Verbena officinalis extract for the prevention and / or treatment of hair loss

Abstract

The present invention relates to an extract of Verbena officinalis and compositions containing this extract of Verbena officinalis for application in the fields of cosmetics and dermatology in the hair field, more particularly for the prevention and / or treatment of alopecia or promoting hair growth.

Description

Title of the invention: Extract of Verbena officinalis for the prevention and / or treatment of hair loss TECHNICAL FIELD OF THE INVENTION

[0001] The present invention relates to an extract of Verbena officinalis and compositions containing this extract of Verbena officinalis for application in the fields of cosmetics and dermatology in the hair field, more particularly for the prevention and / or treatment of alopecia or promoting 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 upon the spirit of research. Many theories have attempted to clarify the etiology of hair loss in cases of baldness, alopecia, alopecia areata, etc., by attributing them to seborrhea, 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. Their distribution is established during growth in utero 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: - A growth phase (anagen): the cells of the dermal papilla (fibroblasts) send a signal to the stem cells of the follicle which allows their proliferation. Cells will transform and envelop the dermal papilla to form the hair matrix. 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, 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. - A regression phase (catagen): the matrix dies and as a result the dermal papilla is no longer in contact with this matrix. There is no longer any exchange between the cells. The follicle and the dermal papilla rise towards the epidermis. - A resting phase (telogen): the cells of the dermal papilla and the follicle are intact and inactive. The hair falls out. For a new one to grow hair grows, the cycle must be restarted.

[0004] The term alopecia refers to partial or general hair loss. Many factors can be involved in alopecia, such as genetics, age, gender, illness, stress, hormonal problems, side effects of medication, and scarring. It is possible to distinguish several forms of alopecia:

[0005] - Hereditary androgenetic alopecia: it is the most common. Early hair loss hair loss occurs in genetically predisposed individuals and particularly affects men. It manifests itself by a progressive and localized reduction in hair volume, or even baldness, and affects 50% of men over the age of 50;

[0006] - Postmenopausal alopecia: this is the most common cause of baldness in women. In women, hair loss is more diffuse and widespread 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;

[0007] - Acute or reactive alopecia or telogen effluvium: it can be linked to a chemotherapy treatment, stress, childbirth, significant nutritional deficiencies, iron deficiency, hormonal disorders, it is a simultaneous and diffuse loss of a significant amount of hair;

[0008] - Scarring alopecia: it can be caused by skin problems (tumor, burn, alopecia), acute irradiation, lupus erythematosus or parasites (ringworm, lichen);

[0009] - Alopecia areata: it appears to be of autoimmune origin and is characterized by a affected in more or less wide patches and in one or more places;

[0010] - Congenital alopecia: rare, it corresponds to an absence of roots or to hair abnormalities (mutations).

[0011] Alopecia, in particular androgenetic 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 cessation of 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.

[0012] To date, various products have been proposed to combat alopecia, allowing in particular to induce or stimulate hair growth. Most combine several active ingredients likely to provide a beneficial action 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 piroctonolamine, undecycline derivatives, cyclopiroxolamine; 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 now the standard treatment for androgenic alopecia.

[0013] Furthermore, compositions comprising very diverse active agents are proposed for hair regrowth, these active agents being able, for example, to be 2,4-diamino pyrimidine 3-oxide derivatives such as those described in patent application EP0522964.

[0014] 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.

[0015] Verbena officinalis L. (Verbenaceae) is a perennial herb native to Europe commonly known as common vervain. It is widely naturalized outside its native range, for example in North America.

[0016] Verbena officinalis is a perennial plant with a fusiform, yellowish root. It is an erect or ascending plant, always more or less perennial. The stems are slightly hairy, quadrangular, canaliculate alternately on two opposite sides, branched then erect. The leaves are opposite, short-petiolate or even sessile, obovate, lanceolate, with unequal, deeply incised or crenate lobes. The bluish or lily-colored flowers are small, sessile and arranged in long, loose, filiform spikes, arranged in a terminal panicle. Each flower has at the base a green tongue or bract less than 3 mm long (shorter than the calyx). The funnel-shaped corolla has a protruding tube, with an almost flat limb and 5 almost equal lobes. The fruit is a brown dehiscent capsule consisting of four achenes which separate when ripe.It is a common plant found everywhere in embankments, rubble, at the edge of roads, especially in villages, on old walls, in uncultivated places, except in very high mountains. Today it is widespread in all temperate regions of the world.

[0017] Verbena officinalis has a rich phytochemical composition. The main groups of secondary metabolites determining its biological activities are mainly glycosylated iridoids, including verbenalin (approx. 6% in the dry plant), aucubin and hastatoside (approx. 0.6% dry plant), as well as glycosylated phenylpropanoids and caffeic acid derivatives: verbascoside (approx. 3% dry plant) and isoverbascoside (approx. 0.3% dry plant) and eukovoside. In addition, numerous flavonoids have been identified in verbena extracts, including common compounds in the plant kingdom such as kaempferol, luteolin, as well as specific flavonoids such as scutellarein and pedalitin. Also noteworthy is the presence of phenolic acids: chlorogenic, ferulic, protocatechuic, rosmarinic, and derivatives of dicaffeoylquinic acid.Finally, the presence of terpenic compounds such as citral, limonene, cineole, carvone, diterpenic compounds such as carmosol, carnosic acid, rosmanol, sesquiterpenic compounds such as caryophyllene oxide, a-curcumin and triterpenic compounds with ursolic acid and its derivatives is described in Verbena officinalis. (Kubica P et al., Planta Med 2020; 86: 1241-1257).

[0018] Many pharmacological studies conducted more recently in vitro and in vivo have sought to establish new important applications for extracts of Verbena officinalis, by demonstrating antioxidant, antimicrobial, anti-inflammatory, anticancer, neuroprotective, analgesic or anticonvulsant activities. (Kubica P et al., Planta Med 2020; 86: 1241-1257)

[0019] However, to date no bibliographic data mentions that an extract of Verbena officinalis may have an activity in the prevention and / or treatment of alopecia and in the promotion of hair regrowth. Summary of the invention

[0020] The present invention relates to an extract of Verbena officinalis for use in the prevention and / or treatment of alopecia as well as a cosmetic or dermatological composition comprising at least one extract of Verbena officinalis and at least one dermatologically or cosmetically acceptable excipient for use in the prevention and / or treatment of alopecia.

[0021] Other aspects of the invention are as described in the claims and hereinafter. DETAILED DESCRIPTION OF THE INVENTION

[0022] Surprisingly, the inventors discovered that an extract of Verbena officinalis is capable of inhibiting the apoptosis of cells of the outer epithelial sheath. This extract also exhibits pharmacological activities of interest for stimulating the growth of blood vessels, by acting in particular on the VEGF target. In addition, this extract of Verbena officinalis also exhibits pharmacological activities to promote hair growth, particularly by acting on the JAK7 STAT pathway.

[0023] The invention therefore relates to the prevention and / or treatment of hair loss insofar as the extract of Verbena officinalis acts on the biological functions or mechanisms at the origin of hair growth. Definitions

[0024] In the present invention, the plant Verbena officinalis L. may be abbreviated to Verbena officinalis. By "verbena extract" is meant the product of extraction of all or part of the plant Verbena officinalis.

[0025] By "aerial parts of verbena" is meant all the aerial parts of the plant, namely the stem, leaves, flowers, fruits and seeds.

[0026] By "extraction product" is meant the product obtained after extraction of all or part of the plant, with a solvent, called extraction solvent, that is to say a product present in the extraction solvent which may then optionally be in a concentrated or dry form after partial or total evaporation of the extraction solvent. It may be a dry extract.

[0027] For the purposes of this document, 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 the extraction of Verbena officinalis.

[0028] By "approximately" is meant in the present invention that the value concerned may be 10% lower or higher, in particular 5%, in particular 1%, compared to the indicated value. This definition applies in particular when defining the content by weight of iridoids (verbenaline for example), compared to the weight of the dry extract.

[0029] By "hydrophilic solvent" is meant a solvent chosen from the group consisting in particular of water, subcritical water, water-miscible alcohols such as for example ethanol, C3 to C5 glycols, glycerol, acetone, and mixtures thereof.

[0030] For the purposes of the present invention, the term "moderately polar solvent" means a solvent chosen from the group consisting in particular of C1 to C5 alcohols, glycols such as propylene glycol, butylene glycol, butanediol, or pentylene glycol, glycerol, acetone, alkyl esters such as ethyl acetate, isopropyl acetate, water-miscible solvents (a hydro-alcoholic mixture or an acetone / water mixture for example), a supercritical CO2 and ethanol mixture. This group also includes alternative solvents of the hydrotropic type (amphiphilic molecules soluble in water and which, from a sufficient concentration, can extract moderately polar compounds as described in the characterization of the extract) or ionic liquid type such as Natural Deep Eutectic Solvents (NaDES) formed from a mixture in well-defined ratios of hydrogen bond donor compounds and hydrogen bond acceptor compounds.

[0031] By "apolar solvent", it is meant, within the meaning of the present invention, a solvent chosen from heptane, hexane, 2-Methyltetrahydrofuran (MTHF), limonene, halogenated hydrocarbons (chloroform, dichloromethane), supercritical CO2.

[0032] By “hair and body hair” is meant hair, body hair, eyebrows, eyelashes and / or fur, preferably hair.

[0033] By “phanère” is meant hair, body hair, eyebrows, eyelashes, and / or nails, preferably hair.

[0034] 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. The consequences of alopecia are a temporary or permanent and partial or total absence of hair and / or body hair.

[0035] By the term "treating" alopecia is meant stopping alopecia, reducing alopecia and / or alleviating alopecia. Thus, "treating" alopecia includes limiting hair loss and / or promoting hair growth, increasing hair follicle density and / or regulating the phases of the hair follicle cycle.

[0036] By the term "preventing" alopecia, we mean reducing the risk of the onset of alopecia, or slowing the progression of alopecia in a mammal, preferably humans, who are likely to develop alopecia.

[0037] By the term “limit” we mean to slow down, reduce, diminish and / or stop.

[0038] By the term “promote” is meant increase, augment, favor, amplify and / or accelerate.

[0039] In the present invention, the term "cosmetically or dermatologically acceptable" is intended to mean that which is useful in the preparation of a cosmetic, 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.

[0040] By “topical application” is meant an application to the skin, in particular to the scalp, the mucous membranes, and / or the appendages, in particular to the hair and / or the scalp. DESCRIPTION OF THE METHODS OF IMPLEMENTATION Verbena extract

[0041] The invention relates to a verbena extract for use in the prevention and / or treatment of alopecia.

[0042] In the context of the present invention, the verbena extract is obtained from one or more parts of the Verbena officinalis plant chosen from the aerial parts such as the stem, the leaves, the flowers and the fruit.

[0043] In a particular embodiment of the invention, the extract is obtained from a culture of Verbena officinalis cells.

[0044] According to a particular embodiment of the invention, the extract is a hydroalcoholic extract, in particular a hydro-ethanolic extract.

[0045] According to a preferred embodiment, the extract according to the invention can be obtained by a process as described below.

[0046] The Verbena officinalis plant or part of the plant can be fresh or dried, whole, cut or crushed and then subjected to an extraction step.

[0047] A process for preparing an extract according to the invention comprises a step of extracting all or part of the Verbena officinalis plant using a hydrophilic to apolar solvent, preferably a moderately polar solvent.

[0048] In one embodiment of the invention, the extraction solvent may be chosen from ethyl acetate, isopropyl acetate, a supercritical CO2 and ethanol mixture, a C3 to C5 glycol, a C1 to C5 alcohol, an alcohol / water mixture, a hydrotropic solution, a NaDES or a mixture thereof.

[0049] Advantageously, it will be an ethanol / water mixture or a hydrotropic solution.

[0050] More advantageously, it will be an ethanol / water mixture.

[0051] Even more advantageously, this ethanol / water mixture will be characterized by an ethanol / water proportion of 9:1 to 1:9 (v / v).

[0052] And even more advantageously, the moderately polar solvent is an ethanol / water mixture in the proportion 7:3 (v / v).

[0053] The extraction can be carried out with stirring, in a dynamic recirculation system, or statically, at reflux, at room temperature, or at a temperature between room temperature and reflux. It can be assisted by ultrasound, microwaves, flash detent or extrusion, in a plant weight / solvent volume ratio that can vary from 1 / 3 to 1 / 30, for a period of 1 minute to 48 hours. The extraction can be repeated 2 to 3 times. Advantageously, the extraction of Verbena officinalis is assisted by ultrasound, with a plant weight / solvent volume ratio of between 1 / 5 and 1 / 30 for 5 to 120 minutes.

[0054] The marc can then be separated from the extract by centrifugation or filtration in order to recover a clear liquid phase free of particles. The liquid phase representing the extract can then be more or less concentrated by evaporation of the extraction solvent, up to the point of obtaining a dry extract.

[0055] A support may be added during the concentration step so as to obtain an extract containing 1 to 75% by weight of dry extract. The support may be maltodextrin, lactose, silica, acacia or arabic gum, glycerin, a glycol, a vegetable oil, a hydrotrope, a water / solubilizer or water / surfactant mixture, or any other cosmetically acceptable support which solubilizes the extract, preferably of biosourced origin such as, for example, biosourced glycols (1,2-pentanediol; 1,3-butanediol; 1,3-propanediol, etc.), esterified fatty acids and also hydrotropes such as, for example, alkyl glycosides (Sepiclear, Apyclean, APXC4, etc.). Advantageously, the Verbena officinalis extract is standardized on maltodextrin support or on a glycerin / water mixture.

[0056] The Verbena officinalis extract may be decolorized to remove chlorophyll, for example on activated carbon or by concentration / precipitation / filtration. Advantageously, the Verbena officinalis extract is decolorized by concentration / precipitation / filtration.

[0057] The extract of Verbena officinalis can be characterized by the presence of at least one phytochemical family among glycosylated iridoids, glycosylated phenylethanoids, or flavonoids.

[0058] The extract of Verbena officinalis may have a content of 0.1 to 60%, particularly 1 to 30%, more particularly 2 to 20% or even more particularly approximately 10% by weight of glycosylated iridoids relative to the weight of the dry extract.

[0059] The extract of Verbena officinalis may have a content of 0.01 to 60%, particularly 1 to 40%, or even more particularly approximately 10% by weight of glycosylated phenylethanoids relative to the weight of the dry extract.

[0060] The extract of Verbena officinalis may have a content of 0.01 to 50%, particularly 0.01 to 20%, or even more particularly approximately 5% by weight of flavonoids relative to the weight of the dry extract.

[0061] The extract of Verbena officinalis as described above makes it possible to prevent or treat alopecia, which can in particular be chosen from the group consisting of androgenetic alopecia, reactive alopecia, postmenopausal alopecia and alopecia areata.

[0062] Preferably, the extract of Verbena officinalis makes it possible to treat or prevent androgenetic alopecia.

[0063] The extract helps to limit hair loss and / or body hair and / or to promote hair growth and / or increase the density of hair follicles and / or obtain more covering hair and / or promote follicular regeneration. Dermatological or cosmetic compositions

[0064] The present invention also relates to a dermatological or cosmetic composition comprising at least one extract of Verbena officinalis and at least one dermatologically or cosmetically acceptable excipient for its use in the prevention and / or treatment of alopecia.

[0065] Alopecia may in particular be chosen from the group consisting of androgenetic alopecia, reactive alopecia, postmenopausal alopecia and alopecia areata, preferably it is androgenetic alopecia.

[0066] The composition makes it possible to limit hair loss and / or body hair and / or to promote hair growth and / or to increase the density of hair follicles and / or to obtain more covering hair and / or to promote follicular regeneration.

[0067] Advantageously, the extract included in the dermatological or cosmetic composition is as described previously.

[0068] Advantageously, the cosmetic or dermatological composition comprises an extract obtained by hydroalcoholic extraction, that is to say a hydroalcoholic extract of Verbena officinalis.

[0069] The cosmetic or dermatological composition may comprise from 0.001 to 5% by weight, preferably 0.002 to 2% by weight, preferably from 0.005 to 1% by weight, even more preferably from 0.01 to 0.5% by weight of dry extract of Verbena officinalis, relative to the total weight of the composition.

[0070] The invention preferably relates to a cosmetic or dermatological composition presented in a clean form and suitable for topical application, in particular to the scalp and / or hair.

[0071] 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.

[0072] 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 leave-in spray.

[0073] We thus distinguish between formulated products which can be rinsed and formulated products which do not require rinsing.

[0074] 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.

[0075] The composition according to the invention, administered for example topically, generally contains, in addition to the extract as described above, a physiologically acceptable medium, generally based on water or solvent, for example alcohols, ethers or glycols. It may also contain surfactants, complexing agents, preservatives, stabilizing agents, emulsifiers, thickeners, gelling agents, humectants, emollients, trace elements, essential oils, perfumes, colorants, moisturizing agents or thermal waters, etc.

[0076] From the first administrations, for example topically or orally, of the composition according to the invention, the hair will regain strength and vitality and regrowth will be encouraged.

[0077] The extract as described above or the composition as described above can therefore be used to promote hair regrowth.

[0078] According to one embodiment of the invention, the extract of Verbena officinalis as described above or the cosmetic or dermatological composition comprising at least one extract of Verbena officinalis and at least one cosmetically or dermatologically acceptable excipient as described above, is intended to be applied topically.

[0079] The invention also relates to a cosmetic process consisting of applying to the hair and body hair a composition comprising the extract of Verbena officinalis described above.

[0080] The subject of the invention is a Verbena officinalis extract or a dermatological composition comprising at least one Verbena officinalis extract and at least one dermatologically acceptable excipient, as defined above, for their use in the prevention and / or in the treatment of alopecia.

[0081] The invention also relates to the use of an extract of Verbena officinalis or of a dermatological composition comprising at least one extract of Verbena officinalis and at least one dermatologically acceptable excipient, as defined above, for the manufacture of a pharmaceutical or dermatological composition intended for the promotion of hair growth by the prevention of hair loss.

[0082] The invention also relates to the use of an extract of Verbena officinalis or of a dermatological composition comprising at least one extract of Verbena officinalis and at least one dermatologically acceptable excipient, as defined above, for promoting hair regrowth.

[0083] The invention also relates to a method for promoting hair growth, in particular for preventing and / or treating alopecia, comprising administering to a patient in need thereof an effective amount of an extract of Verbena officinalis or of a cosmetic or dermatological composition comprising at least one extract of Verbena officinalis and at least one cosmetically or dermatologically acceptable excipient, as defined above.

[0084] The invention also relates to a cosmetic use of an extract of Verbena officinalis as described above or of the cosmetic or dermatological composition as described above, for hair and / or scalp care, and / or to promote hair growth, and / or to increase the density of hair follicles and / or to obtain more covering hair and / or to promote follicular regeneration and / or to combat alopecia, said alopecia being able to be chosen from androgenetic alopecia, postmenopausal alopecia and telogen effluvium.

[0085] The invention also relates to a non-therapeutic cosmetic method for hair and / or scalp care, and / or for limiting hair and / or body hair loss and / or for promoting hair growth, and / or for increasing the density of hair follicles and / or for obtaining more covering hair and / or for promoting follicular regeneration and / or for combating alopecia, said alopecia being able to be chosen from androgenetic alopecia, postmenopausal alopecia, and telogen effluvium, comprising the application to the hair and / or scalp of an extract of Verbena officinalis as described above or of a cosmetic composition as described above.

[0086] The invention also relates to a non-therapeutic cosmetic method for preventing and / or treating alopecia, in particular for promoting hair growth, for example androgenetic alopecia, postmenopausal alopecia, and telogen effluvium, comprising the use of an extract of Verbena officinalis or of a cosmetic or dermatological composition comprising at least one extract of Verbena officinalis and at least one cosmetically or dermatologically acceptable excipient, as defined above. EXAMPLES

[0087] The following examples illustrate the invention without limiting its scope. Examples 1 to 5 illustrate different types of extraction of aerial parts of Verbena officinalis.

[0088] Example 1: Extraction with 70% ethanol assisted by ultrasound

[0089] 34 grams of crushed dry aerial parts of Verbena officinalis are put into contact with 340 milliliters of 70% ethanol then extracted under the action of ultrasound (1000 W, 20 kHz) with stirring for 5 minutes. The extract is then filtered under vacuum on Buchner. Then the extract is decolorized by preconcentration then icing at 4°C for 12 hours and filtered again. The solvent is then evaporated to obtain 3.54 grams of a powder with a mass yield of 10.4%. The dry extract obtained contains 7.3% by weight of Verbenalin (glycosylated iridoid characteristic of Verbena officinalis).

[0090] Example 2: Microwave-assisted 90% ethanol extraction

[0091] 1.5 grams of crushed dry aerial parts of Verbena officinalis are put into contact with 15 milliliters of 90% (v / v) ethanol and then extracted using microwaves (850 W, 50°C max) for 30 minutes. The extract is then vacuum filtered on a Buchner funnel. The solvent is evaporated to obtain a dry extract in powder form with a mass extraction yield of 6.6% and a Verbenalin content of 9.4%. Example 3: Extraction with 50% ethanol at reflux

[0092] 5 grams of crushed dry aerial parts of Verbena officinalis are put in contact with 50 milliliters of 50% (v / v) ethanol and then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel. The solvent is then evaporated to obtain 0.9 grams of a powder with a mass yield of 18%. The dry extract obtained contains 5.3% by weight of Verbenalin.

[0093] Example 4: Extraction with refluxing ethyl acetate

[0094] 5 grams of crushed dry aerial parts of Verbena officinalis are put in contact with 50 milliliters of ethyl acetate and then extracted at reflux with stirring for 60 minutes. The extract is then filtered under vacuum on a Buchner funnel. The solvent is then evaporated to obtain 0.25 grams of a powder with a mass yield of 5%. The dry extract obtained contains 8.5% by weight of Verbenalin.

[0095] Example 5: Ultrasonically Assisted 2-Methyltetrahydrofuran (MTHF) Extraction

[0096] 5 grams of crushed dry aerial parts of Verbena officinalis are put in contact with 50 milliliters of MTHF in a beaker and then extracted under the action of ultrasound (1000 W, 20 kHz) while stirring for 1 minute. The extract is then filtered under vacuum on a Buchner funnel. The solvent is then evaporated to obtain 0.17 grams of a powder with a mass yield of 10.4%. The dry extract obtained contains 3.4% by weight of Verbenalin.

[0097] Example 6: Evaluation of a verbena extract on the inhibition of 15-dPGJ2

[0098] The hair growth cycle is a highly regulated biological mechanism during in which different compartments of the hair follicle (dermal papilla, follicle-resident stem cells, hair follicle matrix keratinocytes) maintain close relationships through a variety of molecular exchanges. Recently, it has been shown that prostaglandin D2 (PGD2) is overexpressed on bald areas compared to hairy areas on the scalp of men with androgenetic alopecia (Garza, Luis et al., Science Translational Medicine 2012, vol 4, no 126, pl26ra34). Consistent with these observations, DP2, a receptor for PGD2, is overexpressed in the outer epithelial sheath of keratinocytes in the bald area.

[0099] The metabolite 15-deoxy-12,14-prostaglandin (15d-PGJ2) is a non-enzymatic compound derived from PGD2 that has been identified as a pro-apoptotic factor, i.e., it induces apoptosis in outer epithelial sheath cells and entry into the catagen phase (Joo, Hyun Woo, et al., Naunyn-Schmiedeberg's Archives of Pharmacology 2016, vol. 389, no7, p809613).

[0100] In this study, it is demonstrated that the metabolite 15d-PGJ2 induces apoptosis and consequently causes hair loss.

[0101] The purpose of this example is to evaluate whether a verbena extract can inhibit the apoptosis pathway induced by the metabolite 15d-PGJ2.

[0102] The experiments are carried out on keratinocytes from the outer epithelial sheath of hair follicles from three human donors. The cells are seeded in 96-well plates and cultured for 24 hours with the necessary supplements. The medium is then replaced and the cells are treated for 24 hours with the products to be tested (verbena extract at a concentration of 3, 10 or 30 pg / ml diluted in DMSO) and the metabolite 15-dPGJ2 at 5 pM. After 24 hours of incubation, the assay on the cell lawns of caspase 3 / 7 activity is measured by luminescence (Promega G6411) following the manufacturer's instructions. All experimental conditions are carried out with n=3 donors and n=3 technical replicates.

[0103] With verbena extracts diluted in DMSO, the cell model was validated and cytotoxicity verified. There is no difference when the cells are treated with DMSO at 0.1% v / v and when they are not treated. Thus, it is concluded that DMSO at the tested concentration does not modify cell viability; it is used in the following experiments as a control.

[0104] Next, caspase 3 / 7 activity was measured after treatment with the metabolite 15d-PGJ2 and in combination with a known caspase inhibitor such as Z-VAD-FMK (Sigma).

[0105] [Tables 1] Tested Compound Mean % Apoptosis vs. 15d-PGJ2 SEM % Inhibition vs. 15d-PGJ2 Statistics DMSO (Control) 17.62 + / - 0.65 100 < 0.0001 15d-PGJ2 (5pM) 100 + / - 1.25 - - 15d-PGJ2 5pM + Z-VAD-FMK 20pg / ml 13.32 + / - 0.71 105 < 0.0001

[0106] SEM: standard error of the mean

[0107] The metabolite 15d-PGJ2 induces apoptosis in the outer epithelial sheath cells. When the compound Z-VAD-FMK is added the percentage of apoptosis decreases significantly and is similar to the control value. The addition of the compound Z-VAD-FMK shows a significant decrease in apoptosis induced by 15d-PGJ2 which reflects the decrease in hair loss.

[0108] [Tables2] Tested compound Mean % apoptosis vs. 15d-PGJ2 SEM % inhibition vs. 15d-PGJ2 Statistics Verbena extract 3 pg / ml + 15d-PGJ2 5pM 67.34 5.17 40 0.0012 Verbena extract 10 pg / ml + 15d-PGJ2 5pM 60.04 3.85 49 0.0001 Verbena extract 30 pg / ml + 15d-PGJ2 5pM 37.48 3.05 76 < 0.0001

[0109] The verbena extract obtained according to example 1 is tested with three different concentrations 3, 10 and 30 pg / ml. The decrease in apoptosis is observed for the 3 verbena extracts and in a concentration-dependent manner.

[0110] Verbena extract induces inhibition of l5d-PGJ2-induced apoptosis in outer epithelial sheath cells in a concentration-dependent manner (40% inhibition at 5pg / mL and 76% inhibition at 30pg / ml compared to the untreated control).

[0111] Therefore, verbena extract acts on the prostaglandin D2 factor and helps limit hair loss by inhibiting apoptosis in the cells of the outer epithelial sheath, which helps promote hair growth.

[0112] Example 7: Evaluation of a verbena extract on the inhibition of the VEGF pathway

[0113] It is known that dermal papilla cells of hair follicles produce VEGF (Lachgar (1996) J Invest Dermatol, 106(1): 17-23). ​​VEGF is a growth factor of endothelial cells; it thus promotes the supply of oxygen and nutrients to the hair follicle. The level of expression of this protein varies during the hair growth cycle: largely produced in the anagen phase (growth phase), its expression decreases when the hair follicle enters the catagen phase (regression phase). The growth factor VEGF promotes the creation of new blood vessels and thus stimulates endothelial cells. Thus, this growth factor when induced stimulates the growth of blood vessels and consequently promotes hair regrowth.

[0114] The purpose of this example is to evaluate whether a verbena extract can stimulate the production of the VEGF factor.

[0115] The experiments are performed on human hair follicle dermal papilla cells (HFDPC) from three different donors.

[0116] The cells are seeded in 96-well plates and cultured for 24 hours in a culture medium (HFDPC medium (Promocell, C-26500) supplemented with the appropriate supplements (Promocell, C-39625)), which is then replaced by a test medium (HFDPC medium from Promocell without supplement). The test medium contains or does not contain (control) the tested compounds or the reference (manganese salt of pyrrolidone carboxylic acid (PCA)) and the cells are incubated for 24 hours.

[0117] The culture supernatants are then collected for VEGF assay. All experimental conditions were carried out in n=3.

[0118] [Tables3] Tested Compound Mean [VEGF] (pg / ml) SEM % induction vs. untreated Statistics Untreated (Control) 175.09 12.25 - PCA Manganese Salt 500pM (Reference Compound) 12.7 20.23 118 p<0.001 Verbena Extract 10pg / ml 17.21 10.33 23 NS Verbena Extract 30pg / ml 21.24 14.03 53 p<0.05

[0119] A significant induction of VEGF release is observed in the presence of PCA manganese salt which is an internal reference (+118% compared to the untreated control). This result was expected, it allows this test to be validated.

[0120] The verbena extract obtained according to example 1 induces an increase in the release of VEGF in dermal papilla cells in a concentration-dependent manner (+23% at 10pg / mL and +53% at 30pg / ml compared to the untreated Ctrl).

[0121] Verbena extract induces the production of VEGF factor and promotes hair regrowth by stimulating the growth of blood vessels.

[0122] Example 8: Evaluation of a verbena extract on the JAK-STAT pathway

[0123] The purpose of this example is to confirm the inhibitory activity of a Verbena officinalis extract of the JAK-STAT signaling pathway on a cellular model, the follicular keratinocytes of the outer epithelial sheath of hair follicles (ORS Outer Root Sheath cells). In this model, interleukin IL-6 is used to activate the JAK1 / 2-STAT3 pathway by activating IL-6 and GP130 receptors which are both expressed at the level of this epithelial sheath of hair follicles. IL-6 is a cytokine which acts as an inhibitor of the hair growth cycle, its overexpression in a transgenic mouse model leads to delayed hair growth. Furthermore, in androgenetic alopecia IL-6 would be overexpressed in the cells of the dermal papilla under the influence of androgens (Kwack et al., 2012, J Invest Dermatology 132(1) 43-9). IL-6 has also been reported to delay hair follicle growth in humans.

[0124] The study is carried out on follicular keratinocytes of the outer epithelial sheath of hair follicles (ORS Outer Root Sheath model). The keratinocytes are cultured in 96-well plates in a suitable medium (CnT-PR from Cellntec). 48 hours later, the cells are washed with an alkaline phosphate buffer (PBS) and the medium is changed to KSFM (standard maintenance medium for human primary keratinocytes). After 4 hours, the cells are treated for 1 hour with the compounds to be tested (verbena extract at 10, 30 and 100 pg / ml diluted in DMSO) and the reference compound (Tofacitinib at 5 pM diluted in DMSO). The verbena extract tested is an extract according to Example 1 of the present invention. Then the IL-6 stimulation treatment (Stim IL6) is carried out at 100 ng / ml for 15 minutes.

[0125] All experimental conditions are carried out with n=3 donors and n=3 technical replicates.

[0126] The activation level of the JAK / STAT signaling pathway is estimated by evaluating the phosphorylation level of the STAT3 protein. Statistical analysis is performed by a parametric test after verifying normality and equivalence of variance, otherwise a non-parametric test is chosen.

[0127] [Tables4] Compound tested Average ratio pSTAT3 / STAT3 vs. Stim IL6 SEM % JAK7 pathway inhibition STAT Statistics DMSO (control) 0.006 0.004 99.3 p<0.0001 Stim IL6 0.594 0.101 0.0 - Stim IL6 + Tofacitinib (5pM) 0.010 0.007 97.5 p<0.0001 Stim IL6 + Verbena Extract 10pg / mL 0.361 0.062 40.4 0.0001 Stim IL6 + Verbena Extract 30pg / mL 0.327 0.062 49.5 p<0.0001 Stim IL6 + Verbena Extract 100pg / mL 0.181 0.010 67.6 p<0.0001

[0128] Treatment of keratinocytes with IL-6 induced a significant increase in the level of STAT3 phosphorylation, this increase was statistically significant (P<0.0001). The reference compound Tofacitinib tested at 5pM significantly reduced tively the STAT3 phosphorylation level of 97.5% p<0.0001. These expected results validate the test.

[0129] The verbena extract obtained according to example 1 at the three concentrations tested significantly and very markedly inhibits the level of STAT3 phosphorylation induced by stimulation by IL-6. It therefore acts in a concentration-dependent manner, and from 100g / mL (p=0.0001).

[0130] The inventors thus highlight the benefit of a verbena extract in hair loss by lengthening the hair life cycle, by significantly inhibiting the activation of the JAK / STAT pathway induced by IL6 in the cells of the outer epithelial sheath.

Claims

Claims

1. Extract of Verbena officinalis for use in the prevention and / or treatment of alopecia.

2. Extract for use according to claim 1 in which the alopecia is selected from the group consisting of androgenetic alopecia, reactive alopecia, postmenopausal alopecia and alopecia areata.

3. Extract for its use according to claim 2 characterized in that the alopecia is androgenic alopecia.

4. Extract for use according to any one of claims 1 to 3, to promote hair regrowth.

5. Extract for use according to any one of claims 1 to 4, to limit hair loss and / or hairs and / or to promote hair growth and / or to increase the density of hair follicles and / or to obtain more covering hair and / or to promote follicular regeneration.

6. Extract for use in any one of claims 1 to 5, characterized in that said extract is in a form suitable for being applied topically.

7. Extract for use according to any one of claims 1 to 6, characterized in that the extract of Verbena officinalis is a hydroalcoholic extract of Verbena officinalis.

8. Cosmetic or dermatological composition comprising at least one extract of Verbena officinalis and at least one dermatologically or cosmetically acceptable excipient for its use in the prevention and / or treatment of alopecia.

9. Cosmetic or dermatological composition for its use according to claim 8, characterized in that it comprises 0.001 to 5% by weight of dry extract of Verbena officinalis relative to the total weight of the composition.

10. Cosmetic or dermatological composition for use according to any one of claims 8 or 9, characterized in that the extract of Verbena officinalis is a hydroalcoholic extract of Verbena officinalis.

11. Cosmetic or dermatological composition for use according to any one of claims 8 to 10, characterized in that the composition is in a form suitable for topical application.

12. Cosmetic or dermatological composition for use according to any one of claims 8 to 11, characterized in that the composition makes it possible to limit hair loss and / or hairs and / or to promote hair growth and / or to increase the density of hair follicles and / or to obtain more covering hair and / or to promote follicular regeneration.