimiting purposes, according to preferred embodiments thereof, with particular reference to the figures of the accompanying drawings.

Brief description of the figures

Figure 1 shows a bar graph showing the results of the cytotoxicity assay (MTT) of the extracts on human keratinocytes according to the present invention. Specifically, the peptidic extract of Scabiosa arvensis cells was tested at a concentration of 0.0006% (0.006 mg/ml), the peptidic extract of somatic embryos of Lotus japonicus at a concentration of 0.0002% (0.002 mg/ml), the ethanolic extract of artichoke at a concentration of 0.002% (0.02 mg/ml), the ethanolic extract of coffee at a concentration of 0.003% (0.03 mg/ml), and the water-soluble extract from prickly pear pads at a concentration of 0.0005% (0.005 mg/ml); additionally, it was tested the mixture containing both the ethanolic extract of coffee and the water-soluble extract from prickly pear pads.

Figure 2 shows a bar graph showing the effect of recombinant GDF11 protein (rGDFl l), tested at concentrations of 10 and 100 ng/ml, on the expression of SOX9 and IGF- 1 genes, which are essential for maintaining sternness and the hair growth phase respectively, in human dermal papilla cells. The values reported in the graph are expressed as percentages relative to the untreated control sample, arbitrarily set at 100%. TGF-p i growth factor, tested at a concentration of 2.5 ng/ml, was used as a positive control. The bars represent standard deviations, while asterisks indicate significant variations.

Figure 3A shows an immunofluorescence analysis in an in vitro model of dermal papilla spheroids to evaluate the effect of recombinant GDF11 protein, tested at a concentration of 10 ng/ml, on the expression of the Noggin protein. Noggin protein plays an essential role in follicle regeneration and growth. The spheroid cell nuclei are stained blue using DAPI (4',6-diamidino-2-phenylindole) dye, while the protein of interest is detected in green, recognized by a primary antibody against a Noggin protein epitope and a secondary antibody, that binds to the primary antibody, labeled with a green-emitting fluorophore.

Figure 3B shows an immunofluorescence analysis in an in vitro model of dermal papilla spheroids to evaluate the effect of recombinant GDF11 protein, tested at a concentration of 10 ng/ml, on the expression of [3- catenin. [3-catenin plays an essential role in inducing and maintaining the hair growth phase. The spheroid cell nuclei are stained blue using the DAPI dye, while the protein of interest is detected in red, recognized by a primary antibody against [3-catenin and a secondary antibody, that binds to the primary antibody, labeled with a red-emitting fluorophore. Figure 4 shows a bar graph reporting 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 TO (start of treatment) and at 7 days posttreatment (T7), expressed as a percentage relative to TO, which is arbitrarily set at 100%.

Figure 5 shows a bar graph reporting the effect of the extracts, according to the present invention, on the production of GDF1 1 in human dermal papilla cells (HFDPC). The measurement was performed using an ELISA assay with a specific antibody against the GDF11 protein. The effect of Minoxidil and TGF-p i factor on GDF11 production was also evaluated: Minoxidil is a drug used for treating androgenetic alopecia and TGF-p i factor is used as a positive control of the assay. The bars represent standard deviations, while asterisks indicate significant variations.

Figure 6A shows a bar graph reporting the effect of the extracts, according to the present invention, on the expression of the SOX9 gene. This gene is crucial for maintaining the sternness of dermal papilla cells. Additionally, the graph also includes the effects of the TGF-p i factor and Minoxidil. The bars represent standard deviations, while asterisks indicate significant variations.

Figure 6B shows a bar graph reporting the effect of the extracts, according to the present invention, on the expression of the IGF- 1 gene. This gene is crucial for maintaining the hair growth phase in dermal papilla cells. Additionally, the graph also includes the effects of the TGF- p i factor and Minoxidil. The bars represent standard deviations, while asterisks indicate significant variations.

Figure 7A shows a bar graph reporting the effect of the extracts, according to the present invention, on the expression of the GDF- 11 gene in human dermal papilla cells under oxidative stress conditions. To induce the generation of free radicals, the cells were treated with hydrogen peroxide (H2O2, 100 pM) for 1 hour, allowed to grow for 7 hours in the culture medium, and then treated again with 100 pM H2O2 for an additional hour. Subsequently, the cells were left to grow overnight in their culture medium. The treatment was then repeated the next day with two more cycles of H2O2, interspersed with 7-hour incubation in the culture medium. At the end of the second H2O2 cycle, the cells were treated with the extracts according to the present invention for 24 hours. TGF-p i factor and Minoxidil were used as positive controls. The values reported in the graph are expressed as percentages relative to the H2O2- stressed sample, which was arbitrarily set at 100%. The bars represent standard deviations, while asterisks indicate significant variations.

Figure 7B shows a bar graph reporting the effect of the extracts, according to the present invention, on the expression of the SOX9 gene in human dermal papilla cells under oxidative stress conditions. To induce the formation of free radicals, the cells were treated with hydrogen peroxide (H2O2, 100 pM) for 1 hour, allowed to grow for 7 hours in the culture medium, and then treated again with 100 pM H2O2 for an additional hour. Subsequently, the cells were left to grow overnight in their culture medium. The treatment was repeated the next day for another full cycle, at the end of which the cells were treated with the extracts according to the present invention for 24 hours. Recombinant GDF11 protein, TGF-p i factor, and Minoxidil were used as positive controls. The values reported in the graph are expressed as percentages relative to the H2O2-stressed sample, which was arbitrarily set at 100%. The bars represent standard deviations, while asterisks indicate significant variations.

Detailed Description

The applicant has discovered that the GDF11 protein is particularly interesting for counteracting hair follicle atrophy and therefore it can be used to identify extracts or products that, by activating it, can be used in hair care, especially for stimulating hair growth.

Recombinant GDF11 (rGDFl l) treatment, similar to TGF-p i growth factor treatment, can activate the S0X9 transcription factor in dermal papilla cells, enhancing their vitality. It also boosts the expression of the IGF- 1 factor in these cells, which is crucial for the elongation and maintenance of the hair growth phase.

The Applicant has also observed a surprising effect of exogenous GDF11 treatment on 3D dermal papilla spheroid models — three-dimensional (3D) cellular aggregates that more effectively replicate the hair follicle microenvironment compared to standard two-dimensional (2D) cell cultures.

The Applicant discovered that treating 3D spheroid models with exogenous GDF11 had a surprising effect on proteins related to the activation, regeneration, and growth of hair follicles and hair growth. Specifically, treatment with recombinant GDF11 protein strongly increased the expression of Noggin and P-catenin proteins in this model. Noggin is involved in hair follicle regeneration, and its loss is linked to a decrease in follicle number and slowed follicle growth, while [3-catenin is involved in the initial phase of the hair lifecycle, promoting the induction and duration of the growth phase (anagen) .

Additionally, the applicant discovered that applying recombinant GDF11 treatment to ex vivo hair follicle explants, from male patients undergoing follicle transplantation using the “Follicular Unit Extraction” (FUE) technique, leads to hair shaft elongation. The observed elongation is more significant than in untreated control explant samples.

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

In this regard, the Applicant found that an extract rich in peptides and sugars from plant cell cultures of Scabiosa arvensis, as well as an extract (rich in peptides and sugars) from plant cell cultures enriched in somatic embryos of Lotus japonicus, a hydroalcoholic artichoke extract, and a combination of water-soluble extract from prickly pear pads and hydroalcoholic extract from coffee beans, for example as a mixture comprising such extracts, are particularly interesting for hair treatment and for promoting the proliferation of hair follicle cells and the hair growth phase.

These plant extracts can stimulate GDF11 expression in dermal papilla cells as effectively as or even better than the control used in the assay, which is the TGF-P 1 growth factor. Additionally, they perform better than Minoxidil, a well-known hair treatment that works by promoting vasodilation of capillaries at the hair follicle and stimulating cell proliferation, thereby countering follicle atrophy (or miniaturization).

Moreover, the extract derived from prickly pear pads and the extract derived from coffee beans individually activate GDF11 production, but they show a better (synergistic) effect when used in combination.

In addition to activating GDF11 expression, the aforementioned extracts can also stimulate genes that are also activated in dermal papilla cells by recombinant GDF11 treatment, such as the transcription factor SOX9 and IGF- 1.

Specifically, a combination of extracts, including the water-soluble extract from prickly pear pads and the hydroalcoholic extract from coffee beans, demonstrated a surprising effect when compared to individual extracts on both the genes analyzed: the extracts were found to be effective in influencing SOX9 gene expression only when tested together as a mixture, rather than when tested individually, and they unexpectedly showed a synergistic effect on IGF- 1 expression.

The applicant discovered that exposing dermal papilla cells to oxidative stress by treating them repeatedly with 100 gM H2O2, which simulates a senescence condition, greatly reduces the expression of the GDF11 factor. However, using the extracts from the current invention can reverse this condition and restore GDF11 expression to levels similar to unstressed cells. The effect of the current invention's extracts is even better than that of the positive control TGF-p i growth factor and Minoxidil.

Finally, the Applicant found that, like recombinant GDF11 protein treatment, the extracts of the present invention can restore the expression of the SOX9 transcription factor, associated with a stem cell marker, in dermal papilla cells where a senescence process was mimicked through repeated treatments with 100 gM H2O2.

The extract, rich in peptides and sugars, from Scabiosa arvensis plant cell cultures can be obtained through a preparation process comprising the following steps: a) homogenizing Scabiosa arvensis plant cell cultures in a saline aqueous solution to obtain a homogenate; b) separating the solid part from the liquid part of this homogenate; c) treating the solid part with proteolytic enzymes in an acidic solution to hydrolyze the cell wall proteins and the glycosidic bonds, obtaining the peptide and sugar-rich extract.

Homogenization refers to the fragmentation treatment of plant material in a suitable container such as a ceramic mortar with a ceramic pestle previously cooled, or for larger volumes, metal containers with metal blades, using either laboratory or industrial blenders or presses can be used.

Preferably, the Scabiosa arvensis plant cell cultures are obtained by taking plant tissue from Scabiosa arvensis plants, inducing callus formation from this tissue on a solid substrate, harvesting these calluses, and setting up liquid cultures from them.

Preferably, step b) of separating the solid part from the liquid part of the homogenate is performed by centrifugation, sedimentation, or filtration. In a preferred embodiment of the invention, the procedure also includes, before step c) of treating the solid part with proteolytic enzymes, washing the solid part separated in step b) with distilled water to remove cytoplasmic component residues.

In another preferred embodiment of the invention, the procedure also includes, before step c) of treating the solid part with proteolytic enzymes, washing the solid part separated in step b) with distilled water to remove cytoplasmic component residues followed by treating the solid part with a heated EDTA solution, preferably brought to a boil, e.g., at around 100°C.

The EDTA solution can be an aqueous solution with, for example, an EDTA concentration of 2 mM, and the treatment time for the solid part of the homogenate with the heated EDTA solution can vary from 10 to 30 minutes, preferably 20 minutes, to remove pectins and starch from the cell walls through calcium chelation (which promotes the binding of pectins and starch in the cell wall) by EDTA.

The peptide and sugar-rich extract obtained by the above process can be used in its current form or can be dried using established methods (e.g., lyophilization or spray drying) to produce a powder.

The present invention also relates to the use of a peptide and sugar-rich extract derived from Scabiosa arvensis plant cell cultures, where the extract is obtained using the above-described procedure, for hair growth treatment.

The extract from plant cell cultures enriched in somatic embryos of Lotus japomcus can be obtained using the preparation method described in European patent EP 3288644, in the name of the Applicant, the content of which is incorporated herein by reference.

This method includes the following steps: a) Inducing plant cell cultures of Lotus japonicus in suspension to form somatic embryos; b) Homogenizing the obtained somatic embryos in a saline aqueous solution to obtain a homogenate; c) Separating the solid part from the liquid part of the obtained homogenate; d) Treating the solid part with proteolytic enzymes in an acidic solution to hydrolyze the cell wall proteins of the somatic embryos, obtaining a peptide and sugar-rich extract.

The plant cell cultures can be obtained by harvesting plant tissue from the Lotus plant, inducing callus formation from this tissue on a solid substrate, collecting these calluses, and setting up liquid cultures from them.

Preferably, step a) is performed by adding phytohormones to these plant cultures, such as Tidiazuron (TDZ) and Benzylaminopurine (BAP).

The saline aqueous solution in step b) is generally a buffered solution, such as a phosphate-buffered saline (PBS) at pH 7.4.

Preferably, step c) of separating the solid part of the homogenate from the liquid part is performed by centrifugation, resulting in the separation of a supernatant (liquid part) and a pellet (solid part) .

In a preferred embodiment of the invention, the procedure also includes, before step d) of treating the solid part with proteolytic enzymes, washing the solid part of the homogenate separated in step c) with distilled water to remove cytoplasmic component residues.

In another preferred embodiment of the invention, the procedure additionally includes, before step d) of treating the solid part with proteolytic enzymes, washing the solid part of the homogenate separated in step b) with distilled water to remove cytoplasmic component residues, followed by treating the solid part with a heated EDTA solution, preferably brought to a boil, for example at around 100°C.

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

The extract obtained by the described procedure can be used in its current form or can be dried using known methods (e.g., lyophilization or spray drying) to obtain a powder.

The present invention also relates to the use of an extract derived from Lotus japonicus plant cell cultures enriched in somatic embryos for hair growth treatment, where the extract is obtained using the abovedescribed procedure.

The present invention also relates to a method for preparing a hydroalcoholic artichoke extract, which comprises the following steps: i) Grinding artichoke flower heads; ii) Adding cold ethanol to the ground product and proceeding with subsequent homogenization, obtaining a homogenate; iii) Agitating the suspension at room temperature; iv) Separating the solid part from the liquid part of the homogenate, which constitutes the hydroalcoholic artichoke extract; v) Filtering the obtained hydroalcoholic extract.

Preferably, the artichoke heads used belong to the Cynara scolymus species, the violet variety.

Preferably, in step i), the artichoke heads intended for grinding are first frozen at a temperature between -20°C and -40°C, preferably around - 30° C. Additionally, preferably, before performing step i), the artichoke heads, stripped of stems and outer leaves, are washed in water and then in a 3% to 5% sodium bicarbonate solution, preferably 5%, for 20 minutes to 60 minutes, for example, 30 minutes. In the washing step, the weight/ volume ratio between the artichokes and the sodium bicarbonate solution can range from 1:3 to 1:5, preferably a ratio of 1:5. The preliminary washing step advantageously ensures that the extract is sterile, uncontaminated, and free from bacteria that could metabolize and degrade the active components in the final extract. After washing, the artichoke heads are rinsed with sterile distilled water to remove bicarbonate residues and then dried, for example, under a horizontal laminar flow hood, and subjected to step i) of grinding. It is preferable to cool the artichoke heads in a freezer to a temperature between -20°C and -40°C, preferably around -30°C, before grinding.

Preferably, in step i), the artichokes are ground while they are still frozen and without solvent using a blade homogenizer operating at 1000-3000 rpm, preferably 1500 rpm, for a while between 3 and 15 minutes, preferably 3 minutes. This results in a more homogeneous matrix.

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

The ethanol added in step ii) is cold, i.e. it has a temperature between - 30°C and - 10°C, preferably -20°C.

Preferably, in step ii) ethanol is added to the ground product which is at a temperature between -30°C and - 10°C, preferably -20°C.

Preferably, in step ii), the ground product undergoes two cycles of homogenization with the addition of the cold ethanol, each cycle of homogenization being performed using a blade homogenizer operating at a speed of 3000-5000 rpm, preferably 3800 rpm, for a time between 3 and 15 minutes.

Advantageously, this allows for better homogenization of the matrix in a small extraction volume; furthermore, using ethanol in the above percentages, rather than pure ethanol (96%), optimizes extraction yield and allows for more efficient extraction of polyphenols.

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

Preferably, in step iii), the homogenate is left in agitation for 1 to 4 hours, preferably for 2 hours, at a temperature between 20°C and 30°C, preferably 25°C, using a mixer.

Preferably, in step iv), separation is performed by centrifugation at a speed of 4000-6000 rpm, preferably 5000 rpm, for a time between 5 and 15 minutes, preferably for 10 minutes.

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

The aforementioned extract can be evaporated using rotary evaporators (maximum temperature 25°C) to remove excess ethanol, which can be harmful to cells.

Subsequently, it can be dried using established methods such as freeze- drying or spray drying, resulting in a powdered form. Freeze-drying results in a more stable extract, thereby extending its shelf life.

The water-soluble prickly pear extract preferably used in combination with the hydroethanolic coffee extract for the use according to the present invention, is obtained by the preparation process described in Italian patent application No. 102021000020309 in the name of the Applicant, the content of which is incorporated herein by reference..

This process includes the following steps: i) Subjecting previously cleaned prickly pear pads to a steam treatment; ii) Peeling the prickly pear pads obtained from step i); iii) Homogenizing the peeled prickly pear pads, obtaining a homogenate; iv) Separating the solid part from the liquid part of said homogenate, the liquid part constituting the water-soluble prickly pear extract.

Preferably, in step i) the steam treatment is conducted at a pressure of up to 4.5 bar, preferably between 3 bar and 4.5 bar, for a time ranging from 5 minutes to 30 minutes, preferably between 10 minutes and 20 minutes.

Preferably, in step iii) the peeled prickly pear pads are homogenized while still frozen, preferably at a temperature of about -30°C.

Preferably, in step iii) the peeled prickly pear pads undergo a first homogenization in the absence of solvent in a rotary blade homogenizer operating at a speed between 2000 rpm and 3500 rpm for a period of 1 to 30 minutes, preferably 3 minutes, followed by a second homogenization in the presence of solvent in a rotary blade homogenizer operating at a speed between 3500 rpm and 4500 rpm, more preferably at 3800 rpm, for a period of 5 to 30 minutes, preferably 3 minutes.

Preferably, in step iii) the solvent is chosen from an aqueous solution or a saline aqueous solution or water, more preferably in a weight ratio between the homogenate obtained in step iii) and solvent ranging from 1: 1 to 1:3. Preferably, step iv) is carried out by centrifugation or filtration.

The hydroethanolic coffee extract, used preferably in combination with the water-soluble prickly pear extract for the use according to the invention, is obtained by a process comprising the following steps: i) Grinding green coffee beans, obtaining a ground product, ii) Adding cold ethanol having a temperature between -30°C and - 10°C, preferably -20°C to the ground product, and then proceeding with subsequent homogenization to obtain a homogenate in the form of a suspension of a solid part in a liquid part, iii) Agitating the suspension at room temperature, iv) Separating the solid part of said homogenate from the liquid part, which constitutes the hydroethanolic coffee beans extract, iv) Filtering the hydroethanolic extract obtained in step iv) .

Preferably, the coffee beans used belong to the species Coffea arabica, variety Santos, are decaffeinated with high-pressure carbon dioxide (CO2) and are not roasted.

Preferably, in step i) the coffee beans are at a temperature between -20°C and -40°C, preferably about -40°C.

Preferably, in step i) grinding is performed in a rotary blade homogenizer operating at a speed of 3000-5000 rpm, preferably 3800 rpm, for a time between 3 and 15 minutes.

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

Preferably, in step ii) ethanol is added to the ground product which is at a temperature between -20°C and - 10°C, preferably - 15°C. The ethanol added in step ii) is cold, i.e. it has a temperature between - 30°C and - 10°C, preferably -20°C.

Preferably, in step ii) the homogenization is performed in a rotary blade homogenizer operating at a speed of 3000-5000 rpm, preferably 4000 rpm, for a time between 3 and 15 minutes.

Preferably, in step iii) the homogenate is agitated for a period of 1 to 4 hours, preferably for 2 hours, at a temperature between 20°C and 30°C, preferably 25°C, using a mixer.

Preferably, in step iv) the separation is performed by centrifugation at a speed of 5000-7000 rpm, preferably 6300 rpm, for a time between 5 and 15 minutes, preferably 10 minutes.

Preferably, in step v) filtration is carried out using qualitative filter paper with a porosity of 60-68 micrometers while microfiltration uses Stericap™ PLUS units (Millipore) with a porosity of 0.22 gm.

The aforementioned extract can be evaporated using rotary evaporators, until obtaining a suspension with a dry substance percentage of 80% ± 4% by weight.

The present invention also relates to a process for preparing a hydroethanolic coffee extract as described above and the use in combination of a water-soluble extract derived from prickly pear pads and a hydroethanolic extract derived from coffee beans for hair growth treatment, where the aforementioned extracts are obtained by their respective processes described above.

The combination of these extracts, particularly for topical application to the scalp, can be simultaneous, separate, or sequential.

In the case of simultaneous use, the aforementioned extracts can be mixed to form a mixture or composition containing such extracts, which is then applied topically. Preferably, in the combined use of the aforementioned extracts according to the invention, the weight ratio between the hydroethanolic coffee extract and the water-soluble prickly pear pads extract can range from 10: 1 to 3: 1, preferably this ratio is 6: 1. In particular, they are used in the form of a mixture or composition containing the aforementioned extracts in the above ratios. Preferably, in the use of the mixture or composition containing the aforementioned extracts in the above ratios, the concentration of the hydroethanolic coffee extract should be at least 0.003% by weight of the mixture or composition.

The present invention further relates to a pharmaceutical or cosmetic composition containing at least one plant extract as an active ingredient that activates the GDF11 growth factor for the described use in stimulating hair growth, wherein the at least one plant extract in the pharmaceutical or cosmetic composition is selected from:

1. An extract, rich in peptides and sugars, derived from plant cell cultures of Scabiosa arvensis;

2. An extract, rich in peptides and sugars, derived from plant cell cultures enriched in somatic embryos of Lotus japonicus;

3. A hydroethanolic extract from artichoke heads of Cynara scolymus; and / or

4. A water-soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans.

The present invention also relates to a pharmaceutical or cosmetic composition comprising as an active ingredient an extract, rich in peptides and sugars, derived from cell cultures of Scabiosa arvensis obtained by the respective process described above.

The present invention also relates to a pharmaceutical or cosmetic composition comprising as an active ingredient an extract, rich in peptides and sugars, derived from plant cell cultures enriched in somatic embryos of Lotus japonicus obtained by the respective process described above.

The present invention further relates to a pharmaceutical or cosmetic composition comprising as an active ingredient a hydroethanolic extract from artichoke, obtained by the respective process described above.

The present invention also relates to a composition comprising as an active ingredient a water-soluble extract derived from prickly pear pads combined with a hydroethanolic extract of coffee beans, obtained by the respective processes described above.

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

Moreover, these compositions may include additional components such as carriers, solvents, excipients, and/or cosmetically and/or pharmaceutically acceptable auxiliaries known in the art.

Preferably, the solvent is a hydrophilic solvent, preferably chosen from water and aqueous saline solutions, or one or more organic solvents compatible with cosmetic and/or pharmaceutical formulations, more preferably chosen from alcohols, glycerol, organic acids, amides, amines, aldehydes, or ketones, or a combination of the two types of solvents, if miscible with each other.

Carriers that can be used in the above compositions include liposomes, preferably multilamellar liposomes, cyclodextrins, and silicates.

Finally, the invention relates to a pharmaceutical or cosmetic treatment method for stimulating hair growth, including topical application to the scalp in need of treatment of a pharmaceutically or cosmetically effective amount of a composition as described above, i.e., comprising at least one of the aforementioned plant extracts. By way of not limiting examples of the present invention, below are some examples relative to the preparation of extracts according to the present invention and experiments that demonstrated the biological activity of the GDF1 1 protein and the extracts of the present invention in vitro cell models, three-dimensional spheroid models and ex vivo hair follicles.

Examples

Example 1: Method for preparing the extract derived from plant cell cultures of the species Scabiosa arvensis according to the present invention

Below are the steps of the method for preparing a peptide- and sugar-rich extract from plant cell cultures of Scabiosa arvensis according to the present invention.

Step of cell culture preparation

Starting from leaf pieces of young seedlings of the species Scabiosa arvensis, plant callus cultures were established on a solid medium. Specifically, the whole leaves of Scabiosa arvensis were sterilized with 70% ethanol (v/v in water) for 15 minutes, followed by 1% sodium hypochlorite (v/v in water) for another 15 minutes. After being washed 3 times with water to remove the alcohol and hypochlorite, the leaves were cut to obtain pieces of 5 mm x 5 mm size and placed on a solid “B5 Gamborg medium” substrate, containing “plant agar” (7.5 mg/L), myoinositol (500 mg/L), sucrose (30 g/L), and adjusted to pH 5.7 using KOH (0.1 N). The autoclave-sterilized culture medium was then supplemented with 2,4-Dichlorophenoxyacetic acid (1 mg/L), adenine (1 mg/L), and kinetin (0.01 mg/L). After approximately 5 weeks of incubation at 20°C in the dark, the calluses were obtained and then transferred to a liquid medium to establish the cultures.

Step of growth of liquid cultures

Once reached a size of about 1 cm in diameter (weight of about 50 mg), the calluses were taken and placed in flasks containing 50 ml of the same medium described above without agar. The flasks were placed in the dark on an orbital shaker with 120 rpm shaking speed, and after approximately 10 days, calluses started to disintegrate and form homogeneous cell cultures, consisting of single cells or small cell aggregates.

Step of cell harvesting

When the culture reached a density of about 150 g/L in a 2 L flask, the cells were collected through low-porosity filters (80- 100 pm), in order to remove the culture medium. The cells were subsequently washed in sterile distilled water and frozen at -80 ° C.

Preparation of peptide and sugar extract

500 g of frozen cells were broken mechanically and subsequently homogenized in the presence of PBS (NaCl 136 mM, KC1 2.7 mM, NaH₂PO₄ 12 mM, KH₂PO₄ 1.76 mM, pH 7.4) at a 1:2 ratio v/w. The resulting homogenate was centrifuged at 8,500 rpm for 15 minutes at 4°C to precipitate the insoluble components. The pellet (containing the cell walls) was then treated with 2 volumes of a 2 mM EDTA solution and heated to 100°C for 20 minutes. After cooling, the extract was filtered through cellulose filters with a pore size of 80- 100 pm, washed and filtered again to remove residual EDTA. Subsequently, the obtained pellet was resuspended in 2 volumes of a 0.1 N HC1 solution and then boiled (100°C) for 1 hour to hydrolyze and dissolve all the sugars from the cell walls glycoproteins.

After boiling, the sample was cooled (on ice) and then enzymatically digested at 37°C for 16 hours with protease. At the end of the enzymatic digestion, the suspension was centrifuged to obtain a transparent solution and adjusted to pH 6.5 with 10 N NaOH: it constitutes the extract rich in peptide and sugar-rich derived from the cell walls of Scabiosa arvensis. Example 2: Method for preparing the extract derived from cultures of cells enriched in Somatic Embryos from the species Lotus japonicus according to the present invention

Starting from leaf pieces of young seedlings of the species Lotus japonicus, first, plant callus cultures were established on a solid medium, and subsequently, liquid cell cultures were induced to form somatic embryos. Once the desired density was reached, the somatic embryo cultures were collected and processed to obtain the extracts.

The method used is as follows: a) Callus Preparation: Whole leaves are taken from plants of Lotus japonicus and sterilized with 70% ethanol (in water v/v) for 15 minutes and 1% sodium hypochlorite (in water v/v) for additional 15 minutes. After being washed 3 times in water to remove alcohol and hypochlorite, the leaves are cut into pieces of about 0.5 cm² each with a sterile blade. All the leaf fragments are placed on a solid B5 Gamborg medium containing: plant agar 7.5 mg/L, 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. After about 5 weeks of incubation at 20°C in the dark, calluses are obtained due to the proliferation of the leaf cells. The calluses are excised and then transferred to a fresh culture medium every 3-4 weeks. b) Cell culture preparation: When the calluses reach a diameter of about 1 cm (about 50 mg of weight), they are picked up and dispersed in flasks containing 50 ml of AB1 liquid culture medium (B5 Gamborg 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). The flasks are placed in the dark on an orbital shaker with 100 rpm shaking speed. After about 10 days, the calluses break up and proliferate to form cell suspension cultures. c) Somatic embryos preparation: The plant cell cultures are resuspended in a fresh AB2 liquid growth medium (B5 Gamborg medium containing: myo-inositol 500 mg/L, sucrose 30 g/L) with the addition of phytohormones (Thidiazuron (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 to induce the formation of somatic embryos in the cell cultures (somatic embryos enriched cell culture). d) Somatic embryo cell culture growth: The induction of somatic embryos is conducted for 4 weeks, adding fresh culture medium every week, until reaching a level of differentiation into somatic embryos of 70-90%. e) Somatic embryo collection: the separation of the embryos from the growth medium can be carried out by centrifugation at 2000 g, sedimentation, or filtration through membranes with pores smaller than 100 microns. The embryos are then frozen at -80°C to preserve their chemical and physical properties. f| Somatic embryo homogenization: frozen somatic embryos are broken mechanically (homogenized) in the presence of phosphate-buffered saline (PBS) (NaCl 136 mM, KC1 2.7 mM, NaH₂PO₄ 12 mM, KH₂PO₄ 1.76 mM, pH 7.4) in the ratio of 1:2 weight/ volume. This step can be carried out in a suitable container such as a ceramic mortar and pestle, previously cooled, or for larger volumes, larger containers may be used, including metal ones, where the plant material can be homogenized with metal blades, using either laboratory or industrial blenders or presses.

Once obtained a homogenate lysate by homogenizing the embryos, the sample is centrifuged, for example at 4000 rpm for about 15 minutes at 4°C, to precipitate the insoluble components. g) Obtaining the peptide and sugar extract from cell walls: The pellet (containing the cell walls) obtained in step f) is further washed with distilled water to remove the residues of the soluble fraction and filtered again. The pellet is weighed, suspended and boiled with 2 volumes (ratio w/v) of a 2 mM EDTA solution for 20 minutes. After cooling, the extract is filtered through a filter cloth, washed and filtered again to remove residual EDTA. Subsequently, the obtained pellet is boiled under a chemical hood with 2 volumes of a 0.1 N HC1 solution for 1 hour to hydrolyze the sugar bonds. After boiling, the sample is suspended again, cooled in ice and then enzymatically digested at 37° C for 16 hours with protease, using 1 mg of enzyme per ml of suspension. At the end of the enzymatic digestion, the suspension is centrifuged or filtered again to obtain a transparent solution: it constitutes the hydrophilic extract rich in peptides and sugars derived from the cell walls.

Example 3: Preparation of water-soluble cactus extract

The water-soluble cactus extract according to the present invention was produced using the following method:

1.2 kg of freshly harvested cactus pads were washed with running water to remove any soil residues. The pads were then subjected to high- pressure steam (up to 4.5 bar) for about 15 minutes, then peeled to remove the spines and dried with absorbent paper under a horizontal laminar flow hood. The pretreated pads were then weighed (approximately 1 kg) and transferred to a freezer at -30°C.

Next, the frozen cactus pads were first homogenized at 2000 rpm for 3 minutes at room temperature without any solvent. Afterward, the material underwent a second homogenization at 3800 rpm for 3 minutes at room temperature, with the addition of an aqueous or saline solution (phosphate-buffered saline, or PBS) in a 1: 1 ratio.

The resulting homogenate was centrifuged at 6300 rpm for about 15 minutes at 4°C to precipitate the insoluble components. The supernatant obtained from centrifugation was collected and freeze-dried to obtain a water-soluble cactus extract according to the present invention.

Example 4: Preparation of hydroethanolic coffee extract The coffee beans, used according to the present invention, were obtained from Kimbo Caffe S.p.A (Naples). They belong to the Coffea arabica species, Santos variety, and were decaffeinated using a high-pressure CO2 method. Additionally, this coffee is characterized by being green, i.e. unroasted, which typically occurs at 200°C and deactivates many of the active molecules present in the product.

Upon receipt, the decaffeinated green coffee beans were transferred to a freezer at -40°C. The hydroethanolic extract from these coffee beans, according to the present invention, was produced using the following method:

500 g of still-frozen coffee beans were subjected to an initial homogenization at 3800 rpm for 3 minutes at room temperature, without any solvent. This was followed by two additional cycles of homogenization at 4000 rpm for 3 minutes at room temperature, with the addition of cold 96% ethanol in a 1:2 (weight/ volume) ratio.

The resulting suspension was incubated with agitation for 2 hours at 25°C using a mixer. The obtained suspension was centrifuged at 6300 rpm for 10 minutes, and the resulting supernatant was filtered using vacuum pumps with qualitative filter paper (60-68 micrometers) to remove residual solid matter. It was then further microfiltered using Stericap™ PLUS filtration units (0.22 pm porosity, Millipore), again with the aid of vacuum pumps.

The microfiltered extract was evaporated in glass flasks using rotary evaporators (maximum temperature reached by the sample was 25°C), resulting in a hydroethanolic coffee extract according to the present invention.

Example 4a: Preparation of a mixture of hydroethanolic coffee bean extract and water-soluble cactus pads extract

A mixture (composition), according to the present invention, was prepared by combining hydroethanolic coffee beans extract obtained according to Example 4 and water-soluble cactus pads extract obtained according to Example 3, in a 6: 1 ratio. The mixture was used in subsequent test examples using the following method:

Both extracts were separately dissolved at a 10% concentration in water with continuous agitation for 5 minutes and then centrifuged to remove the insoluble residue. The resulting supernatants were recovered and used for in vitro assays; the supernatants from the coffee bean and cactus pads extract were mixed in the culture medium to be added to the cells during the tests described in the subsequent examples.

Example 5: Preparation of hydroethanolic artichoke extract

Artichoke heads, Cynara scolymus var. violetto, purchased from a company in Puglia that had all relevant organic certifications, were stripped of their stems and outer leaves and washed thoroughly in running water before being soaked in a 5% sodium bicarbonate solution (1:5 weight/ volume ratio) for 30 minutes. After treatment, the artichokes were rinsed in sterile distilled water to remove any bicarbonate residues and then dried under a horizontal laminar flow hood. The pretreated artichoke heads were weighed (approximately 500 g) and transferred to a freezer at -30°C.

The hydroethanolic artichoke extract according to the present invention was produced using the following method:

500 g of still-frozen artichoke heads were subjected to initial homogenization at 1500 rpm for 3 minutes at room temperature, without using any solvent. Then, they were subjected to two additional homogenization cycles at 3800 rpm for 3 minutes each, with the addition of cold 80% ethanol in a 1: 1 (weight/ volume) ratio. After homogenization, a further 80% ethanol was added in a 1:4 (homogenate weight/ solvent volume) ratio, achieving a final extraction ratio of 1:5. The resulting suspension was incubated with agitation for 2 hours at 25°C on a stirring plate. Next, the obtained suspension was centrifuged at 5000 rpm for 10 minutes, and the supernatant was then filtered using vacuum pumps with qualitative filter paper (60-68 micrometers) to remove the residual solid matter.

The extract was then evaporated in glass flasks using rotary evaporators (the maximum temperature reached by the sample was 25°C) to remove excess ethanol and subsequently freeze-dried to obtain a hydroethanolic artichoke extract according to the present invention.

Example 6: Cytotoxicity Assay

To ensure that the concentrations of the extracts, subject of the present invention, used in subsequent assays were not toxic to growing cells, a cytotoxicity assay was conducted.

This assay is based on the use of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] for the first time described by Mosmann in 1983. It is based on the capacity of the enzyme mitochondrial dehydrogenase of the viable cells to hydrolyze the MTT tetrazolium ring (light yellow) and to form crystals of formazan (dark blue color). These crystals are impermeable to cell membranes and accumulate in the cytoplasm of metabolically active cells. The number of live and healthy cells is thus directly proportional to the level of formazan product.

HaCaT cells (immortalized human keratinocytes), in the initial number of 1 x 10⁴ per well, were grown in 96-well plates in DMEM (Dulbecco’s Modified Eagle Medium) (Lonza), supplemented with 10% fetal bovine serum, for about 8 hours. After treatment for approximately 48 hours with 0.0006% peptide and sugar-rich extract derived from Scabiosa arvensis, 0.0002% peptide and sugar-rich extract derived from Lotus japonicus, 0.002% hydroethanolic artichoke extract, 0.003% hydroethanolic coffee beans extract, 0.0005% water-soluble cactus pads extract, and a mixture containing 0.003% hydroethanolic coffee beans extract and 0.0005% water-soluble cactus pads extract, the cells were washed in PBS and incubated with 100 pl/ well of reaction buffer containing: 10 mM Hepes, 1.3 mM CaCh, 1 mM MgSCU, 5 mM glucose, and 0.5 mg/ml MTT colorimetric substrate in PBS buffer at pH 7.4. After 3 hours of incubation at 37° C, 5% CO2, 100 pl of solubilizing solution containing 10% Triton-XlOO and 0.1 N HC1 in absolute isopropanol was added to each well. After 16 hours, the colorimetric reaction was measured at 595 nm with a Victor3 plate reader. The MTT results, shown in Figure 1, indicated that the concentrations of the tested extracts did not cause any toxicity to the cells.

Example 7: Analysis of SOX9 and IGF- 1 gene expression in human dermal papilla cells

Human dermal papilla cells (HFDPC), in the initial number of 8 x 10⁴ per well, were grown in 6-well plates in "Human Follicle Growth Medium" (C- 26501, Promocell), supplemented with appropriate additives for 20 hours. The following day, treatments were added in fresh medium for 24 hours. Specifically, recombinant GDF-11 protein (rGDFl l) at two concentrations (10 ng/ml and 100 ng/ml) and TGF-p i at 2.5 ng/mL were tested. Additionally, in a subsequent experiment, the following extracts were tested: Scabiosa arvensis peptide and sugar-rich extract at 0.0006%, Lotus japonicus peptide and sugar-rich extract at 0.0002%, hydroethanolic artichoke extract at 0.002%, hydroethanolic coffee beans extract at 0.003%, water-soluble cactus pads extract at 0.0005%, and the combination of these two latter extracts. In this experiment, in addition to TGF-p i, Minoxidil at 1 gM was also tested.

After the treatments, cells were washed with PBS, collected in lysis buffer, and subjected to the RNA extraction procedure using the "GenElute™ Total RNA Purification" kit from Merck. The RNA samples were treated with DNase I (Ambion) at 37°C for 30 minutes to remove genomic DNA contaminants. 2 gl of each sample were loaded onto a 1% agarose gel in the presence of denaturing loading dye and quantified using specific RNA marker (ThermoScientific) as a reference. For quantification, IBright Analysis software (ThermoScientific) was used. 500 ng of total RNA was reverse transcribed using the Reverse Transcriptase enzyme (ThermoScientific) . Semi-quantitative RT-PCR reactions were performed using internal standards as the universal 18S primer/ competimer pair (Ambion) in a 4:6 ratio for SOX9 and 3:7 for IGF- 1.

The PCR products were separated on a 1.5% agarose gel, visualized using the iBright instrument (ThermoScientific) and analyzed using the iBright Analysis software (Thermo Fisher Scientific).

The values shown in the graphs represent the ratio of the band intensity relative to the gene in analysis and that of the band relative to the 18S standards, thus getting a value related to the actual expression of the gene of interest. The values were then converted into a percentage (%), by establishing the value obtained from the untreated control as 100%.

The primer sequences used for amplification were as follows:

. Hs SOX9 forward (for): SEQ ID NO 1;

• Hs SOX9 reverse (rev): SEQ ID NO 2;

. Hs IGF- 1 forward (for): SEQ ID NO 3;

• Hs IGF- 1 reverse (rev): SEQ ID NO 4.

The results shown in Figure 2 indicate that treating dermal papilla cells with rGDF- 11 at both concentrations results in an 80% and 66% increase in SOX9 gene expression and a 63% and 38% increase in IGF- 1 gene expression, respectively; these increases were either equal to or greater than those produced by the positive control, TGF-p i.

Regarding the extracts of the present invention, results shown in Figure 6A reveal that all extracts, like rGDFl l, boosted SOX9 gene expression. Specifically, the peptide and sugar-rich extract from Scabiosa arvensis cells increased SOX9 gene expression by approximately 110%, the peptide and sugar-rich extract from Lotus japonicus embryonic cells by 25%, the artichoke extract by roughly 80%, and, notably, the combination of coffee beans extract and cactus pads extract by 28%, in contrast, the two extracts tested individually did not have an impact on SOX9 gene expression.

Figure 6B demonstrates the efficacy of treatments with the extracts of the present invention on increasing IGF- 1 gene expression. The Scabiosa arvensis peptide and sugar-rich extract and the artichoke extract led to an approximately 35% increase in IGF- 1 gene expression, while the peptide and sugar-rich extract from Lotus japonicus embryonic cells and the combination of coffee bean extract and cactus pads extract resulted in an approximate 50% increase. Treatments with individual extracts also showed efficacy, but the combined effect was synergistic, unexpected, and greater than the sum of the effects produced by the two individual extracts.

Example 8: Analysis of Noggin and Q-Catenin protein expression in an in vitro dermal papilla spheroid model

In recent years, significant progress has been made in developing three- dimensional models that mimic tissue characteristics. These 3D spheroid-based models are successfully used in screening to identify potential products or compounds because they more accurately reproduce the tissue microenvironment compared to conventional two- dimensional (2D) cell cultures. They consist of both surface-exposed cells and cells located at a hypoxic core, like in vivo conditions.

Dermal papilla cell aggregates, when seeded into a low-attachment round-bottom well plate, form individual spheroids that can be used as hair follicle spheroid models.

Generation of human dermal papilla spheroids

To create a more complex cellular system, 3D dermal papilla spheroid cultures were constructed to assess the impact of GDF- 11 on the production of proteins related to hair follicle development. 3 ^x 10³ dermal papilla cells (Hair Follicle Dermal Papilla Cells, HFDPC) were seeded in 96-well round-bottom U-plates for 48 hours in 50 pl of “Human Follicle Growth Medium” (C-26501, Promocell), with supplements added as needed to facilitate spheroid formation. At the end of the incubation period, it was observed under a microscope that the dermal papilla cell aggregates had developed into distinct spheroids.

Immunofluorescence Analysis - Analysis of Noggin and Beta-Catenin Proteins

The obtained spheroids were collected and centrifuged at 1500 rpm for 3 minutes. After being washed twice in PBS buffer, they were fixed in 4% paraformaldehyde for 1 hour. Following three additional PBS washes, the spheroids were permeabilized with a 0.1% Triton-XlOO solution in phosphate buffer for 15 minutes. After another PBS wash, the spheroids were incubated in a “blocking” solution (6% BSA, 5% goat serum, 20 mM MgCk, and 0.2% Tween) for 1 hour at room temperature. Then, after a PBS wash, the spheroids were incubated overnight at 4°C with shaking, either with the primary mouse antibody against Noggin (Noggin Monoclonal Antibody, OTI 1C1, Thermo Fisher Scientific) or with the primary mouse antibody against P-Catenin (Beta-Catenin Monoclonal Antibody, 6F9, Thermo Fisher Scientific). Following three washes in PBS, the spheroids were incubated for 1 hour at room temperature with secondary anti-mouse antibodies, Alexa-Fluor-488 for Noggin or Alexa- Fluor-588 for P-Catenin. Nuclei were stained with DAPI (4’,6-diamidino- 2-phenylindole) at 1 gg/ml in PBS for 10 minutes. Finally, images were acquired with a ZEISS ISM700 confocal microscope and analyzed using Imaged software.

As shown in Figure 3, treatment of the spheroids with recombinant rGDF- 11 protein at a concentration of 10 ng/ml for 24 hours led to increased production of both Noggin protein, which plays a crucial role in follicle regeneration, and [3-Catenin, which promotes the induction and duration of the anagen phase of the hair growth cycle. Example 9: Analysis of hair shaft length in hair follicle explants

Follicles from male patients undergoing follicle transplantation using the Follicular Unit Extraction (FUE) technique were used to analyze the effects of recombinant GDF11 protein treatment on hair shaft length. All patients provided informed consent for the use of follicles for research purposes, and follicular units were collected from areas with higher follicle density.

The follicles were examined under an optical microscope, separated to each other and only those in the anagen phase, which is the active growth phase of the hair, showing a more expanded bulb shape, were chosen for the study. These selected follicles were then cultured in E-Williams medium (12551032, Gibco-Fisher Scientific) supplemented with 2 mM Glutamine, 10 ng/ml hydrocortisone (Merck), 10 gg/ml insulin (Merck), and 1% penicillin and streptomycin (Gibco). Following a 24-hour culture period, the follicles were treated with recombinant GDF11 protein at 100 ng/ ml concentration for seven days. The culture medium and treatments were changed every other day during this period. At the end of the treatment, the bulbs were observed under an optical microscope and images were acquired and analyzed using Imaged software.

Figure 4 shows that treating with rGDFl 1 protein led to an approximate 15% increase in hair shaft length over 7 days, compared to an approximate 8% increase observed without treatment.

Example 10: Analysis of GDF11 production

8 ^x 10³ dermal papilla cells (Hair Follicle Dermal Papilla Cells, HFDPC) per well were grown in 96-well plates using appropriate culture medium and necessary supplements (C-26501, Promocell). The next day extracts to be tested, along with TGF-p i and Minoxidil as positive controls, were added. The tested concentrations of the extracts were as follows: 0.0006% for the peptide and sugar-rich extract from Scabiosa arvensis cells, 0.0002% for the peptide and sugar-rich extract from somatic embryos of Lotus japonicus, and 0.002% for the hydroethanolic extract of artichoke. For the combination, 0.003% concentration of the hydroethanolic extract from coffee beans and 0.0005% of the water-soluble extract from Ficus carica pads were used. Both individual and combined extracts were tested. After 6 hours, the cells were washed with PBS and fixed in 4% paraformaldehyde for 10 minutes. The cells were then washed with washing buffer (PBS lx, 0.5 mM CaCk, 1 mM MgCk, 0.1% Triton) three times and incubated with a blocking buffer containing 0.5% non-fat dried milk (NFDM, sc-2334, Santa Cruz Biotechnology Inc) for 30 minutes with shaking. After a wash with the washing buffer, the cells were incubated with the primary anti-GDFl l antibody (Abeam, ab 124721) (dilution 1: 1000) in washing buffer containing 0.5% NFDM. After 2 hours of shaking, the plates were washed three times with washing buffer and incubated with secondary anti-rabbit peroxidase-conjugated antibody (1706515, Biorad) (dilution 1:5000) in washing buffer containing 0.5% NFDM. After 1 hour of incubation, the plate was washed three times with washing buffer. The chemiluminescent reaction was developed using the QuantaRed™ Enhanced Chemifluorescent HRP substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 15 minutes, absorbance was measured at 490 nm.

As shown in Figure 5, treatments with the extracts of the present invention induced GDF- 11 production: specifically, the peptidic extract from Scabiosa arvensis cells and the hydroalcoholic extracts from artichoke and coffee induced about a 50% increase in GDF11 production, the peptidic extract enriched in somatic embryos of Lotus japonicus and the water-soluble extract from Opuntia ficus-indica pads induced about a 30% increase, while the combination of coffee extract and prickly pear extract showed an approximately 85% increase, equivalent to the sum of the effects produced by the individual extracts.

Example 11: Analysis of GDF11 and SOX9 gene expression in human dermal papilla cells under oxidative stress conditions

8 ^x 10⁴ dermal papilla cells (Hair Follicle Dermal Papilla Cells, HFDPC) per well were grown in 6-well plates in “Human Follicle Growth Medium” (C-26501, Promocell), with appropriate supplements for 20 hours. The next day, the cells were treated with hydrogen peroxide (H2O2, 100 pM) for 1 hour to induce the formation of free radicals, allowed to grow for 7 hours in culture medium, and then treated again with 100 pM H2O2 for another hour. Finally, the cells were allowed to grow overnight in their culture medium.

The treatment was repeated with two additional cycles of H2O2, interspersed with 7 hours of incubation with culture medium, on the following day. After the second H2O2 cycle, the cells were treated with the extracts of the present invention for 24 hours.

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

The sequences of the primers used for amplification were as follows:

. Hs SOX9 forward (for): SEQ ID NO 1

• Hs SOX9 reverse (rev): SEQ ID NO 2

. Hs GDF11 forward (for): SEQ ID NO 5

• Hs GDF11 reverse (rev): SEQ ID NO 6

The results shown in Figure 7A indicate that oxidative stress conditions, induced by repeated H2O2 treatment, led to an approximately 80% reduction in GDF11 expression compared to non-stressed control cells. However, treatment with the extracts of the present invention was able to reverse this condition and restore expression to a level similar to that of non-stressed control cells: specifically, treatment with the peptide and sugar-rich extract from Scabiosa arvensis and the combination of coffee bean extract and prickly pear pads extract increased GDF11 gene expression by about 65%, the peptide and sugar-rich extract from somatic embryos of Lotus japonicus by approximately 50%, and the artichoke extract by 80%. Positive controls, TGF-p i and Minoxidil, showed an increase equivalent to or less of the effects produced by the extracts of the present invention.

The results shown in Figure 7B display the effect on SOX9 gene expression: SOX9 was reduced by about 150% compared to non-stressed control cells, and treatments with the extracts of the present invention were able to counteract this effect by increasing SOX9 gene expression; the effect is like that produced by the recombinant GDF11. Specifically, treatment with the peptide and sugar-rich extract from Scabiosa arvensis, like rGDFl l, led to an approximately 65% increase in SOX9 gene expression, the hydroethanolic artichoke extract to an approximately 50% increase, the somatic embryo extract from Lotus japonicus to an approximately 90% increase and the combination of coffee bean extract and prickly pear pads extract to an approximately 115% increase.

Literature

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2. Weger N and Schlake T (2005) IGF- 1 Signalling Controls the Hair Growth Cycle and the Differentiation of Hair Shafts. Journal of Investigative Dermatology Volume 125, Issue 5, Pages 873-882.

3. J. L. Bueno, M. Ynigo, C. de Miguel, 1 R. M. Gonzalo-Daganzo, A. Richart, C. Vilches,C. Regidor, J. A. Garcia-Marco, E. Flores-Ballester & J. R. Cabrera (2016). Growth differentiation factor 11 (GDF11)- a promising anti-ageing factor - is highly concentrated in platelets. Vox Sanguinis, Nov; 111(4):434-436.

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6. Loffredo FS, Steinhauser ML, Jay SM, Gannon J, Pancoast JR, Y alamanchi 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 and Lee RT1 (2013). Growth differentiation Factor 11 is a circulating factor that reverses age-related cardiac hypertrophy. Cell, 153 (4): 828-839.

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Claims

1. Use of at least one plant extract capable of activating the 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 cultures of plant cells belonging to the Scabiosa arvensis species;

- an extract, rich in peptides and sugars, derived from cultures of plant cells enriched in somatic embryos belonging to the Lotus japonicus species;

- a hydroethanolic extract from artichoke heads belonging to the Cynara scolymus species; and / or

- a water-soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans.

2. Use according to claim 1, wherein said extract, rich in peptides and sugars, from cultures of Scabiosa arvensis plant cells is obtained by a preparation process comprising the steps of: a) homogenizing cultures of Scabiosa arvensis plant cells in a saline aqueous solution, thus obtaining a homogenate; b) separating the solid part of said homogenate from the liquid part; c) treating the aforementioned solid part with proteolytic enzymes in acid solution, so as to hydrolyse the proteins of the cell walls and the glucosidic bonds, thus obtaining said extract rich in peptides and sugars.

3. Use according to claim 1, wherein said extract from cultures of plant cells enriched in somatic embryos belonging to the Lotus japonicus species is obtained by a preparation process comprising the steps of: a) inducing cultures of Lotus japonicus plant cells in suspension so as to form somatic embryos; b) homogenizing the somatic embryos obtained in a saline aqueous solution, thus obtaining a homogenate; c) separating the solid part of said homogenate from the liquid part, d) treating the aforementioned solid part with proteolytic enzymes in acid solution, so as to hydrolyse the cell wall proteins of said somatic embryos, thus obtaining an extract rich in peptides and sugars.

4. Use according to claim 1, wherein said prickly pear water soluble extract is obtained by a preparation process comprising the following steps: i) subjecting previously cleaned prickly pear pads to a steam treatment; ii) peeling the prickly pear pads obtained from step i); iii) homogenizing the peeled prickly pear pads, thus obtaining a homogenate; iv) separating the solid part of said homogenate from the liquid part, which constitutes said prickly pear water-soluble extract.

5. Process for the preparation of a coffee hydroethanolic extract comprising the following steps: i) Subjecting green coffee beans to grinding, thus obtaining a ground product, ii) Adding cold ethanol having at temperature between -30° and - 10° C, preferably -20°C, to the aforementioned ground product, and proceeding with a subsequent homogenization, thus obtaining a homogenate in the form of a suspension of one solid part in one liquid part, iii) Shaking the suspension at room temperature iv) Separating the solid part of said homogenate from the liquid part, which constitutes said hydroethanolic extract of coffee beans, v) Filtering the hydroethanolic extract obtained in step iv) .

6. Use of a water-soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans for hair growth treatment, wherein said water-soluble extract derived from prickly pear pads is obtained by the process according to claim 4 and said hydroethanolic extract derived from coffee beans is obtained by the process according to claim 5.

7. Process for the preparation of an artichoke hydroethanolic extract comprising the following steps: i) Subjecting the artichoke flower heads, preferably belonging to the Cynara scolymus species, violet variety, to grinding, ii) Adding cold ethanol having at temperature between -30° and - 10° C, preferably -20°C, to the obtained ground product and proceeding with a subsequent homogenization, thus obtaining a homogenate, iii) Shaking the suspension at room temperature, iv) Separating the solid part of said homogenate from the liquid part, which constitutes said artichoke hydroethanolic extract, v) Filtering the obtained hydroethanolic extract.

8. Process according to claim 7 wherein, prior to carrying out step i), said artichoke heads are deprived of the stem and outer skins and are subjected to a washing step in water and subsequently in a sodium bicarbonate solution 3% to 5%, preferably 5%, for a time period comprised between 20 minutes and 60 minutes, preferably 30 minutes, the weight/ volume ratio between the artichoke heads and the sodium bicarbonate solution being comprised between 1:3 and 1:5, preferably 1:5.

9. Process according to claim 7 or 8, wherein said artichoke heads are ground at room temperature and in the absence of solvent with the aid of a blade homogenizer operating at a speed of 1000-3000 rpm, preferably 1500 rpm, for a time period between 3 and 15 minutes, preferably 3 minutes.

10. Process according to anyone of claims 7 to 9, wherein in step ii) ethanol is added at a percentage 70% to 80%, preferably 80% in a homogenate weight/ solvent volume ratio comprised between 1: 1 and 1:2, preferably in a 1: 1 ratio, and / or wherein said ground product is subjected to two homogenization cycles with the addition of the aforementioned cold ethanol, each homogenization cycle being carried out with the aid of a blade homogenizer operating at a speed of 3000-5000 rpm, preferably 3800 rpm, for a time period between 3 and 15 minutes.

11. Use of an artichoke hydroethanolic extract for hair growth treatment, wherein said artichoke hydroethanolic extract is obtained by the process according to claim 10.

12. Pharmaceutical or cosmetic composition comprising at least one plant extract as an active ingredient capable of activating the GDF11 growth factor for use in hair growth treatment, wherein said at least one extract is selected from:

- an extract, rich in peptides and sugars, derived from cultures of plant cells belonging to the Scabiosa arvensis species;

- an extract, rich in peptides and sugars, derived from cultures of plant cells enriched in somatic embryos belonging to the Lotus japonicus species;

- a hydroethanolic extract from artichoke heads belonging to the Cynara scolymus species; and / or

- a water-soluble extract derived from prickly pear pads in combination with a hydroethanolic extract derived from coffee beans.

13. Pharmaceutical or cosmetic composition according to claim 12 comprising, as an active ingredient:

- an extract, rich in peptides and sugars, derived from cultures of Scabiosa arvensis cells obtained by the process according to claim 2, - an extract, rich in peptides and sugars, derived from cultures of plant cells enriched in somatic embryos belonging to the Lotus japonicus species obtained by the process according to claim 3,

- an artichoke hydroethanolic extract obtained by the process according to anyone of claims 7- 10, and/or - a water-soluble extract derived from prickly pear pads obtained by the process according to claim 4 in combination with a hydroethanolic extract derived from coffee beans obtained by the process according to claim 5.