Silybum marianum (L.) Gaertn. achene extract to promote hair growth

Abstract

The present invention relates to an extract of achenes of Silybum marianum (L.) Gaertn., as well as to compositions containing this extract for application in the fields of cosmetics and dermatology to promote hair growth.

Description

Title of the invention: Extract of achenes of Silybum marianum (L.) Gaertn. for promoting hair growth TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to an extract of achenes of Silybum marianum (L.) Gaertn., as well as to compositions containing this extract for application in the fields of cosmetics and dermatology to promote hair growth. STATE OF THE ART

[0002] Hair care, not only for cosmetic purposes but also to prevent hair loss and to regenerate it, has always called for research. Many theories have attempted to clarify the etiology of hair loss in cases of baldness, alopecia, alopecia areata, etc., attributing them to an increase in tissue tension on the cranial sphere, reduced blood flow, or certain endocrine or nervous disorders.

[0003] The hair follicle is a mini-organ anchored in the skin up to the hypodermis, whose main function is the production of a hair shaft. The distribution of hair follicles is established during in utero growth and their number is genetically determined. The hair follicle is a dynamic structure that produces hair during the cycle of growth and tissue remodeling. This cycle is divided into three phases:

[0004] - A growth phase (anagen), the cells of the dermal papilla (fibroblasts) send a signal to the stem cells of the bulb which allows their proliferation. Cells will transform and envelop the dermal papilla to form the sulfur matrix of the hair. They divide and differentiate into follicular keratinocytes, cells responsible for the structure of the hair. For the hair to be well structured, these keratinocytes need sulfur proteins, vitamin B6 and various minerals such as zinc and magnesium. The duration of this phase determines the length of the hair and depends on the proliferation and differentiation of the matrix cells at the base of the follicle.

[0005] - A regression phase (catagen), the matrix dies and therefore the papilla The dermal layer is no longer in contact with this matrix. There is no longer any exchange between the cells. The follicle and the dermal papilla move up towards the epidermis.

[0006] - A resting phase (telogen), the cells of the dermal papilla and the bulb are intact and inactive. The hair falls out. For new hair to grow, the cycle must be restarted.

[0007] Hair is constantly renewed and of the 100,000 to 150,000 hairs in a head of hair, the majority are in the growth phase. There is a normal and physiological loss of hair of around 60 to 100 per day for healthy hair. Beyond this, the loss is said to be pathological, whether it is occasional or established.

[0008] The term alopecia refers to the partial or general lack of hair on the head. Effluvium is excessive hair loss.

[0009] Many factors can be involved in alopecia such as genetic factors, age, sex, diseases, stress, hormonal problems, side effects of medications, scars. It is possible to distinguish several forms of alopecia:

[0010] - Hereditary androgenetic alopecia, it is the most frequent; early hair loss Hair loss occurs in genetically predisposed individuals and particularly affects men. It manifests itself as a reduction in hair volume, or even baldness, and affects 50% of men over the age of 50.

[0011] - Postmenopausal alopecia, this is the most common cause of baldness in Women. Hair loss is more diffuse and extensive in women than in men. Diffuse female alopecia is a disorder that often begins at menopause and affects approximately 40% of women over the age of 70. The term diffuse illustrates that, unlike in men, hair loss affects the entire scalp in a homogeneous manner.

[0012] - Acute or reactive alopecia, it can be linked to drug treatment (including anticancer treatments), stress, childbirth, significant nutritional deficiencies, iron deficiency, hormonal disorders, it is a simultaneous and diffuse loss of a significant amount of hair.

[0013] - Scarring alopecia, it can be caused by skin problems (tumor, burn, alopecia), acute irradiation, lupus erythematosus or parasites (ringworm, lichen).

[0014] - Alopecia areata, it seems to be of autoimmune origin and is characterized by a affected in more or less large patches and in one or more places.

[0015] - Congenital alopecia, rare, it corresponds to an absence of roots or to hair abnormalities (mutations).

[0016] The term alopecia also covers a whole family of hair follicle disorders resulting in permanent, partial or general hair loss.

[0017] We distinguish between diffuse hair loss and localized hair loss.

[0018] Diffuse hair loss: alopecia areata, telogen effluvium, anagen effluvium, alopecia areata. Among diffuse hair loss, the most common are alopecia areata (male and female androgenetic alopecia) and effluvium telogen (after high fever, pregnancy, medication or a strict diet).

[0019] Localized hair loss: androgenetic alopecia, alopecia areata, cicatricial alopecia, tumors. Localized hair loss is observed in the context of male androgenetic alopecia (gulfs, tonsure), alopecia areata, alopecia induced by traction (trichotillomania, braiding and straightening) or cicatricial alopecia (central centrifugal cicatricial alopecia, postmenopausal frontal fibrosing alopecia). Skin tumors and growths are also accompanied by localized hair loss (sebaceous hamartoma, basal cell carcinoma, squamous cell carcinoma).

[0020] Alopecia is essentially linked to a disruption in hair renewal which initially leads to an acceleration in the frequency of cycles at the expense of hair quality and then of hair quantity. The most frequent phenomenon is a reduction in the duration of the growth phase (anagen phase) linked to a halt in cell proliferation. The consequence is a premature induction of the catagen phase and a greater number of hair follicles in the telogen phase and therefore greater hair loss. To combat alopecia, it is therefore necessary to restart the hair cycle, for example by activating the anagen phase.

[0021] To date, various products have been proposed to combat alopecia, and in particular to induce or stimulate hair growth. Most of them combine several active ingredients capable of providing a beneficial effect on the biological parameters involved in hair loss.Among the most commonly encountered active ingredients, we can cite as examples: vitamins such as vitamins A, E, B5, B6, C, H, and PP; trace elements such as zinc, copper, magnesium, silicon; protein derivatives such as peptides, sulfur amino acids (methionine, cystine, cysteine ​​or derivatives); essential oils or extracts of plant origin of a lipophilic or hydrophilic nature, the list of which is not exhaustive; antifungal agents such as piroctone olamine, undecylenic derivatives, cyclopriroxolamine; chemically synthesized molecules known for their specific action on androgen receptors or on the activity of 5-a reductases. Minoxidil or 2,4-diamino-6-piperidinopyrimidine 3-oxide is today the reference in the treatment of androgenetic alopecia. Despite the many theories put forward about its mechanism of action, the latter is not clearly elucidated.Furthermore, its effectiveness remains limited, because even if a stabilization of hair loss is observed in many clinical cases, a resumption of the alopecia process is observed as soon as treatment is stopped. Its restrictive daily use is probably the cause of undesirable side effects noted in patients using it in the long term such as localized skin reactions or . systemic effects. Furthermore, compositions comprising a wide variety of active ingredients are proposed for hair regrowth, these active ingredients being, for example, 2,4-diaminopyrimidine 3-oxide derivatives such as those described in patent application EP0522964. Clinical studies have demonstrated that PGF2a analogues have the property of causing hair and eyelash growth in humans and animals (Johnstone, Am J Opht, 124(4), 544-547, 1997). In humans, tests carried out on the scalp have shown that a prostaglandin E2 analogue, viprostol, has the property of increasing hair density. Application WO98 / 33497 describes pharmaceutical compositions containing prostaglandins or prostaglandin derivatives intended to promote hair growth.

[0022] Thus, despite the many options currently available, consumers still need new products to promote hair regrowth, which are natural and environmentally friendly, while being as effective as chemical active ingredients.

[0023] Recent advances in understanding the biology of the hair cycle involve early and late type stem cells (Mesler et al. Cell Reports 19, 809-821, 2017). Furthermore, these early stem cells have as progeny cells that produce certain keratins with functional properties essential in preventing hair loss. Among these keratins, K75 is the first to be produced, and its genetic deficiency in humans leads to pathological hair loss. This protein is characteristic of the cells forming the companion layer of the inner root sheath of hair follicles (Gu and Coulombe J Invest Dermatol, 2007, 127(5):1061-73). In the study described by Sperling et al. (J Cutan Pathol 2010, 37:243-248), it is shown that K75 expression decreases sharply during the process of early desquamation of the inner root sheath of hair follicles.This desquamation being a histological marker of alopecia, it turns out that this K75 protein undoubtedly plays a key role in the mechanisms of alopecia.

[0024] The scientific name Silybum marianum (L.) Gaertn. refers to a plant belonging to the Asteraceae family, annual or biennial, with a robust stem, which can reach more than one meter in height. Its large, shiny, alternate leaves, without stipules, are marbled with white and edged with hard, pointed spines. The flowers are grouped in terminal heads, often solitary. They are surrounded by large, spiny bracts with very sharp tips. The tubular, five-lobed flowers are purplish-purple in color. The fruits are shiny achenes, black or marbled with yellow, topped with a pappus with ring-shaped, toothed bristles at their base. The common name of this plant is Milk Thistle.

[0025] The achene (often erroneously called seed in the literature) of Silybum marianum (L.) Gaertn. and its preparations are traditionally used orally in the symptomatic treatment of functional digestive disorders attributed to hepatic origin.

[0026] The main active ingredient of the achene of Silybum marianum (L.) Gaertn. is si-lymarin, a mixture of several flavonolignans (mainly silybin, isosilybin, silychristin, and silydianin). The achenes contain up to 3% by weight of si-lymarin. They also consist of oil (20-30% by weight), mucilages and proteins.

[0027] Silymarin has been the subject of numerous studies (in vitro, in vivo and clinical) which have demonstrated its antioxidant, hepatoprotective, digestive and anti-inflammatory properties.

[0028] Currently, extracts of Silybum marianum (L.) Gaertn. achenes titrated in silymarin are present in several pharmaceutical preparations intended for the treatment of various hepatic and biliary disorders, such as Legalon®.

[0029] Silybin (major flavonoid of silymarin) has also been the subject of a very recent study in hair growth (Cheon et al., J Microbiol Biotech 29(2), 321-329, 2019) via the Akt Wnt / [3-catenin] signaling pathway.

[0030] Silybum marianum (L.) Gaertn. oil, rich in omega 6 and vitamin E, is mainly used in cooking. It is conventionally obtained from the achenes by cold pressing. Studies on the antioxidant and hepatoprotective properties of such milk thistle oil, administered orally, have however been carried out in vivo on rats or mice (Zhu et al. Pharmacogn Mag, 10 (Suppl 1) S92-S99, 2014).

[0031] Application WO2018 / 002338 describes an extract of Silybum marianum (L.) Gaertn. achenes low in silymarin which has interesting properties for the treatment of acne, seborrhea, rosacea or seborrheic dermatitis.

[0032] Thus, to date no bibliographic data mentions that an extract of Silybum marianum (L.) Gaertn. achenes low in silymarin can have an activity in hair regrowth and therefore be useful in the prevention and / or treatment of alopecia. Summary of the invention

[0033] Unexpectedly and surprisingly, the applicants have demonstrated that an extract of Silybum marianum (L.) Gaertn. achenes low in silymarin exhibits pharmacological activities of interest in the field of treatment and / or prevention to combat hair loss, in particular by promoting hair growth.

[0034] Indeed, the inventors have demonstrated that an extract of Silybum marianum (L.) Gaertn. achenes low in silymarin induces an increase in the expression of the Keratin 75 protein in the follicles. Thus, the extract of Silybum marianum (L.) Gaertn. achenes, low in silymarin, will help delay and prevent hair loss and prolong the hair's life cycle.

[0035] According to a first aspect, the invention relates to an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for its use in promoting hair growth and in particular in treating or preventing alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0036] The invention also relates to the use of an extract of Silybum marianum (L.) Gaertn. achenes comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract to promote hair growth and in particular to treat or prevent alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0037] The invention also relates to the use of an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for the preparation of a cosmetic or dermatological composition intended to promote hair growth and in particular to treat or prevent alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0038] The invention also relates to a method for promoting hair growth and in particular for treating or preventing alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata, comprising administering to a person in need thereof an effective amount of an extract of Silybum marianum (L.) Gaertn. achenes comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract.

[0039] According to a second aspect, the present invention relates to a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract, with at least one dermatologically or cosmetically acceptable excipient for its use in promoting hair growth and in particular in treating or preventing alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0040] The invention also relates to the use of a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of si- lymarin relative to the weight of the dry extract, with at least one dermatologically or cosmetically acceptable excipient, to promote hair growth and in particular to treat or prevent alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0041] The invention also relates to the use of a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of si-lymarin relative to the weight of the dry extract, with at least one dermatologically or cosmetically acceptable excipient, for the preparation of a medicinal product intended to promote hair growth and in particular to treat or prevent alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata.

[0042] The invention also relates to a method for promoting hair growth and in particular for treating or preventing alopecia such as androgenetic alopecia, reactive alopecia, postmenopausal alopecia or alopecia areata, comprising administering to a person in need thereof an effective amount of a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract, with at least one dermatologically or cosmetically acceptable excipient. DETAILED DESCRIPTION OF THE INVENTION Definitions

[0043] In the present description, the plant Silybum marianum (L.) Gaertn. may be abbreviated to Silybum marianum.

[0044] For the purposes of the present invention, the term "silymarin" means a purified extract of achenes of Silybum marianum (L.) Gaertn. comprising predominantly (at least 95% by weight) a mixture of the following four flavonolignans: silybin, isosilybin, silychristin and silydianin (Kuki et al., Chromatographia 75:175-180, 2012). A silymarin content of less than 0.2% by weight therefore means that the total quantity of silymarin constituents is less than 0.2% by weight. Such a content can be determined in particular by HPLC (High Performance Liquid Chromatography) or UPLC (Ultra High Performance Liquid Chromatography) by calculating the total area of ​​the peaks corresponding to all the constituents of silymarin, in particular by using a reference silymarin sample, which can be obtained for example from Sigma Aldrich, to determine the position of these peaks.

[0045] By "silybin", also called silibinin in the art, is meant, within the meaning of the present invention, the four diastereoisomers silybin A, silybin B, 2,3-cis-silybin A and 2,3-cis-silybin B.

[0046] For the purposes of the present invention, the term “isosilybin” means the two diastereoisomers isosilybin A and isosilybin B.

[0047] For the purposes of the present invention, the term “silychristin” means the two diastereoisomers silychristin A and silychristin B.

[0048] By "approximately" is meant in the present description that the value concerned may be lower or higher by 10%, in particular by 5%, in particular by 2%, more particularly by 1%, than the indicated value.

[0049] For the purposes of the present invention, the term "dry extract" means an extract free of extraction solvent or carrier, or containing only non-significant traces thereof. Such a dry extract thus contains only material from Silybum marianum (L.) Gaertn. It may also contain non-significant traces of extraction solvent.

[0050] By "organic solvent immiscible with the oil derived from achenes of Silybum marianum (L.) Gaertn.", is meant, within the meaning of the present invention, an organic solvent which is not capable of mixing, or only partially, with the oil derived from achenes of Silybum marianum (L.) Gaertn., so that the mixture of the organic solvent and the oil derived from achenes of Silybum marianum (L.) Gaertn. gives a heterogeneous mixture in which at least two distinct phases can be observed.

[0051] For the purposes of the present invention, the term "fatty acid" means a carboxylic acid R1-CO2H whose chain RI is a linear or branched hydrocarbon chain, saturated or comprising C=C double bonds, the carboxylic acid comprising from 16 to 22 carbon atoms (including the carbon atom of the carboxylic acid function).

[0052] By “free” fatty acid (including linoleic acid) is meant, for the purposes of the present invention, a fatty acid not bound to other molecules (for example to glycerol or derivatives thereof to give glycerides or to an alcohol to give a fatty ester).

[0053] For the purposes of the present invention, the term "tocopherol" means α-tocopherol, β-tocopherol, γ-tocopherol and δ-tocopherol.

[0054] For the purposes of the present invention, the term "C1 to C3 alcohol" means an alcohol R2-OH whose chain R2 is a saturated, linear or branched hydrocarbon chain comprising 1 to 3 carbon atoms. It may be methanol, ethanol, n-propanol or isopropanol, in particular methanol, ethanol or isopropanol. Preferably, it will be ethanol or isopropanol, in particular ethanol.

[0055] For the purposes of the present invention, the term "room temperature" means a temperature of from 15 to 40°C, preferably from 20 to 30°C, in particular approximately 25°C.

[0056] By “hair and body hair” is meant hair, body hair, eyebrows, eyelashes and / or fur, preferably hair.

[0057] By "hair growth" is meant the growth of hair and / or body hair as defined above, preferably hair.

[0058] By "alopecia" is meant the total or partial loss of hair and / or body hair, for example linked to the reduction of hair growth and / or the acceleration of hair and / or body hair loss. This term includes but is not limited to androgenetic alopecia, postmenopausal alopecia, reactive alopecia, cicatricial alopecia, alopecia areata, and congenital alopecia. Preferably, it will be androgenetic alopecia, postmenopausal alopecia, reactive alopecia or alopecia areata. The consequences of alopecia are a temporary or permanent and partial or total absence of hair and / or body hair.

[0059] By the term "treating" alopecia is meant stopping alopecia, reducing alopecia and / or alleviating alopecia. Thus, "treating" alopecia includes limiting hair and / or body hair loss and / or promoting hair and / or body hair growth, increasing hair follicle density and / or regulating the phases of the hair follicle cycle.

[0060] The term "preventing" alopecia means reducing the risk of alopecia occurring, or slowing the progression of alopecia in a mammal, preferably humans, who are likely to develop alopecia.

[0061] By the term “limit” we mean to slow down, reduce, diminish and / or stop.

[0062] By the term “promote” is meant increase, augment, favor, amplify and / or accelerate.

[0063] In the present invention, the term "cosmetically or dermatologically acceptable" is intended to mean that which is useful in the preparation of a cosmetic or dermatological composition, which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for cosmetic or dermatological use, in particular by topical application to the hair and / or scalp.

[0064] By “topical application” is meant an application to the skin, in particular to the scalp, mucous membranes, hair and / or body hair, in particular to the hair and / or scalp. Extract according to the invention

[0065] The extract according to the invention is an extract of Silybum marianum achenes comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract.

[0066] According to a particular embodiment, the extract according to the invention contains at least 3% by weight, preferably at least 4% by weight of free linoleic acid relative to by weight of the dry extract. In particular, the extract according to the invention contains between 3% and 25% by weight, for example between 3 and 20% by weight, in particular 3% and 15% by weight, in particular between 4% and 10% by weight, in particular between 4% and 6% by weight, for example approximately 5% by weight of free linoleic acid relative to the weight of the dry extract. The silymarin / free linoleic acid mass ratio of the extract according to the invention may be in particular less than 0.07, in particular less than 0.05 and in particular less than 0.01.

[0067] The extract according to the invention may further comprise between 10% and 70%, in particular between 10% and 50%, more particularly between 10% and 30% by weight, in particular between 15% and 25% by weight of free fatty acids relative to the weight of the dry extract.

[0068] According to a particular embodiment, the extract according to the invention contains at least 0.01% by weight, in particular at least 0.05% by weight of tocopherols relative to the weight of the dry extract. In particular, the extract according to the invention contains between 0.01% and 2% by weight, more particularly between 0.01% and 1% by weight, even more particularly between 0.01% and 0.5% by weight, in particular between 0.05% and 0.2% by weight, for example approximately 0.1% by weight of tocopherols relative to the weight of the dry extract. The silymarin / tocopherols mass ratio of the extract according to the invention may in particular be less than 1, in particular less than 0.1.

[0069] According to another particular embodiment, the extract according to the invention contains between 3% and 25% by weight, for example between 3% and 20% by weight, in particular between 3% and 15% by weight, in particular between 4% and 10% by weight, in particular between 4% and 6% by weight, for example approximately 5% by weight of free linoleic acid relative to the weight of the dry extract; and between 0.01% and 2% by weight, in particular between 0.01% and 1% by weight, even more particularly between 0.01% and 0.5% by weight, in particular between 0.05% and 0.2% by weight, for example approximately 0.1% by weight of tocopherols relative to the weight of the dry extract. In particular, the silymarin / free linoleic acid mass ratio of the extract according to the invention may be in particular less than 0.07, in particular less than 0.01 and the silymarin / tocopherols mass ratio of the extract according to the invention may be in particular less than 1, in particular less than 0.1.

[0070] Preferably, the extract according to the present invention will be a dry extract.

[0071] According to a preferred embodiment, the extract according to the invention can be obtained by a process according to the invention described below.

[0072] Process for preparing the extract according to the invention

[0073] A process for preparing an extract according to the invention comprises in particular a step of extracting an oil from achenes of Silybum marianum (L.) Gaertn. by an extraction solvent comprising, in particular consisting of, a hydrotropic aqueous solution, subcritical water or an organic solvent immiscible with the oil from of achenes of Silybum marianum (L.) Gaertn. optionally mixed with water.

[0074] According to a particular embodiment, the extraction solvent comprises, in particular is constituted by, an organic solvent immiscible with the oil from achenes of Silybum marianum (L.) Gaertn. optionally mixed with water.

[0075] The organic solvent immiscible with the oil from achenes of Silybum marianum (L.) Gaertn. may in particular be a C1 to C3 alcohol.

[0076] The extraction solvent may in particular be a C1 to C3 alcohol, possibly mixed with water.

[0077] The organic solvent immiscible with the oil from achenes of Silybum marianum (L.) Gaertn., in particular a C1 to C3 alcohol such as methanol, ethanol or isopropanol, in particular ethanol or isopropanol, preferably ethanol, may be used in a mixture with water, in particular in an organic solvent / water volume ratio of 70 / 30 to 100 / 0, in particular 80 / 20 to 100 / 0, for example approximately 80 / 20 or 90 / 10.

[0078] The extraction solvent may in particular be chosen from methanol, a methanol / water mixture, ethanol, an ethanol / water mixture, isopropanol and an isopropanol / water mixture, in particular an ethanol / water mixture or an isopropanol / water mixture, preferably an ethanol / water mixture.

[0079] According to a particular embodiment, the extraction solvent will be methanol, an ethanol / water mixture in a volume ratio of approximately 80 / 20 or 90 / 10 or an isopropanol / water mixture in a volume ratio of approximately 90 / 10.

[0080] The step of extracting the oil from Silybum marianum achenes will be carried out in particular by mixing the oil from Silybum marianum achenes with the extraction solvent for 1 to 12 hours and in particular at a temperature of between 15 and 25°C, in particular around 20°C. The quantity of extraction solvent used to carry out this extraction will advantageously be from 0.5 to 3g, in particular from 1 to 3g per 1g of oil from Silybum marianum (L.) Gaertn. achenes.

[0081] An extraction phase and a lipid phase will then be obtained at the end of this extraction. The extraction phase will advantageously be separated from the lipid phase and recovered before being dried (in particular by evaporation of the extraction solvent), partially or totally, in particular under vacuum, to eliminate more or less the extraction solvent and obtain either the dry extract if the solvent is totally eliminated, or the concentrated extract which is diluted in residual solvent.

[0082] The oil from Silybum marianum achenes may advantageously be obtained by extraction from Silybum marianum (L.) Gaertn. achenes (the achenes may be whole or in pieces), in particular by pressure, advantageously by cold pressing (i.e. without heating), at room temperature).

[0083] According to one embodiment according to the invention, the method according to the invention will include the following two successive stages:

[0084] (i) Extraction of an oil from achenes of Silybum marianum (L.) Gaertn., and

[0085] (ii) Extraction of the oil from achenes of Silybum marianum (L.) Gaertn. with an extraction solvent, comprising, in particular consisting of, a hydrotropic aqueous solution, subcritical water or an organic solvent immiscible with the oil from achenes of Silybum marianum (L.) Gaertn. optionally mixed with water.

[0086] According to a preferred embodiment according to the invention, the method according to the invention will comprise the following successive steps:

[0087] (i) Optionally extraction of an oil from achenes of Silybum marianum (L.) Gaertn.,

[0088] (ii) Extraction of the oil from achenes of Silybum marianum (L.) Gaertn with an extraction solvent, comprising, in particular consisting of a hydrotropic aqueous solution, subcritical water or an organic solvent immiscible with the oil from achenes of Silybum marianum (L.) Gaertn. optionally mixed with water,

[0089] (iii) Recovery of the extraction phase obtained in step (ii), and

[0090] (iv) Partial or total drying of the extraction phase to give a concentrated extract or dry according to the invention.

[0091] Step (i) will advantageously be carried out by cold pressing the achenes of Silybum marianum (L.) Gaertn., whole or in pieces.

[0092] Step (ii) will advantageously be carried out with an extraction solvent as defined above, and in particular chosen from methanol, a methanol / water mixture, ethanol, an ethanol / water mixture, isopropanol, and an isopropanol / water mixture.

[0093] The extraction solvent may in particular be an organic solvent immiscible with the oil obtained from achenes of Silybum marianum (L.) Gaertn, in particular a C1 to C3 alcohol such as methanol, ethanol or isopropanol, optionally mixed with water, in particular in an organic solvent / water volume ratio of between 80 / 20 and 100 / 0, in particular between 85 / 15 and 95 / 5, in particular approximately 90 / 10. An advantageous extraction solvent is an isopropanol / water mixture in a volume ratio of approximately 90 / 10.

[0094] The extraction step (ii) may be carried out by mixing the oil from Silybum marianum achenes with the extraction solvent for 1 to 12 hours and in particular at a temperature of 15 to 25°C, in particular approximately 20°C. The quantity of extraction solvent used to carry out this extraction will advantageously be 0.5 to 3g, in particular 1 to 3g per 1g of oil from Silybum marianum (L.) Gaertn. achenes.

[0095] This extraction step (ii) makes it possible to obtain at the end an extraction phase of interest and a lipid phase.

[0096] Step (iii) will advantageously be carried out by separating the extraction phase from the lipid phase.

[0097] Step (iv) will advantageously be carried out under vacuum. Cosmetic and dermatological applications

[0098] According to a first aspect, the present invention relates to an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention for its use in promoting hair growth.

[0099] The invention also relates to an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention for its use in the treatment or prevention of alopecia.

[0100] The extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention can be used as an accompaniment to a hair transplant, in particular during micro-transplantation of follicular units. A follicular unit represents a group of hairs, assembled together naturally in the scalp in small bundles, which can contain from one to four hairs. The use of an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention is perfectly suitable in particular as an accompaniment to a laser hair transplant.

[0101] The extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention can also be used as an accompaniment to a treatment based on platelet-rich plasma (PRP). It is a concentrate of platelets, and therefore rich in growth factors, which is injected into the scalp.

[0102] According to a second aspect, the present invention relates to a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention, with at least one dermatologically or cosmetically acceptable excipient for its use in promoting hair growth.

[0103] The invention also relates to a dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention, with at least one dermatologically or cosmetically acceptable excipient for its use in the treatment of alopecia.

[0104] The dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention, with at least one dermatologically or cosmetically acceptable excipient can be used as an accompaniment to a hair transplant, in particular a laser hair transplant.

[0105] The dermatological or cosmetic composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract according to the invention, with at least one dermatologically or cosmetically acceptable excipient can also be used in conjunction with a treatment based on platelet-rich plasma (PRP).

[0106] Advantageously, the extract included in the dermatological or cosmetic composition is as described previously.

[0107] The invention preferably relates to an extract and a cosmetic or dermatological composition according to the invention presented in a form which is specific and suitable for topical application, in particular to the scalp and / or hair.

[0108] The cosmetic or dermatological composition according to the invention can thus be presented in the forms which are usually known for topical administration, that is to say in particular lotions, shampoos, balms, mousses, gels, dispersions, emulsions, sprays, serums, masks or creams, with excipients allowing in particular penetration in order to improve the properties and accessibility of the active ingredient.

[0109] Advantageously, the composition according to the invention may be presented in the forms which are usually known for topical administration to the hair and scalp, that is to say in particular a shampoo, a conditioner, a hair cream, a hair lotion, a mask or a spray, in particular without rinsing.

[0110] We thus distinguish between formulated products that can be rinsed and formulated products that do not require rinsing.

[0111] Preferably, the composition according to the invention has a light texture which also allows optimal penetration without greasing the hair and / or body hair, or the scalp.

[0112] These compositions generally contain, in addition to the extract according to the present invention, a physiologically acceptable medium, generally based on water or solvent, for example alcohols, ethers or glycols. They may also contain surfactants, complexing agents, preservatives, stabilizing agents, emulsifiers, thickeners, gelling agents, humectants, emollients, trace elements, essential oils, perfumes, colorants, moisturizing agents, etc.

[0113] According to a particular embodiment, the composition according to the invention comprises, as a cosmetically or dermatologically acceptable excipient, isopropanol, polyethylene glycol (PEG) or a mixture thereof. Thus, advantageously, the composition according to the present invention will comprise, in particular will consist of, an extract according to the invention, isopropanol and PEG. The isopropanol / PEG mass ratio will advantageously be between 1 / 2 and 2 / 1, in particular between 1 / 1.5 and 1.5 / 1, in particular will be approximately 1 / 1.

[0114] The PEG may in particular have a number-average molecular mass of between 200 and 600 g / mol, in particular of between 200 and 500 g / mol, in particular of between 200 and 400 g / mol, for example of between 250 and 350 g / mol, in particular of approximately 300 g / mol. It may thus in particular be PEG 300.

[0115] In another particular embodiment of the invention, the composition according to the invention is characterized in that it is presented in a form suitable for oral administration.

[0116] The composition according to the invention may then be presented in the forms which are usually known for oral administration, that is to say in particular tablets, capsules, powders, granules and oral solutions or suspensions. When preparing a solid composition in the form of tablets, the main active ingredient may be mixed with a cosmetic or dermatological vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic, silica or the like. The tablets may be coated with sucrose or other suitable materials or they may be treated in such a way that they have a prolonged or delayed activity and that they continuously release a predetermined quantity of active ingredient. A preparation in capsules may be obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard capsules.

[0117] Advantageously, the composition according to the present invention comprises 0.01 to 15% by weight, preferably 0.1 to 10% by weight, of an extract according to the invention relative to the total volume of the composition. EXAMPLES

[0118] Example 1: preparation of extracts

[0119] Process according to the invention giving an isopropanolic extract 90 (extract I) comprising a low content of silymarin.

[0120] - Cold pressing of Silybum marianum achenes to obtain an oil from achenes of Silybum marianum,

[0121] - Extraction of oil from Silybum marianum achenes by an iso- mixture propanol / water (90 / 10 v / v) with 1 gram of isopropanol / water mixture per gram of oil for 2 hours at 20°C,

[0122] - Recovery of the isopropanolic phase, and

[0123] - Evaporation of the solvent.

[0124] Methanolic extracts (extract M) and ethanolic extracts (extract E) were obtained in a similar manner by replacing the isopropanol / water mixture (90 / 10 v / v) with methanol and an ethanol / water mixture (90 / 10 v / v) respectively. The extraction of the oil from Silybum marianum achenes is carried out respectively with 3 volumes of methanol and 3 volumes of the ethanol / water mixture (90 / 10 v / v) for 1 volume of oil for 2 hours at 20°C.

[0125] These different extracts were characterized by UPLC (ultra high performance liquid chromatography) or by GC-MC (gas chromatography coupled with mass spectrometry) according to the protocols detailed below. Protocol for evaluating the extracts obtained

[0126] Protocol 1: Evaluation of silymarin content by UPLC

[0127] Preparation of the sample and the control:

[0128] - Silymarin control: prepare a solution of silymarin at 5 mg in 10 ml of a methanol / water mixture (60:40) (v / v).

[0129] - Sample: heat the dry extract to be analyzed (extract M, E or I) to 35°C under stirring until a homogeneous and clear solution is obtained. Weigh exactly 200 mg of the extract, solubilize it in 10 ml of a methanol / dichloromethane mixture allowing the total solubilization of the extract and homogenize the solution. This mixture goes from the methanol / dichloromethane ratio (1:1) v / v) to pure methanol.

[0130] Analytical conditions:

[0131] Column: Acquity BEH Shield Cl8 150mm x 2.1 mm - l.7pm (Waters)

[0132] Mobile phase:

[0133] - A: water + 0.1% formic acid

[0134] - B: Acetonitrile + 0.1% formic acid

[0135] Gradient according to Table 1 below:

[0136] [Tables 1] T (min) A (%) B (%) 0 90 10 15 60 40 20 0 100 39.5 0 100 40 90 10 45 90 10

[0137] Column temperature: 40°C

[0138] Flow rate: 0.4 ml / min

[0139] Detection: 287 nm

[0140] Injection volume: 1 μL.

[0141] Protocol 2: Evaluation of linoleic acid content by UPLC

[0142] Preparation of the sample and the control:

[0143] - Linoleic acid control: prepare a 10 mg linoleic acid solution in 10 ml of a methanol / dichloromethane mixture (1:1) (v / v).

[0144] - Sample: heat the dry extract to be analyzed (extract M, E or I) to 35°C under Stir until a homogeneous and clear solution is obtained. Weigh exactly 50 mg of the extract, dissolve it in 1 ml of a methanol / dichloromethane mixture allowing the total solubilization of the extract and homogenize the solution. This mixture goes from the methanol / dichloromethane ratio (1:1) (v / v) to pure methanol.

[0145] Analytical conditions:

[0146] Column: Acquity BEH Shield C18 150mm x 2.1 mm - l.7pm (Waters)

[0147] Mobile phase:

[0148] - A: water + 0.1% formic acid

[0149] - B: Acetonitrile + 0.1% formic acid

[0150] Gradient according to Table 2 below:

[0151] [Tables2] T (min) A (%) B (%) 0 50 50 1 50 50 10 0 100 15 0 100 15.5 50 50 20 50 50

[0152] Column temperature: 40°C

[0153] Flow rate: 0.4 ml / min

[0154] Detection: 215 nm

[0155] Injection volume: 1 μL.

[0156] Protocol 3: Evaluation of fatty acid content by GC-MS

[0157] Sample preparation:

[0158] Heat the dry extract to be analyzed to 35°C while stirring until a clear, homogeneous liquid is obtained.

[0159] Solubilize 20 mg of the extract in 800 μl of a methanol / dichloromethane mixture (1:1) (v / v).

[0160] Add 200 μl of the derivatizer N,O-Bis(trismethylsilyl)trifluoroacetamide (BSTFA) + Trimethylchlorosilane (TMCS) (99:1) (Supelco-Sigma Alrich).

[0161] Vortex for 1 minute.

[0162] Gas chromatography (GC) conditions:

[0163] Column: DB-5ms (Agilent Technologies); 30m x 0.25mm; 0.25pm

[0164] Injection: T = 300°C; Mode = Split; Split ratio = 100:1

[0165] Oven: temperature gradient (°C)

[0166] - Initial temperature = 150°C

[0167] - Gradient = 7°C / min until final temperature = 340°C

[0168] - Keep at 340°C for 10 minutes

[0169] Carrier gas flow rate: Iml / min

[0170] Detection: MS-EI, T=300°C, Scan thne=0.2 sec; Full Scan Start Mass = 40; Full Scan End Mass = 600.

[0171] Injection volume: IpL Results

[0172] The extract I of Silybum marianum achenes according to the invention, low in silymarin, mainly contains substances having a retention time of between 13 and 30 minutes by UPLC.

[0173] The silymarin content of extracts I was determined by UPLC after calibration with control solutions of commercial silymarin (Sigma Aldrich).

[0174] Extract I contains 0.06% by mass of silymarin.

[0175] The contents of free fatty acids and more particularly linoleic acid in extract I were determined by UPLC and by CPG and are presented in Table 3 below.

[0176] [Tables3] Components % by weight Free fatty acids (mainly palmitic, oleic and linoleic acids) 24.6 of which linoleic acid 5.1

[0177] The inventors were able to demonstrate that the UPLC and CPG profiles of the 3 achene extracts according to the invention obtained by methanolic extraction (extract M), ethanolic 90 (extract E) and isopropanolic 90 (extract I) are similar.

[0178] The various analyses carried out by UPLC and GC-MS made it possible to highlight the following characteristics:

[0179] - The isopropanolic extract 90 of achenes according to the invention (extract I) does not contain almost no silymarin, especially polar flavonolignans.

[0180] - The UPLC and CPG profiles of the 3 achene extracts according to the invention obtained for example methanolic traction, ethanolic 90 and isopropanolic 90 are very close.

[0181] Example 2: Quantification of Keratin 75 protein in forehead follicles of human subjects

[0182] The purpose of this example is to evaluate the effects of an extract of Silybum marianum (L.) Gaertn. achenes comprising less than 0.2% by weight of silymarin on the expression of the Keratin 75 protein in humans. Indeed, such pharmacological activity is of interest in the field of treatment and / or prevention of hair loss, in particular by promoting their growth. Methods

[0183] A topical preparation of extract I prepared in Example 1 was formulated at 1.75% or 7.0% (w / v) of the dry extract, in an isopropanol / PEG 300 (1:1) (w / w) mixture, and then put into a spray for application twice daily for the duration of the treatment.

[0184] 13 healthy volunteers were followed for 4 to 12 weeks depending on their availability. This study was conducted in accordance with the Declaration of Helsinki, with written consent from each subject. This was a single-center, longitudinal, observational study. Subjects were followed in a private practice with close monitoring by the same staff throughout the study, supplemented by ongoing digital contact and exchange with the subjects. Weekly monitoring using digital facilities was installed. Follicle sampling was performed at each visit.

[0185] Cyanoacrylate follicle samples were taken from the forehead of the subjects according to Piérard et Coll (Scientific World Journal, 2014 ID 463634, 2014) using a drop of ethyl-2-cyanoacrylate adhering to a microscope glass slide.

[0186] Protein extraction:

[0187] The biopsies are detached from the glass slide in distilled water for 30 minutes at room temperature, are cut into small pieces and then homogenized in an extraction buffer of 2 mM Tris-HCl, pH 6.8, 3% SDS, 10 mM sodium pyrophosphate, 5 mM EDTA, and 2 mM sodium vanadate. The samples are heated at 50°C for 3 hours. The extracts are centrifuged at 10,000 rpm for 10 minutes. The determination of proteins in the supernatants is carried out by the method according to Bradford (Anal Biochem 72:248-254, 1976) using the Bio-Rad reagent (Cressier, Switzerland). The sebocyte cultures are washed with a PBS buffer and then the cells are lysed in a specific buffer (50 mM Tris-HCl pH 7.4, NaCl 150mM, 1mM EDTA, 1% Triton™ X-100). The suspensions were sonicated and then analyzed by the Bradford method.

[0188] Protein samples are analyzed by Western Blot using a standard procedure and the following antibody: rabbit anti-human polyclonal IgG K75, 1:5000 dilution (Fisher Scientific, Reinach, Switzerland).

[0189] Proteomic analysis was performed by electrophoresis (SDS-PAGE), followed by Coomassie staining. The stained protein bands were collected and analyzed on the proteomic platform of the University of Geneva. Results

[0190] The level of expression of the K75 protein (densitometric unit of the western blot bands) in the hair follicles of the forehead of human subjects is represented in Table 4 below before and after treatment with an extract according to the invention.

[0191] [Tables4] Subject Before treatment After treatment Treatment duration (weeks) Extract concentration (%) Subject 1 55.50 89.77 6 1.75 Subject 2 110.20 132.50 4 7.0 Subject 3 195.26 272.12 4 7.0 Subject 4 124.56 126.62 4 7.0 Subject 5 83.01 127.12 6 1.75 Subject 6 66.04 83.89 7 1.75 Subject 7 59.14 73.25 6 7.0 Subject 8 97.11 114.00 8 1.75 Subject 9 66.60 136.03 8 7.0 Subject 10 38.25 92.34 12 7.0 Subject 11 40.38 59.03 8 1.75 Subject 12 28.68 90.09 4 1.75 Subject 13 34.63 45.53 8 7.0 Mean 76.87 110.95 Standard deviation 46.3 56.2 Test TP=0.0003

[0192] The inventors clearly demonstrate that treatment with an extract according to the invention induces a statistically significant increase (p<0.001) in the expression of the K75 protein. It is interesting to note that this increase is observed in all patients regardless of the concentration tested or the duration of treatment carried out. Indeed, from 4 weeks of treatment, the increase in the expression of the K75 protein already seems very marked.

[0193] Example 3: quantification of Keratin 75 proteins on the scalp of a patient with alopecia areata

[0194] In this severe hair disorder, it is observed that the expression of K75 is extremely low. The patient is treated with the same extract at a concentration of 7%, and in the same way as in Example 2. The results obtained, namely the level of expression of the K75 protein (densitometric unit of the Western blot bands) in the hair follicles of the patient suffering from alopecia areata, before and after treatment with an extract according to the invention, are presented in Table 5 below.

[0195] [Tables5] Subject Duration of treatment (weeks) Before treatment After treatment Subject 1 6 3.1 23.1

[0196] The inventors thus demonstrate that a treatment comprising an extract according to the invention makes it possible to significantly increase the level of expression of K75 in a patient suffering from alopecia areata. It is interesting to note that at the end of the treatment, hair regrowth could be observed.

[0197] All of these results allow the inventors to demonstrate the interest of an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2% by weight of silymarin in promoting hair regrowth.

Claims

Claims

1. Extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for its use in promoting hair growth.

2. Extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for its use according to claim 1, characterized in that said extract is presented in a form suitable for topical application.

3. Extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for its use according to any one of claims 1 and 2, characterized in that said extract makes it possible to prevent or treat alopecia.

4. Extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract for its use according to claim 3, characterized in that the alopecia is chosen from the group consisting of androgenetic alopecia, reactive alopecia, postmenopausal alopecia and alopecia areata.

5. Dermatological composition comprising at least one extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract, with at least one dermatologically acceptable excipient for its use in promoting hair growth.

6. Dermatological composition for use according to claim 5, characterized in that it is in a form suitable for topical application.

7. Dermatological composition for use according to any one of claims 5 and 6, characterized in that the composition comprises 0.01 to 15% by weight of an extract of achenes of Silybum marianum (L.) Gaertn. comprising less than 0.2%, preferably less than 0.1% by weight of silymarin relative to the weight of the dry extract, relative to the total weight of the composition.

8. Dermatological composition for use according to any one of claims 5 to 7, characterized in that the com- position helps prevent or treat alopecia.

9. Dermatological composition for use according to claim 8, characterized in that the alopecia is chosen from the group consisting of androgenetic alopecia, reactive alopecia, postmenopausal alopecia and alopecia areata.