WO2023110882A1

WO2023110882A1 - Use of an aqueous-alcoholic extract of aerial parts of oenothera biennis to induce and/or stimulate the growth of human keratin fibres and/or to slow down the loss thereof

Info

Publication number: WO2023110882A1
Application number: PCT/EP2022/085642
Authority: WO
Inventors: Sébastien THIBAUT; Saliou NGOM
Original assignee: L'oreal
Priority date: 2021-12-15
Filing date: 2022-12-13
Publication date: 2023-06-22
Also published as: FR3130163A1

Abstract

The invention relates to a non-therapeutic cosmetic use of an aqueous-alcoholic extract of aerial parts of Oenothera biennis to induce and/or stimulate the growth of human keratin fibres such as head hair, eyelashes, body hair and eyebrows and/or to slow down the loss thereof, and in particular to treat androgenic alopecia. The invention also relates to a method for the non-therapeutic cosmetic treatment of human keratin fibres and/or the scalp, comprising the application of a cosmetic composition comprising said extract on said fibres such as head hair, eyelashes, body hair and eyebrows, and/or on the scalp, to induce and/or stimulate the growth and/or to slow down the loss of said fibres.

Description

Use of an aqueous-alcoholic extract of aerial parts of Oenothera biennis to induce and/or stimulate the growth of human keratin fibres and/or to slow down the loss thereof

The present invention relates to the field of caring for the scalp and keratin fibres, more particularly to the field of the growth of keratin fibres, and the prevention of the loss thereof.

The present invention relates to a non-therapeutic cosmetic use of an aqueous-alcoholic extract of aerial parts of Oenothera biennis in order to induce and/or stimulate the growth and/or slow down the loss of said fibres.

The invention also relates to a method for the non-therapeutic cosmetic treatment of human keratin fibres and/or the scalp using a cosmetic composition comprising said aqueous- alcoholic extract of aerial parts of Oenothera biennis in order to induce and/or stimulate the growth and/or slow down the loss of said fibres.

The growth of the hair and its renewal are mainly determined by the activity of the hair follicles and their matrix environment. Their activity is cyclical and essentially comprises three phases, namely the anagen phase, the catagen phase and the telogen phase.

The anagen phase (active or growth phase), which lasts several years and during which the hairs lengthen, is followed by a very short and transient catagen phase which lasts a few weeks, and then by a telogen phase or resting phase which lasts a few months. At the end of the resting period, the hairs fall out and another cycle begins again. The head of hair is thus undergoing constant renewal and, of the approximately 150 000 hairs which make up a head of hair, approximately 10% are at rest and will be replaced in the months to come. The natural loss of the hair can be estimated, on average, at a few hundred hairs per day for a normal physiological state. This constant physical renewal process undergoes a natural change during the course of ageing: the hairs become finer and their cycles shorter. In addition, various causes may bring about substantial temporary or permanent hair loss. Hair loss, in particular alopecia, is essentially due to disruptions in hair renewal. These disruptions lead in a first step to acceleration of the frequency of the cycles at the expense of the quality of the hairs, and then of their quantity. Progressive miniaturization of the bulbs takes place, in conjunction with isolation of these bulbs by gradual thickening of the perifollicular collagen matrix and also of the outer connective sheath. Revascularization around the hair follicle is thus made more difficult cycle after cycle. The hairs regress and become miniaturized until they are no more than an unpigmented down, and this phenomenon leads to gradual thinning of the head of hair. The number and mean diameter of the hair follicles that constitute the head of hair are affected. Certain areas are preferentially affected, especially the temporal or frontal regions in men, and diffuse alopecia of the crown is observed in women.

The term "alopecia" also covers an entire family of hair follicle disorders of which the final consequence is partial or general permanent hair loss. It may more particularly be a case of androgenic alopecia. In a large number of cases, early hair loss occurs in genetically predisposed individuals, which is known as andro-chrono-genetic alopecia; this form of alopecia especially concerns men. It is known, furthermore, that certain factors, such as hormonal imbalance, physiological stress or malnutrition, can accentuate the phenomenon. In addition, loss or impairment of the hair can be in connection with seasonal phenomena.

Loss of hair density and hair loss are often experienced as distressing by persons thereby affected, especially when they are still young.

Pharmacological active agents such as minoxidil, latanoprost, fluridil, spironolactone and combinations thereof are known.

Other products belonging to the cosmetic field exist. Among these, examples that may be mentioned include aminexil® and stemoxydine®.

However, consumers are increasingly in search of effective natural products, which would delay the process of "loss of hair density" or "excessive hair loss".

Thus, there is a need to provide natural active agents that make it possible to effectively prevent and/or treat loss of hair density.

The applicant has surprisingly found that an aqueous-alcoholic extract of aerial parts of evening primrose (Oenothera biennis) makes it possible to modulate the degree of expression of a series of genes associated with the HIF-1 biological pathway, this pathway being known in the literature for its involvement in hair loss (F Juchaux, T Sellathurai, V Perrault, F Boirre, P Delannoy, K Bakkar, J Albaud, A Gueniche, A Cheniti, S Dal Belo, L Souverain, M Le Balch, S Commo, S Thibaut, J F Michelet. "A combination of pyridine-2, 4-dicarboxylic acid diethyl ester and resveratrol stabilizes hypoxia-inducible factor 1 -alpha and improves hair density in female volunteers", Int J Cosmet Sci. 2020 Apr; 42(2): 167-173). Thus, the aqueous-alcoholic extract of aerial parts of evening primrose (Oenothera biennis) advantageous properties as an active agent for inducing and/or stimulating the growth of human keratin fibres such as head hair, eyelashes, body hair and eyebrows and/or slowing down the loss thereof, and in particular for treating androgenic alopecia. To the knowledge of the applicant, the use of an aqueous-alcoholic extract of aerial parts of evening primrose (Oenothera biennis) in order to induce and/or stimulate the growth of human keratin fibres and/or to slow down the loss thereof has never been disclosed before.

However, the oil extracted from Oenothera biennis seeds has been disclosed in the literature for its anti-hair loss action. Another hand, for those skilled in the art, a fatty plant oil derived from seeds differs from an aqueous-alcoholic extract derived from aerial parts not only in its physical chemistry but also by in its composition. Furthermore, the aqueous of an alcoholic extraction favours the extraction of polar to moderately polar compounds and consequently disfavours the extraction of fatty acids even if these are present in the aerial parts. Moreover, it is known that the seeds are generally naturally rich in fatty substances unlike the aerial parts.

One subject of the invention is thus the non-therapeutic cosmetic use of an aqueous-alcoholic extract of aerial parts of Oenothera biennis to induce and/or stimulate the growth of human keratin fibres such as head hair, eyelashes, body hair and eyebrows and/or to slow down the loss thereof, and in particular to treat androgenic alopecia.

Preferably, the said aqueous-alcoholic extract of aerial parts of Oenothera biennis is used to induce and/or stimulate the growth of human keratin fibres such as head hair, eyelashes, body hair and eyebrows and/or to slow down the loss thereof, in a subject having a loss of keratin fibres density, especially a loss of head hair density.

The aqueous-alcoholic extract of aerial parts of Oenothera biennis additionally makes it possible to obtain improved effects on the head of hair, notably regarding the hair density.

The head of hair appears to be more voluminous, in particular in the case of fine hair.

The present invention also relates to a method for the non-therapeutic cosmetic treatment of human keratin fibres and/or the scalp, comprising the application of a cosmetic composition comprising at least one aqueous-alcoholic extract of aerial parts of Oenothera biennis on said fibres such as head hair, eyelashes, body hair and eyebrows, and/or on the scalp, to induce and/or stimulate the growth and/or to slow down the loss of said fibres.

Preferably, the said cosmetic composition comprising at least one aqueous-alcoholic extract of aerial parts of Oenothera biennis is applied on said fibres such as head hair, eyelashes, body hair and eyebrows, and/or on the scalp, in a subject having a loss of keratin fibres density, especially a loss of head hair density.

Other subjects, characteristics, aspects and advantages of the invention will become even more clearly apparent on reading the description and the example which follows.

In the text which follows, unless otherwise indicated, the limits of a range of values are included in that range, notably in the expressions “between” and “ranging from ... to ...”. Moreover, the expression “at least one” used in the present description is equivalent to the expression “one or more”.

Definition

For the purposes of the present invention, the expression "cosmetic composition" means a composition comprising a physiologically acceptable medium, i.e. a medium that is compatible with the skin.

For the purposes of the present invention, the expression "keratin fibres" means keratin fibres such as head hair, body hair, eyebrows and eyelashes.

For the purposes of the present invention, the expression "to slow down the loss of keratin fibres" means to curb or reduce the loss of keratin fibres.

For the purposes of the present invention, the expression “to stimulate the growth of keratin fibres” means to promote or improve the growth of existing keratin fibres, notably the length of existing keratin fibres.

For the purposes of the present invention, the expression “to induce the growth of keratin fibres” means to promote or improve the growth of new keratin fibres, notably the density of keratin fibres (via the growth of new keratin fibres).

Detailed description

The plant used in the invention may be Oenothera biennis ssp maritima.

Preferably, the aerial parts are chosen from the flowers, leaves and/or stems. In particular, the aerial parts do not include the roots or rhizomes.

Plant extraction is a process that aims to extract certain constituents present in the plants. It is a solid/liquid separation operation: a solid body (the plant, in this case the aerial parts of Oenothera biennis) is brought into contact with a fluid (the extraction solvent, in this case a mixture of water and at least one alcohol). The compounds of interest are then solubilized and contained in the extraction solvent. The solution obtained after filtering the mixture corresponds to the desired extract.

All or part of the extraction solvent can then be removed in order to isolate the plant extract: when it is removed completely, a dry extract is then obtained.

Thus, a "dry extract” is understood to mean an extract obtained after removal of the extraction solvent. Preferably, the dry extract comprises less than 10% by weight, preferably less than 5% by weight, preferably less than 3% by weight, preferably less than 1 % by weight, preferably less than 0.5% by weight, relative to the total weight of extract, of extraction solvent (in this case, alcohol or mixture of water and at least one alcohol). Preferably, the dry extract is free of extraction solvent.

Preferably, according to a first embodiment, the extract according to the invention is a dry extract.

When the extraction solvent is not completely removed, it is then referred to as a fluid extract. Preferably, the fluid extract comprises a content of greater than 10% by weight, relative to the total weight of extract, of extraction solvent (in this case, mixture of water and at least one alcohol).

Preferably, according to a second embodiment, the extract according to the invention is a fluid extract.

An “aqueous-alcoholic extract” is understood to mean an extract obtained with an extraction solvent which is a mixture of water and at least one alcohol.

In particular, the alcohol is a monoalcohol having from 2 to 6 carbon atoms. Preferably, the alcohol is ethanol.

Preferably, the mixture of water and at least one alcohol is a mixture comprising from 20% to 60% by volume of water relative to the total mixture volume, and from 40% to 80% by volume of alcohol relative to the total mixture volume. Preferably, the mixture of water and at least one alcohol is a mixture comprising from 30% to 55% by volume, preferably from 45% to 55% by volume, of water relative to the total mixture volume, and from 45% to 60% by volume, preferably from 47% to 55% by volume of alcohol relative to the total mixture volume.

Preferably, the mixture is a mixture of water and a single alcohol.

More preferentially, the mixture of water and at least one alcohol is a mixture comprising 50% by volume of water relative to the total mixture volume, and 50% by volume of alcohol, preferably ethanol, relative to the total mixture volume (50% mixture).

Preferably, the aqueous-alcoholic extract of aerial parts of Oenothera biennis according to the invention is obtained from the aerial parts which have been pre-ground and extracted using an aqueous-alcoholic mixture (i.e. mixture of water and at least one alcohol having from 2 to 6 carbon atoms, preferably ethanol), preferably a 50% mixture.

The Oenothera genus belongs to the family Onagraceae which comprises around 145 species, growing in the regions of North and South America, and also in Europe. Certain species, in particular Oenothera biennis (or biennial evening primrose), the source of evening primrose oil derived from the seeds, which contains a high percentage of unsaturated fatty acids, in particular gamma-linoleic acid. The defatted seeds of evening primrose are waste products from the pharmaceutical and cosmetic industries and they have been intensively studied for their high content of flavonoids and tannins.

Antioxidant, anti-inflammatory and anti-tumour effects have been attributed to the methanoic extract of the defatted seeds. Conversely, the aerial parts are often waste products of the evening primrose oil industry, which oil is obtained from the seeds, and are rarely reused. Studies regarding the chemical composition and the properties of the extracts prepared from the aerial parts of species of the genus Oenothera are quite limited and show that the aerial parts contain flavonoids, phenolic acids, tannins and triterpenes.

The Oenothera biennis used is a halophyte, i.e. capable of growing in an environment subjected to saline stress.

According to the invention, the biomass used is constituted by dried aerial parts of evening primrose (Oenothera biennis). In particular, the variety Oenothera biennis ssp maritima can be used. Preferably, this plant is cultivated in the North Brittany region (France) by the company. The aerial parts are preferably harvested in full bloom in July.

The aqueous-alcoholic extract of aerial parts of Oenothera biennis according to the invention is obtained by a process comprising the extraction of the aerial parts, preferably which have been pre-ground, in a mixture of water and at least one alcohol, preferably a mono alcohol having from 2 to 6 carbon atoms, preferably ethanol.

The aqueous-alcoholic extract according to the invention is preferably obtained as described in Example 1. In particular, it is obtained by the process comprising the following steps: a. grinding the dried aerial parts of Oenothera biennis to fine particles in a grinder (1-2 mm particle size); b. extracting by macerating in a mixture of water and at least one alcohol, at a temperature of from 15°C to 45°C, in particular from 15°C to 40°C and notably at room temperature, notably by introducing from 10 g to 500 g of plant powder obtained in a, per litre of water and alcohol mixture, in particular from 50 g to 200 g, and more particularly from 70 g to 150 g for instance 100 g of plant powder obtained in a, per litre of water and alcohol mixture, and by maintaining at this temperature for 1 to 5 hours, notably 1 to 3 h, in particular 1 h 30 min to 2 h 30 min, for instance 2 hours. Preferably, the mixture comprises from 20% to 60% by volume, preferably from 30% to 55% by volume, preferably from 45% to 55% by volume, preferably 50% by volume, of water relative to the total mixture volume, and from 40% to 80% by volume, preferably from 45% to 60% by volume, preferably from 47% to 55% by volume, preferably 50% by volume, of alcohol, preferably ethanol, relative to the total mixture volume; then c. filtering the mixture obtained in b, notably on a Whatman GF/C filter with a porosity of 1.2 pm; d. partial or complete evaporation of the alcohol in the filtered mixture obtained in c, typically with a rotary evaporator at 35°C; and e. optionally freeze drying of the aqueous residue obtained in d, typically for 24 hours.

When step d) is a complete evaporation of the alcohol followed by the freeze-drying step e), the extract is obtained in dry form (dry extract), which may optionally be ground and/or packaged, typically in a glass bottle.

The aqueous-alcoholic dry extract according to the invention is preferably in the form of a powder which is soluble in water to 1 .5%, soluble in a 50/50 water/alcohol mixture to 5%, and is insoluble in pure ethanol. It is preferably of light brown colour.

Preferably, the aqueous-alcoholic extract according to the invention comprises at least two, preferably at least three, preferably at least four, preferably at least five molecules chosen from oenothein B, quercetin, ellagic acid, hyperoside, isoquercitrin (or isoquercetin) and luteolin-7-O-glucuronide, and mixtures thereof.

Preferably, the aqueous-alcoholic extract according to the invention comprises the following six molecules: oenothein B, quercetin, ellagic acid, hyperoside, isoquercitrin and luteolin-7-O- glucuronide.

Alternatively, the extract according to the invention may comprise at least two, preferably at least three, preferably at least four, preferably at least five molecules, preferably at least six molecules, preferably at least seven molecules, preferably at least eight molecules, chosen from oenothein B, oenothein A, ellagic acid, gallic acid, quercetin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, and luteolin-7-O-glucuronide.

Still in this particular embodiment, the extract according to the invention comprises the following nine molecules: oenothein B, oenothein A, ellagic acid, gallic acid, quercetin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, and luteolin- 7-O-glucuronide.

Preferably, the extract according to the invention is prepared by mixing between 20 g and 500 g, preferably between 50 g and 250 g, of powder of plant aerial parts per 1 L of water and alcohol mixture (extraction solvent), preferably with 100 g of powder of plant aerial parts per 1 L of water and alcohol mixture (extraction solvent).

Said extract may be present in a content ranging from 0.0001 % to 10% by dry weight, preferably from 0.001% to 5% by dry weight, relative to the total weight of the composition.

Said extract may be present in a content ranging from 0.0001 % to 30% by weight of fluid extract, preferably from 0.001 % to 25% by weight of fluid extract, relative to the total weight of the composition.

The composition preferably contains a physiologically acceptable medium.

This physiologically acceptable medium may more particularly constituted of water and optionally of a physiologically acceptable organic solvent chosen, for example, from lower alcohols including from 1 to 8 carbon atoms and in particular 1 to 6 carbon atoms, for instance ethanol, isopropanol, propanol or butanol; polyethylene glycols containing from 6 to 80 ethylene oxide units; polyols, for instance propylene glycol, isoprene glycol, butylene glycol, glycerol, sorbitol or 1 ,3-propanediol.

It may also be an anhydrous medium, notably an oily medium containing oils and/or fatty substances other than oils.

When the physiologically acceptable medium is an aqueous medium, it has a pH that is compatible with the skin, preferably ranging from 3 to 8 and better still from 4 to 7.

When the composition includes an aqueous or aqueous-alcoholic medium, it is possible to add a fatty (or oily) phase to this medium.

The composition according to the invention is in particular a composition intended for topical application to the scalp and/or keratin fibres.

The composition according to the invention may also comprise one or more additional compounds chosen notably from surfactants, preferably chosen from non-ionic, anionic, cationic and amphoteric surfactants, conditioning agents preferably chosen from cationic polymers, silicones, natural synthetic, polymeric or non-polymeric thickeners, UV screening agents, fillers such as nacres, titanium dioxide, resins and clays, fragrances, peptizers, vitamins, preservatives, acid agents, alkaline agents, reducing agents, oxidizing agents, additional active agents different from the extract according to the invention that are intended to further improve the activity regarding hair regrowth and/or regarding the slowing down of hair loss and that have already been described for this activity, such as nicotinic acid esters, notably including tocopherol nicotinate, benzyl nicotinate and C1-C6 alkyl nicotinates such as methyl or hexyl nicotinates; agents for promoting hair regrowth chosen from the derivatives of pyrimidine-3-oxide such as 2,4-diaminopyrimidine-3-N-oxide (Aminexil), derivatives of pyridine-dicarboxylate or of a salt thereof such as those described in FR2838336 such as the dimethyl ester of pyridine-2,4-dicarboxylate; and a mixture of these compounds.

The above additional compounds may each be present in an amount ranging from 0.01 % to 20% by weight relative to the total weight of the composition.

The composition according to the invention is intended for cosmetic use by topical application to the scalp and/or keratin fibres, and more especially to the scalp, head hair and eyelashes, body hair and eyebrows.

According to the application method, this composition may be in any presentation form normally used in the cosmetic field, such as a lotion, serum, milk, cream, gel, ointment, pomade, powder, balm, patch, impregnated pad, soap, bar or foam. It may be formulated in the form of an emulsion, such as an oil-in-water direct emulsion or a water-in-oil inverse emulsion.

For topical application to the scalp and/or keratin fibres, the composition may especially be in the form of an aqueous or aqueous-alcoholic solution or suspension, an emulsion or dispersion of liquid or semi-liquid consistency obtained by dispersing a fatty phase in an aqueous phase (O/W) or vice versa (W/O), a dispersion or emulsion of soft consistency, an aqueous or aqueous-alcoholic gel, or else microcapsules or microparticles, or vesicular dispersions of ionic and/or non-ionic type.

The composition may also be in the form of a foam or else in the form of an aerosol composition also comprising a pressurized propellant.

Preferably, for a topical application to the scalp and/or keratin fibres, notably the scalp or head hair, the composition may be in the form of a hair lotion, shampoo, conditioner, hair shaping product (lacquer, hair setting product, styling gel), a hair mask, or a foaming cream or gel for cleansing the hair.

According to one preferred embodiment, the composition is in the form of a cream or lotion, a shampoo or conditioner.

The present invention also relates to a method for the non-therapeutic cosmetic treatment of human keratin fibres and/or the scalp, comprising the application of the composition described above, to the fibres and/or the scalp, and more particularly to the head hair, eyelashes, eyebrows, body hair and/or scalp.

In particular the method comprises applying the composition to the scalp and/or the fibres, then, optionally, rinsing, after a composition leave-on time which can range from 1 minute to 30 minutes. The cosmetic composition is preferably not rinsed out.

Finally, a subject of the present invention is said extract as described above or a composition comprising it as described above, for use thereof for inducing and/or stimulating the growth of human keratin fibres such as head hair and eyelashes, eyebrows and body hair and/or slowing down the loss thereof, and in particular for treating androgenic alopecia.

Advantageously, one subject of the present invention is said extract as described above or a composition comprising it as described above, for use thereof in the treatment of alopecia, preferably androgenic alopecia.

The examples that follow serve to illustrate the invention.

Examples

Example 1 - Preparation of the aqueous-alcoholic extract of aerial parts of evening primrose {Oenothera biennis) according to the invention

The extract was prepared according to the following process:

Grinding the dried aerial parts of Oenothera biennis to fine particles in a grinder (1- 2 mm particle size); The aerial parts being the parts exposed to the open air (leaves, stems, flowers) under the normal growth conditions of Oenothera biennis, which excludes the roots in particular.

Extracting by macerating in an ethanol/water (50/50 v/v) mixture at room temperature, by introducing 500 g of powder of aerial parts into 5 litres of extraction solvent (ethanol/water (50/50 v/v) mixture) (corresponding to a ratio of 100 g per 1 litre of extraction solvent), and by maintaining at this temperature for 2 hours, with motor stirring using an impeller system at 250 rpm.

Next, prefiltration on a 50 pm Nitex gauze.

Next, filtration on a Whatman® GF/C filter with a porosity of 1 .2 pm, 0 110 mm.

The extration residue was re-extracted followed by a pre-filtration and a filtration under the same conditions as before and then combining the 2 filtrates.

Evaporation of the alcohol on a rotary evaporator (Buchi Rotavapor® R215, equipped with a Buchi B-491 heating bath, and also a Vacuubrand PC 3001 Vario Pro vacuum pump) at 35-40°C down to a volume of around 20% of the initial volume.

Next, freeze drying the aqueous residue for 24 hours using a Labconco FreeZone 4.5 Plus freeze dryer, coupled to a Vacuubrand RZ-6 vacuum pump in automatic start-up mode from -40°C in the collector. Thus 94.9 g, equivalent to 18.98% of extraction yield, of an aqueous-alcoholic dry extract of aerial parts of Oenothera biennis are obtained as a powder of light brown colour.

Next, homogenizing by grinding with a manual mortar.

Example 2 - Evaluation of the anti-hair loss effectiveness

Inducing and/or stimulating the growth of human keratin fibres and/or slowing down the loss thereof via modulation of the expression of genes associated with the HIF-1 biological pathway.

Principle:

The aim of this study is to evaluate the effects of the 50/50 v/v aqueous-alcoholic extract of aerial parts of Oenothera biennis obtained according to Example 1 on the expression of genes associated with the HIF-1 biological pathway by quantitative RT-PCR.

The aqueous-alcoholic extract of aerial parts of Oenothera biennis was evaluated at 0.1 mg/mL.

The various markers studied are: EGLN3, BNIP3, CA9.

Protocol (experimental conditions):

The tests were carried out on human keratinocytes in culture, seeded in Greiner 48-well plates, coated with bovine collagen I. The plates were prepared according to the following procedure: the solution of bovine collagen I at 0.1 mg/ml was prepared by dilution in a phosphate-buffered saline (PBS) solution of bovine collagen I sold by the company Life Technologies. Each well was immersed with 1 ml of this dilution, which was left at the bottom of the wells for 1 hour at 37°C. At the end of the incubation, the collagen solution was withdrawn and the wells were rinsed twice with 1 ml of PBS. The plates were then stored at 4°C until the time of use. The tests are performed on primary human keratinocytes at a rate of 23 800 cells/cm² of wells coated with bovine collagen I as explained previously, followed by culturing for 72 hours in the presence of 500 pl of KGM medium sold by the company Lonza, supplemented with: 0.1% by weight of gentamicin sulfate/amphotericin mixture sold under the trade name GA-1000 by the company Lonza (CC 3101/CC-4 131), 0.4% by weight of bovine pituitary extract sold under the trade name BPE by the company Lonza, 0.1% by weight of insulin sold by the company Lonza, 0.1% by weight of hydrocortisone and 0.1% by weight of epidermal growth factor (or recombinant human EGF sold by the company Lonza, at 37°C under an atmosphere saturated with water and containing 5% CO2. The cells were then treated with the extract of Oenothera biennis from Example 1 at the highest non-cytotoxic concentration (0.1 mg/mL), for 48 hours under normoxia (21% oxygen). Following this culturing and this treatment, the cell lawns were washed with phosphate- buffered saline (or PBS) and then lysed using a lysis buffer proposed in the kit from the supplier Qiagen. The RNAs were then extracted using the RNeasy isolation kit and the QIAcube station, both sold by the company Qiagen, according to the manufacturer's instructions. The quantity and quality of RNAs were checked using the LabChip® GX bioanalyser from Perkin- Elmer before performing reverse transcription (RT) using the Qiagen kit and according to the recommendations (QuantiTect reverse transcription kit). The cDNA obtained following the RT was then amplified by quantitative real-time PCR calculations using a specific kit sold under the trade name LightCycler® 480 SYBR Green Master Mix by the company Roche (Cat. No. 14123920) and an LC480 thermocycler (Roche). The PCRs were performed in triplicate (n=3).

Priming was performed using specific standard primers sold by the company Qiagen, the references for which are BNIP3/QT00024 178/Hs_BNIP3-l-SG Quantitect Assay Primer; CA9/QT000 11697/ Hs_CA9-l-SG Quantitect Assay Primer; EGNL3/QT00025900/ Hs_EGNL3-l-SG Quantitect Assay Primer; RPL 13A/QT000899 15/ Hs RPL 13A-1-SG Quantitect Assay Primer, and the fluorescent probe of brand name SYBR Green sold by the company specified previously. The PCR was performed in three phases: denaturation phase for 10 minutes at 95°C, amplification phase which is composed of 45 cycles comprising a step of denaturation for 30 seconds at 95°C, a step of hybridization for 30 seconds at 60°C, and a step of elongation at 72°C for 30 seconds, melting phase for ensuring the quality of the hybridizations. The incorporation of SYBR Green into the amplified DNA was measured continuously during the amplification cycles.

These measurements make it possible to obtain curves of fluorescence intensity as a function of the PCR cycles and thus to evaluate the relative expression of each marker from the cycle thresholds (Ct), corresponding to the number of cycles required to correctly detect a fluorescence level. For each marker and for each condition, the relative expression (RE) value was normalized relative to the expression of the reference gene RPL 13. The expression of each gene is normalized by that of the "stable referent gene" (or "housekeeping gene" RPL13A, ribosomal gene).

The results ("fold change" (Fc)) are expressed relative to the control condition. The genes used are sensitive to hypoxia (HIF-1 alpha signalling pathway) and are collated in

Table 1 below.

[Table 1]

Three hypoxia-regulated genes (BNIP3, EGLN3 and CA9) were used in the following examples. Three tests were performed according to the protocol described above for each gene. The results are expressed as a function of the expression of the referent gene RPL13 (control) (1.00) and indicated in the table below as the mean, and also the standard deviation between parentheses.

Results and conclusions: All the results are expressed as fold change (fc) relative to the untreated control after normalization of the relative expressions with respect to the expression of a housekeeping gene (RPL13). The expression of a gene is considered to be stimulated when it is multiplied at least by 1.5.

Table 2 below shows the effects of an aqueous-alcoholic extract of the aerial parts of Oenothera biennis on the expression of a selection of genes in connection with the effects described for hypoxia for an anti-hair loss benefit.

[Table 2]

The results show that the extract of aerial parts of Oenothera biennis at 0.1 mg/mL induces a significant stimulation of the expression of the genes EGLN3, BNIP3, CA9. These genes are described in the literature as being associated with the HIF-1 biological pathway (Pouyssegur J et al., M EDECI NE/SCIENCES 2002; 18: 70-8). When the HIF-1 transcription factor is stabilized (in a hypoxic medium or following a particular treatment), this factor is translocated to the nucleus of the cells and activates a large number of genes, including EGLN3, BNIP3 and CA9, which are associated with the regeneration of tissues such as skin and hair.

The aqueous-alcoholic extract of aerial parts of Oenothera biennis therefore has an advantage for stimulating the growth of human keratin fibres such as head hair, body hair, eyebrows and eyelashes and/or slowing down the loss thereof and/or increasing the hair density.

Example 3 - Cosmetic composition: anti-hair loss hair lotion

The following composition was prepared.

[Table 3]

The composition was applied to the scalp and head hair and makes it possible to obtain a stimulating effect on the growth of head hair.

Claims

Claims Non-therapeutic cosmetic use of an aqueous-alcoholic extract of aerial parts of Oenothera biennis to induce and/or stimulate the growth of human keratin fibres such as head hair, eyelashes, body hair and eyebrows and/or to slow down the loss thereof, and in particular to treat androgenic alopecia. Use according to Claim 1 , in which said extract is obtained by extraction of the aerial parts in a mixture of water and at least one alcohol, preferably a monoalcohol having from 2 to 6 carbon atoms, preferably ethanol. Use according to Claim 1 or 2, in which the aerial parts are chosen from the flowers, the leaves and/or the stems of Oenothera biennis. Use according to any one of the preceding claims, in which the mixture of water and at least one alcohol is a mixture comprising from 20% to 60% by volume of water relative to the total mixture volume, and from 40% to 80% by volume of alcohol relative to the total mixture volume, preferably the mixture of water and at least one alcohol is a mixture comprising from 30% to 55% by volume, preferably from 45% to 55% by volume, of water relative to the total mixture volume, and from 45% to 60% by volume, preferably from 47% to 55% by volume, of alcohol relative to the total mixture volume. Use according to any one of the preceding claims, in which the mixture of water and at least one alcohol is a mixture comprising 50% by volume of water relative to the total mixture volume, and 50% by volume of alcohol relative to the total mixture volume. Use according to any one of the preceding claims, in which said extract is obtained from the aerial parts which have been pre-ground and extracted using an aqueous- alcoholic mixture, preferably a 50% mixture. Use according to any one of the preceding claims, in which said extract is obtained by a process comprising the following steps: a. grinding the dried aerial parts of Oenothera biennis to fine particles in a grinder; b. extracting by macerating in a mixture of water and at least one alcohol, at a temperature of from 15°C to 45°C, in particular from 15°C to 40°C and notably at room temperature, notably by introducing from 10 g to 500 g of plant powder obtained in a, per litre of water and alcohol mixture, in particular from 50 g to 200 g, and more particularly from 70 g to 150 g for instance 100 g of plant powder obtained in a, per litre of water and alcohol mixture, and by maintaining at this temperature for 1 to 5 hours, notably 1 to 3 h, in particular 1 h 30 min to 2 h 30 min, for instance 2 hours; then c. filtering the mixture obtained in b; d. partial or complete evaporation of the alcohol in the filtered mixture obtained in c; and e. optionally freeze drying of the aqueous residue obtained in d. Use according to any one of the preceding claims, in which said extract comprises at least two, preferably at least three, preferably at least four, preferably at least five molecules, preferably at least six molecules, preferably at least seven molecules, preferably at least eight molecules, chosen from oenothein B, oenothein A, ellagic acid, gallic acid, quercetin, quercetin-3-O-galactoside, quercetin-3-O-glucoside, quercetin-3-O-glucuronide, and luteolin-7-O-glucuronide; preferably it comprises the following nine molecules: oenothein B, oenothein A, ellagic acid, gallic acid, quercetin, quercetin-3-O-galactoside, quercetin-3-O- glucoside, quercetin-3-O-glucuronide, and luteolin-7-O-glucuronide. Use according to any one of the preceding claims, in which said extract is a dry extract. Method for the non-therapeutic cosmetic treatment of human keratin fibres and/or the scalp, comprising the application of a cosmetic composition comprising at least one aqueous-alcoholic extract of aerial parts of Oenothera biennis as defined according to any one of Claims 1 to 9 on said fibres such as head hair, eyelashes, body hair and eyebrows, and/or on the scalp, to induce and/or stimulate the growth and/or to slow down the loss of said fibres.