Use of an extract of Populus Nigra as a slimming agent

Abstract

The invention relates to the cosmetic use of a Populus nigra leaf extract as a slimming agent, and more particularly as an anti-adipogenesis and lipolytic agent. The invention also relates to the use of a Populus nigra leaf extract in a slimming composition.

Description

Title of the invention: Use of an extract of Populus nigra as a slimming agent

[0001] The present invention relates to the field of cosmetic skin treatment. It concerns a cosmetic composition comprising an extract of Populus nigra.

[0002] The skin is a superficial covering organ that represents a protective barrier against external aggressions and covers the entire body surface. It is often described as the heaviest organ of the human body. Furthermore, the skin plays an active role in numerous biological and biochemical processes.

[0003] It consists of three superimposed layers or tissues comprising, from the most superficial to the deepest part, the epidermis, the dermis and the hypodermis.

[0004] This latter tissue, also called adipose tissue, represents 15 to 20% of body weight in an average-weight individual. It is attached to the lower part of the dermis by expansions of collagen fibers. The thickness of the hypodermis varies according to age, body location, and sex, and largely determines the body's contours.

[0005] In addition to being a vascularized adipose tissue containing adipocytes whose role is to absorb fats and store them as triglycerides, it is a storage organ in which fats can be mobilized and transformed into energy in the event of prolonged physical exertion. It also serves as a mechanical protection organ against shocks and a thermal protection organ against temperature variations in order to maintain bodily homeostasis. It controls lipid mobilization through two main metabolic activities: lipogenesis (synthesis of fatty acids (FAs) and triglycerides, and storage of excess energy) and lipolysis (hydrolysis of triglycerides when energy is needed).

[0006] Adipose tissue is composed of:

[0007] - of specific cells, adipocytes, which have the ability to store fats (lipogenesis) but also to mobilize them (lipolysis),

[0008] - of an extracellular matrix,

[0009] - of a dense capillary network.

[0010] The development of adipose tissue is characterized by a modification of all the components of the hypodermal tissue; it has repercussions at the level of the more superficial layers. At the level of the dermis, an increase in thickness is observed, probably due to fluid engorgement (water retention) and infiltration of adipose lobules deforming the dermis / hypodermis junction; on the surface the skin takes on a dimpled appearance, this is the famous "orange peel" appearance.

[0011] The development of adipose tissue involves various mechanisms:

[0012] - the reorganization of the extracellular matrix,

[0013] - the differentiation of pre-adipocytes into adipocytes,

[0014] - the increase in the volume of adipocytes.

[0015] Mature adipocytes constitute the majority of the mass of adipose tissue; they result from adipogenesis.

[0016] Adipogenesis consists of the transformation of preadipocytes in adipose tissue into adipocytes. Adipogenesis follows a highly controlled process in which different transcription factors and regulatory proteins act successively to form mature adipocytes. During the first hours of adipogenesis, in particular, a profound modification of the "chromatin landscape" (i.e., opening of DNA and expression of genes) occurs, which coincides with the cooperative binding of numerous early transcription factors (or trans factors), including C / EBP-[3, C / EBP-β and stat5α, or the zinc finger trans factor ZNF-638. The binding of these trans factors allows the binding of other factors during preadipocyte differentiation, in particular, PPAR-y, via binding with a p204 cofactor (Proença et al. 2014, doi: 10.1590 / 141 / -431X20132911).

[0017] In adipocytes, triglycerides (TAGs), the major cellular constituents of adipose tissue, are stored in a single, large lipid inclusion called a "lipid droplet." Unlike vesicular organelles, which are surrounded by a phospholipid bilayer (like cell membranes), lipid droplets are surrounded by only a single phospholipid monolayer that delimits a highly hydrophobic content consisting mainly of triglycerides and sterol esters.

[0018] In adipose tissue, these droplets occupy 80 to 90% of the volume of the adipocyte, and a central position, displacing the nucleus, cytosol and remaining organelles to the periphery of the cell close to the cell membrane.

[0019] For the synthesis of TAGs, three fatty acid molecules are associated with one glycerol molecule. There is very little de novo lipogenesis in human adipose tissue (with the formation of fatty acids from non-lipid substrates). The fatty acids in TAGs are derived from free fatty acids bound to albumin from the bloodstream, or from the hydrolysis of TAGs transported in the blood by lipoproteins.

[0020] Hormonal regulation is very important in lipid metabolism: while insulin increases glucose uptake and stimulates lipogenesis, leptin, conversely, limits lipid storage by inducing, among other things, the release of glycerol of adipocytes and by inhibiting fatty acid synthesis (Proença et al. 2014, doi: 10.1590 / 141 / -431X20132911).

[0021] Lipolysis corresponds to the catabolism of triglycerides stored in adipocytes, that is, their breakdown into non-esterified fatty acids and glycerol. The molecules thus released into the bloodstream can be used by other tissues as an energy substrate (fatty acids) or serve as gluconeogenic precursors (glycerol).

[0022] The mobilization of fatty acids from triglycerides stored in adipose tissue requires the activation of a lipolytic enzyme, hormone-sensitive lipase (HSL). HSL hydrolyzes triglycerides into diglycerides and then diglycerides into monoglycerides; it is the rate-limiting enzyme of lipolysis. Monoglyceride lipase (MGL) then breaks down the monoglycerides into non-esterified fatty acids and glycerol.

[0023] Surprisingly, the Applicant has highlighted the effect of an extract of Populus nigra leaves both on the inhibition of the differentiation of preadipocytes into adipocytes (adipogenesis) and on their destocking (lipolysis).

[0024] Due to its properties, the extract of Populus nigra leaves therefore appears to be a useful active ingredient as a slimming agent.

[0025] The invention therefore relates to the cosmetic use of an extract of Populus nigracommc leaves as a slimming agent.

[0026] According to a preferred embodiment, Populus nigra leaf extract is used in a slimming cosmetic composition.

[0027] The cosmetic use according to the invention is particularly suited to topical application on healthy skin. For the purposes of this invention, healthy skin means skin that does not present any skin pathology and is also in good condition (within the meaning of Article 2.1a) of REGULATION (EC) No 1223 / 2009 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 30 November 2009 on cosmetic products). Cosmetic use therefore means non-therapeutic use.

[0028] The use according to the invention is particularly suitable for topical application on the skin of the face as well as for application on the skin of the body.

[0029] The black poplar or Populus nigra is a tree in the Salicaceae family, reaching 30 m in height. It is common in moist, sunny areas and grows up to 1800 m in altitude across Europe, as far as Central Asia and the coasts of North Africa. It is a very long-lived species, around 200 years and up to 400 years, and a pioneer species of alluvial forests. Like many trees in the Salicaceae family, it has flexible inflorescences—or catkins—that are drooping and appear before the leaves. Its leaves, 5 to 10 cm long, are triangular— Oval, glossy, with small, regular teeth. The new shoot leaves are larger and less wedge-shaped at the base than those of the adult branches.

[0030] Its buds are glossy and its fruits are hairless, containing cottony seeds. Flowering takes place from March to April.

[0031] Populus nigra has high therapeutic potential due to its antioxidant, anti-inflammatory, hepatoprotective, hypouricemic, antitumor, and antimicrobial properties. It was traditionally used to treat arthritis, rheumatism, liver disorders, endothelial dysfunction, respiratory infections, and fever. The leaves and buds are the parts of the plant most commonly used in the form of alcoholic extracts and infusions as potent antioxidant and anti-inflammatory agents. The leaves are primarily antioxidant.Flower buds are the most studied: they are recommended for kidney and bladder diseases, bronchitis and respiratory illnesses, intermittent fevers, rheumatism, hemorrhoids, gout, sciatica, as an antimicrobial, antidiabetic, and healing agent, for dermatological problems, for their antitumor properties, cardiovascular, urate-lowering, and hepatoprotective effects. Flower buds are also said to have beneficial effects on skin aging because they exhibit strong antioxidant, anti-inflammatory, and cell-regenerating activity. They also have a skin-brightening capacity: they form a potent tyrosinase inhibitor and reduce melanin concentration.

[0032] According to a particular embodiment of the invention, the Populus nigra leaf extract is a hydroglycerinated extract obtained by a process comprising the following steps which a person skilled in the art will be able to carry out to perform an extraction from tree leaves:

[0033] - Aqueous extraction of dried leaves of Populus nigra;

[0034] - Solid / liquid separation;

[0035] - Clarification (filter and diatomaceous earth);

[0036] - Concentration;

[0037] - Addition of glycerin;

[0038] - Heat decontamination.

[0039] The extract of Populus nigra leaves thus obtained is an amber to brown liquid with a characteristic odor.

[0040] It has the following analytical characteristics:

[0041] - pH 5 - 7

[0042] - density at 20°C: 1.150 - 1.250 g / ml

[0043] - dry matter: 15-25% by weight

[0044] - refractive index: 1.400 - 1.460

[0045] - Polyphenol content: ≥ 1.0 g / L

[0046] The cosmetic use according to the present invention preferably employs an extract of Populus nigra leaves or a cosmetic composition comprising such an extract.

[0047] The extract according to the invention is an extract of Populus nigra leaves, more particularly said extract is a water-soluble extract.

[0048] Advantageously, the Populus nigra leaf extract is a hydroglycerinated extract.

[0049] Preferably, the cosmetic composition according to the invention comprises from 0.01 to 10% of a Populus nigra leaf extract by weight of the total composition. Advantageously, the composition comprises from 0.01 to 5% of a Populus nigra leaf extract by weight of the total composition.

[0050] The compositions according to the invention may comprise one or more formulation agents or additives of known and conventional use in cosmetic compositions such as, by way of example and without limitation, emollients, colorants, film-forming agents, surfactants, perfumes, preservatives, emulsifiers, oils, glycols, vitamins such as vitamin E, UV filters, etc. Thanks to their knowledge of cosmetics, a person skilled in the art will know which formulation agents to add to the compositions of the invention and in what quantities, depending on the desired properties.

[0051] The compositions according to the invention can be in any form known to those skilled in the art in the field of cosmetology, with no other galenic restriction than a form suitable for application to the face and body. Advantageously, the compositions according to the invention are in the form of a gel, cream, lotion, mask, oil, milk, spray, etc.

[0052] The invention also relates to the use of an extract of Populus nigra leaves or of a cosmetic composition comprising such an extract, to tone and / or firm the skin, reduce dimpling, smooth / reduce skin texture due to cellulite, refine and / or redefine the silhouette, reduce subcutaneous fat deposits, prevent and / or delay the cutaneous manifestations of increased adipose tissue such as cellulite and / or dimpling.

[0053] The following examples relate, firstly, to the effect of a Populus nigra leaf extract on the differentiation of pre-adipocytes into mature adipocytes in 2D, a process called adipogenesis, and secondly, to the effect of a Populus nigra leaf extract on mature human adipocytes in 3D, reflecting its action on lipolysis. Finally, examples of cosmetic formulations using Populus nigra leaf extract illustrate compositions that are the subject of the present invention.

[0054] [Fig. 1] Figure 1 shows the effect of a Populus nigra leaf extract, as described above, on adipogenesis. This figure represents the number of cells after 7 days (end of culture). The statistical analysis consists of an unpaired t-test (in gray) and ordinary one-way ANOVA with Dunnett's multiple comparisons test (in black).

[0055] [Fig.2] Fig.2 shows the effect of an extract of Populus nigra leaves such as described previously regarding lipolysis. This figure represents the release of non-esterified fatty acids (NEFAs) by cultured adipocytes after two hours of treatment. Statistical analysis consists of an unpaired t-test (in gray) and ordinary one-way ANOVA with Dunnett's multiple comparisons tests (in black).

[0056] Significant differences are indicated with asterisks (*) on the graphs according to the following results: * p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001; **** p-value < 0.0001.

[0057] I. Effect of a Populus nigra leaf extract on adipogenesis

[0058] Adipogenesis is the process of cell differentiation by which preadipocytes become adipocytes; its inhibition therefore makes it possible to limit the development of adipose tissue. A. Materials and Methods 1. Cell culture

[0059] Pre-adipocytes are isolated from human subcutaneous adipose tissue after digestion with collagenase.

[0060] The adipogenesis model consists of culturing human pre-adipocytes in 2D under pro-adipogenic conditions. The evaluation criterion is the number of cells quantified by image processing after fluorescent labeling of lipid droplets and cell nuclei.

[0061] Pre-adipocytes are cultured for 24 hours in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) in 96-well plates, then:

[0062] (i) incubated in DMEM / F12 basal medium (condition without differentiation); or

[0063] (ii) incubated in a pro-adipogenic cocktail containing insulin, glucocorticoid, 3-isobutyl-l-methylxanthine (IBMX) and thiazolinedione (condition with differentiation and adipogenesis = positive control); or

[0064] (iii) incubated in a pro-adipogenic cocktail containing insulin, glucocorticoid, 3-isobutyl-l-methylxanthine (IBMX) and thiazolinedione, and treated with the PPARg antagonist (GW9662, 1 pM, M6191, Sigma) (condition without differentiation and without adipogenesis = negative control, condition GW9662).

[0065] The medium is changed every 2-3 days for 7 days. Populus nigra leaf extract is added at three different concentrations: 0.1%; 0.05%; and 0.01% expressed as v / v in condition (ii).

[0066] All conditions are fulfilled in triplicate. 2. Measurement of cell accumulation

[0067] After 7 days of culture, the pre-adipocytes are fixed with 4% paraformaldehyde and then labeled at room temperature with Bodipy and DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) to reveal intracellular lipid droplets and nuclei respectively.

[0068] Cell number quantification is performed by hnactiv-3D with automated fluorescence quantification for lipid droplets and nucleus detection. The data are represented as the number of cells expressed as a percentage relative to the control condition.

[0069] The quantification of the number of cells is obtained by image acquisition and carried out according to the following procedure:

[0070] - Acquisition of microphotographs with an apotome videomicroscope (Cell Insight CX7, ThermoFisher) (7 fields of view per 1 mm2 well composed of Z-Stacks of 10 images spaced 7 pm apart, 3 wells per treated conditions);

[0071] - Quantification of the surface area of ​​the labeling and the intensity of the lipids with the image processing with Image J;

[0072] - Determination of the number of nucleated cells with a detection algorithm shapes based on the size and circularity of the kernels.

[0073] The data are represented as a percentage based on the quantification carried out with the positive control (differentiation and therefore adipogenesis), the number of cells of which is arbitrarily set at 100. B. Results

[0074] Condition GW9662 (negative control / without adipogenesis) was compared to the differentiation condition / with adipogenesis (i.e., in % of the differentiated condition for undifferentiated, GW9662 and Populus nigra leaf extract).

[0075] Internal controls are validated with maintenance of cell number and viability regarding the undifferentiated conditions (95.94%) and GW9662 (91.23%) compared to the differentiation / adipogenesis condition (100%).

[0076] All doses of Populus nigra leaf extract tested induced a decrease in cell number compared to the differentiation condition: 0.1%: 54%; 0.05%: 65.7%; and 0.01%: 74.5%, as illustrated in [Fig. 1]. Since these cell number results are less than 100%, they illustrate the anti-adipogenic effect of the Populus nigra leaf extract, given that these decreases in cell number cell loss is not correlated with cell detachment or cell distress.

[0077] IL The effect of Populus leaf extract will not be on lipolysis

[0078] This test evaluates the hydrolysis and release of non-esterified fatty acids (NEFAs) by mature adipocytes under different conditions. The induction of lipolysis promotes the reduction of adipose tissue. A. Materials and Methods 1. Cell culture

[0079] Mature adipocytes are isolated from subcutaneous adipose tissue after gentle digestion with collagenase. The isolated adipocytes are washed before being encapsulated in a peptide hydrogel to form 3D adipocyte capsules. This 3D encapsulation of adipocytes is described in WO2011 / 148310A1. The cells are incubated for 12–24 hours at 37°C for stabilization. Treatments are initiated on day 0 with a change of medium.

[0080] Conditions:

[0081] - Control cells in the basic medium (untreated reference);

[0082] - Positive control of lipolysis: cells in the basal medium, treated by isoproterenol at 1 pM;

[0083] - Tests: cells in the base medium, treated with the 3 concentrations of the extract of Populus nigra leaf (0.1%; 0.05%; and 0.01% expressed as v / v).

[0084] Culture media are collected after 2 hours of treatment to measure NEFA (Non-Esterified Fatty Acids).

[0085] Each culture condition was carried out in triplicate. 2. Measurement of lipid accumulation

[0086] The culture media from the different conditions are collected after 2 hours of treatment. The concentrations of non-esterified free fatty acids (NEFA) are evaluated by colorimetric tests using specific kits in accordance with the supplier's recommendations (Non Esterified Fatty Acids kit, FA 115, Randox).

[0087] NEFA release from adipocytes is measured after 2 hours of treatment to assess lipolytic activity. The results are expressed as a percentage of the control condition. B. Results

[0088] Lipolytic activity controlled by adrenergic signaling pathways mediated by beta-adrenergic receptors can occur rapidly, within a few hours with, for example, stimulation by isoproterenol, which induces a significant increase in the release of NEFAs after 2 hours of treatment, a sign of lipolysis.

[0089] The positive control (isoproterenol 1 pM) is validated with an increase in the release of NEFA (174% of NEFA in the medium; i.e. +74% lipolysis) compared to the control condition (100%; i.e. 0% lipolysis).

[0090] In this experiment, regardless of the dose used (0.1%; 0.05% and 0.01%), Populus nigra leaf extract significantly increases lipolysis after 2 hours of treatment (0.1%: 119.42% of NEFA released; 0.05%: 117.27% and 0.01%: 117.99%) as illustrated in [Fig.2]. III. Examples of GEL cosmetic formulations (% by weight)

[0091] AQUA 85,911

[0092] CAFFEIN 0,50

[0093] BETAINE 1,00

[0094] GLYCERIN 4,00

[0095] ETHYLHEXYLGLYCERIN 0,20

[0096] PROPANEDIOL 1,50

[0097] PENTYLENE GLYCOL 1,50

[0098] AMMONIUM ACRYLOYLDIMETHYLTAURATE / VP COPOLYMER 1,20

[0099] POLYACRYLATE CROSSPOLYMER-6 / AQUA / t-BUTYL ALCOHOL 0,50

[0100] ESCIN 0,10

[0101] SODIUM HYDROXIDE 0,009

[0102] AQUA / AVENA SATIVA KERNEL EXTRACT / SODIUM BENZOATE 2,00

[0103] EXTRAIT DE POPULUS NIGRA 0,50

[0104] GLYCERIN / AQUA / PANAX GINSENG ROOT EXTRACT 1,00 EMULSION (% en poids)

[0105] ISONONYL ISONONANOATE 3,50

[0106] DICAPRYLYL CARBONATE / TOCOPHEROL 1,50

[0107] C14-22 ALCOHOLS / C12-20 ALKYL GLUCOSIDE 3,00

[0108] CETYL ETHYLHEXANOATE 3,50

[0109] SODIUM ACRYLATES COPOLYMER / HYDROGENATED POLYDECENE / PHOSPHOLIPIDS / POLYGLYCERYL-10 STEARATE / HELIANTHUS ANNUUS (SUNFLOWER) SEED OIL / TOCOPHEROL 2,50

[0110] AQUA 63,35 [OUI] CAFFEINE 1,80

[0112] BUTYLENE GLYCOL 1,50

[0113] SODIUM POLYACRYLATE 0,30

[0114] GLYCERIN 5,00

[0115] POTASSIUM CETYL PHOSPHATE 0,50

[0116] ESCIN 0,15

[0117] AQUA / METHYLSILANOL MANNURONATE / PHENOXYETHANOL / SORBIC ACID 1,50

[0118] EXTRAIT DE POPULUS NIGRA 0,50

[0119] GLYCERIN / AQUA / SCABIOSA ARVENSIS EXTRACT / POTASSIUM SORBATE 1,00

[0120] PARFUM 0,40

[0121] ALCOHOL 10,00

Claims

Demands

1. Cosmetic use of an extract of Populus nigra leaves as a slimming agent.

2. Cosmetic use of an extract of Populus nigra leaves according to claim 1, as an anti-adipogenesis agent.

3. Cosmetic use of an extract of Populus nigra leaves according to claim 1, as an anti-lipolytic agent.

4. Cosmetic use of an extract of Populus nigra leaves in a slimming composition.

5. Use according to any one of the preceding claims, wherein the Populus nigra leaf extract is a water-soluble extract.

6. Use according to any one of the preceding claims, wherein the Populus nigra leaf extract is a hydroglycerinated extract.

7. Use according to any one of the preceding claims, characterized in that said extract is formulated in a cosmetic composition comprising from 0.01 to 10% of a Populus nigra leaf extract by weight relative to the total weight of the composition, preferably 0.01 to 5% of said extract by weight relative to the total weight of the composition.

8. Use according to claim 4 or 7, wherein said composition further comprises one or more formulation agents or additives of known and conventional use in cosmetic compositions such as softeners, colorants, film-forming agents, surfactants, perfumes, preservatives, emulsifiers, oils, glycols, vitamins such as vitamin E, and UV filters.

9. Use according to any one of the preceding claims for topical application to the skin of the face and / or body.

10. Use according to any of the preceding claims to tone and / or firm the skin, reduce dimpling, smooth / reduce skin texture due to cellulite, refine and / or redefine the silhouette, reduce subcutaneous fat deposits, prevent and / or delay the cutaneous manifestations of increased adipose tissue such as cellulite and / or dimpling.