FR2834639A1 - Topically applied dermatological or cosmetic composition, used e.g. for promoting skin regeneration and treating edema, contains flavonoid and vegetable oil containing linoleic and/or alpha-linolenic acid - Google Patents

Abstract

Composition (I) comprises: (A) at least one flavonoid, and (B) at least one vegetable oil containing at least 20 (preferably 30-50) wt.% linoleic acid and/or at least 10 (preferably 25-40) wt.% alpha-linolenic acid.

Description

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 The present invention relates to a novel composition comprising in combination at least one flavonoid and at least one vegetable oil rich in linoleic acid and / or alpha-linolenic acid. The subject of the invention is also such a composition for its use as a dermatological or dermatocosmetic agent, especially as an anti-inflammatory agent and as a regenerating agent for epidermal and dermal tissue, or for its use in the prevention or treatment of oedemas, pressure sores, or disorders of the cutaneous microcirculation, or in the treatment of radiodermites.

 Numerous compositions for the skin, based on peroxidized bodies such as peroxide oils, have been used in the prior art, in particular to facilitate the cellular restructuring of the skin of the face or the body, or to treat the inflammatory condition related to the skin. dry (FR 2 591 104), or to preserve and improve the state of the skin, for example by preventing disturbances of microcirculation (FR 2 750 331).

 The Applicant has surprisingly discovered that the combination of the following two compounds: a bioflavonoid, such as troxerutin, and a vegetable oil rich in linoleic acid and / or alpha-linolenic acid, in compositions for the skin, such as dermatological or dermatocosmetic compositions, made it possible to treat various disorders and skin conditions, and this much more effectively than the compositions based on peroxide compounds used until now.

 It has thus been found that the combination of the two compounds according to the present invention have various advantages, as compared with the use of a peroxygenated oil in a composition for cutaneous application, such as an improvement of the micro-circulatory dynamics, a reduction in induced capillary dilation and secondary interstitial edema, an increase in the thickness of the compact horny layer, a positive modulation on the protection of structural proteins of the epithelium, an increase in total lipids, as well as stimulation of epithelial proliferation.

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 In addition, the flavonoids as well as the vegetable oils prove to be compounds, cosmetically, pharmaceutically and dermatologically acceptable, non-aggressive, neither toxic nor irritating for the skin, hypoallergenic, soothing, moisturizing and anti-inflammatory for the skin. In addition, these compounds are commercially available.

 Thus, the Applicant has shown that the following active ingredients: flavonoid and vegetable oil, to which may be added another compound such as a peptide extract of lupine, could be used in combination in pharmaceutical, dermatological or dermatological cosmetic compositions, especially in as anti-inflammatory agents, as regenerating agents of the epidermal and dermal tissue, in the prevention or treatment of oedemas, bedsores, or disorders of the cutaneous microcirculation, or in the treatment of radiodermites.

 The subject of the present invention is thus a composition comprising in combination: (a) at least one flavonoid, and (b) at least one vegetable oil containing at least 20% by weight of linoleic acid and / or at least 10% by weight of alpha-linolenic acid, relative to the total fatty acids of the oil.

 Linoleic acids (Cl8: 2) are fatty acids of the n-6 family, and alpha-linolenic acids (Cl 8: 3) are fatty acids of the n-3 family.

 The vegetable oil (b) according to the present invention is an oil rich in linoleic acid and / or alpha-linolenic acid.

 For the purposes of the present invention, the term "oil rich in linoleic acid" means oils containing at least 20% by weight, preferably 20 to 50% by weight, of linoleic acid relative to the acids. total fat of the oil. As an example of an oil rich in linoleic acid, mention may be made of sunflower oil or grape seed oil.

 By the term "oil rich in alpha-linolenic acid" is meant in the sense of the present invention, the oils containing at least 10% by weight, preferably 10 to 40% by weight, of alpha-linolenic acid compared to the total fatty acids of the oil.

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 As an example of an oil rich in alpha-linolenic acid, mention may be made of chia oil, linseed oil and rosehip oil.

 In a particular embodiment of the present invention, the vegetable oil (b) contains 30 to 50% by weight of linoleic acid and / or 25 to 40% by weight of alpha-linolenic acid.

 Advantageously according to the present invention, the vegetable oil (b) is Rosehip Muscat oil. This oil may have a linoleic acid content of about 30 to 50% by weight, and an alpha-linolenic acid content of about 25 to 40% by weight.

The Rosehip Muscat oil can be extracted from the seeds of the rose fruit, advantageously by the process of extracting the oil by cold pressing.

 Flavonoids, also known as bioflavonoids, are polyphenols with a 15 carbon base backbone resulting from the fusion of two aromatic units, one of which is derived from benzene and the other from phenylpropane.

In a particular embodiment of the present invention, the flavonoid (a) is a flavanone, of general formula (1):

In another particular embodiment of the present invention, the flavonoid (a) is a flavone of general formula (II):

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The flavones or flavanones that can be used according to the present invention are obtained by chemical synthesis or are natural substances extracted from natural products, in particular from plants. The flavonoids (a) according to the present invention may be in aglycone form or in glycosylated form. The glycosylated forms of the flavonoids according to the present invention, which are the most abundant forms in nature, may comprise one or more monosaccharides, which may especially be rutinose, mannose, rhamnose, glucose, etc. Advantageously according to the present invention, when the flavonoids (a) are in the form of glycolysed, they are substituted with a 0-rutinose group, preferably in position 3 or 7.

 Advantageously according to the present invention, when the flavonoid (a) is a flavanone, said flavonoid is selected from the group consisting of naringenin, naringin, hesperetin, and hesperidin.

 Advantageously according to the present invention, when the flavonoid (a) is a flavone, said flavonoid is selected from the group consisting of rutin, troxerutin, diosmin, diosmetin, chrysin, quercetin and luteolin.

 The flavonoid (a) according to the present invention may also be a flavonol which corresponds to a flavone substituted in the 3-position by a hydroxy group, or a dihydroflavonol which corresponds to a flavanone substituted in the 3-position by a hydroxyl group. The flavonoid (a) may also be a flavanol which corresponds to the following general formula (III), or a flavandiol (leucoanthocyanidin) which corresponds to a flavanol substituted in the 4-position by a hydroxyl group.

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The cathechine or leucocyanidin can thus be used in particular as flavonoid (a) according to the present invention.

The flavonoid (a) according to the present invention may also be a biflavonoid, or an isoflavonoid, such as an isoflavone or isoflavanone.

Isoflavanones and isoflavones correspond respectively to flavanones (1) and flavones (II), but whose phenyl group, numbered from 1 to 6 on formulas (I) and (II) above, is substituted in position 3 , instead of being substituted in position 2.

Advantageously according to the present invention, the flavonoid (a) and the vegetable oil (b) constitute the only active ingredients of the composition.

Advantageously according to the present invention, the composition also contains: (c) at least one peptide extract of lupine. Peptide extracts of lupine (c) according to the present invention can be extracted from various varieties of lupine (lupine).

Advantageously according to the present invention, the extracts (c) are derived from the sweet white lupine (Lupinus albus), of European genus, preferably the Ares variety of low alkaloid content.

In general, the invention comprises lipid lupine flours, or peptide extracts further comprising sugars.

Advantageously according to the present invention, the peptide extract of lupine (c) comprises glutamate peptides of low molecular weight and oligosaccharides.

Advantageously according to the present invention, the peptide extract of lupine (c) is obtained by hydrolysis of the protein fraction of lupine, which contains mainly amino acids, peptides and proteins. The hydrolysis may be carried out by any suitable means, including enzymatic, acidic or basic hydrolysis. Advantageously according to the present invention, when the peptide extract of lupine (c) is hydrolysed, said extract, in the form of an aqueous solution,

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 contains 45 to 55 g / l of dry matter, 24 to 32 g / l of protein and 8 to 14 g / l of total sugars (simple sugars and oligosaccharides). A peptide extract of lupine (c) according to the present invention may be commercially available from Silab (Structurine).

- éventuellement séparation de la fraction protéique, - éventuellement hydrolyse de la fraction protéique et récupération, éventuellement après filtration, de l'extrait protéique. In a particular embodiment of the present invention, the peptide extract of lupine (c) can be obtained according to the process comprising the following steps: - preparation of a delipidated and ground lupine cake or a lupine meal ( micronized lipid), - extraction of soluble protein and osic fractions or precipitation at acidic pH (4 or 5) according to the isoelectric point,

- optionally separation of the protein fraction, - optionally hydrolysis of the protein fraction and recovery, optionally after filtration, of the protein extract.

Preferably, the protein extract has the following amino acid composition (the concentrations given below may vary by more or less than 50%):

<Tb>
<tb> Aspartic <SEP> 5.0 <SEP> g / 100 <SEP> g
<tb> Threonine <SEP> 2. <SEP> 1 <SEP> g / 100 <SEP> g
<tb> Serine <SEP> 2.8 <SEP> g / 100 <SEP> g
<tb> Acid <SEP> Glutamic <SEP> 9.8 <SEP> g / 100 <SEP> g
<tb> Glycine <SEP> 2 <SEP> g / 100 <SEP> g
<tb> Alanine <SEP> 2. <SEP> 1 <SEP> g / 100 <SEP> g
<tb> Valine <SEP> 1. <SEP> 8g / 100 <SEP> g
<tb> Cystine <SEP> 0.4 <SEP> g / 100 <SEP> g
<tb> Methionine <SEP> 0. <SEP> 1 <SEP> g / 100 <SEP> g
<tb> Isoleucine <SEP> 1. <SEP> 9g / 100 <SEP> g
<tb> Leucine <SEP> 3. <SEP> 6 <SEP> g / 100 <SEP> g
<tb> Tyrosine <SEP> 1. <SEP> 9g / 100 <SEP> g
<tb> Phenylalanine <SEP> 1. <SEP> 9g / 100 <SEP> g
<tb> Omithine <SEP> 0. <SEP> 1 <SEP> g <SEP> 1100 <SEP> g
<tb> Lysine <SEP> 2g / 100g
<tb> Histidine <SEP> 1. <SEP> 1 <SEP> g / 100 <SEP> g
<Tb>

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<Tb>
<tb> Arginine <SEP> 5g / 100g
<tb> Pro <SEP> line <SEP> 1. <SEP> 9 <SEP> g <SEP> 1100 <SEP> g
<Tb>

Advantageously according to the present invention, the flavonoid (a) is present at a concentration of between 0, 1 and 20%, even more advantageously between 1 and 10%, relative to the total weight of the composition.

 Advantageously according to the present invention, the vegetable oil (b) is present at a concentration of between 10 and 60%, even more advantageously between 20 and 40%, relative to the total weight of the composition.

 Advantageously according to the present invention, the peptide extract of lupine (c), when it is present in the composition according to the present invention, is present in a concentration of between 0, 1 and 20%, advantageously between 1 and 10%, relative to the total weight of the composition.

 The compositions according to the present invention may be pharmaceutical, dermatological or dermo-cosmetic compositions. The compositions according to the present invention may further contain cosmetologically and / or pharmaceutically acceptable carriers and excipients, as well as conventional cosmetic additives, known to those skilled in the art.

 Advantageously, the compositions according to the present invention are formulated to be administered externally externally, for example in the form of white or colored cream, ointment, milk, lotion, ointment, serum, paste, foam, or aerosol.

 Even more advantageously according to the present invention, the compositions are in the form of an emulsion. In particular, the compositions additionally contain a Pemulen emulsifier polymer. Even more advantageously, the compositions according to the present invention are packaged in the form of a spray.

 Pemulen are high molecular weight polymers with a long hydrophilic portion and a shorter lipophilic portion. Their chemical structure allows them to effectively play the role of emulsifier in oil-in-water emulsions. The lipophilic portion adsorbs at the oil-water interface, and the hydrophilic portion swells in the phase

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 aqueous solution to form emulsions of high stability with a wide range of oils.

 Emulsions made with small amounts of Pemulen are very stable.

Surrounded by an aqueous gel with a high flow threshold, the oil droplets are protected and kept in suspension. By forming oil-in-water (O / W) emulsions, the Pemulen molecules form a layer of gel adsorbed around each oil droplet, the lipophilic portion of the polymer being fixed in the oil phase. Thus, when two droplets approach one another, a repulsive force is generated by the presence of these adsorbed gel layers.

surfactants, de faibles quantités de surfactants pouvant toutefois être ajoutées pour i optimiser certaines caractéristiques de l'émulsion. A l'application, une couche homogène d'huile se forme immédiatement sur la peau. Ce film ne contient aucun résidu de surfactant et résiste à l'immersion dans l'eau. Pemulen are thus advantageously used in the context of the present invention, because they overcome many disadvantages encountered in conventional emulsions. They eliminate the potential irritation that can be associated with

surfactants, however, small amounts of surfactants can be added to optimize certain characteristics of the emulsion. On application, a homogeneous layer of oil is formed immediately on the skin. This film contains no surfactant residues and resists immersion in water.

 These Pemulen polymers also have various other advantages over conventional emulsifiers: they are effective at very low concentration (0.1% to 0.5%), thus reducing formulation costs; they emulsify any oil, even at room temperature; they emulsify the waxes, if they are melted during the preparation; stable emulsions can be made with surfactants which will be used in small quantities to optimize the size of the oil droplets and provide additional emollient or spreading, or other aesthetic properties; they have excellent long-term stability (over two years); the release of the oily phase can be triggered upon contact with surfaces containing electrolytes.

 The present invention also relates to the compositions described above for their use as dermatological or dermato-cosmetic agents.

 The present invention also relates to the compositions described above for their use as anti-inflammatory agents or as agents

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 regenerators of the epidermal and dermal tissue, thus making it possible to increase the cellular renewal of the epidermis.

 The present invention also relates to the compositions described above for their use in the prevention or treatment of oedemas, pressure ulcers, disorders of the cutaneous microcirculation, or ulcers.

 The present invention also relates to the compositions described above for their use in the treatment of radiodermites. Radiodermatitis is particularly characterized by disorders of the cutaneous microcirculation and the regeneration of the epithelium.

The following examples are intended to illustrate the invention without in any way limiting its scope. Unless otherwise specified, the percentages given in the following examples are percentages by weight. In the following examples, rose hip oil was extracted from rose seed by cold pressing and the lupine peptide extract was provided by Silab (Structurine).

Examples of compositions according to the present invention: Example 1: Composition containing Troxerutin (a), Rosier Mucat Oil (b) and Peptide Lupine Extract (c)

<Tb>
<tb> Phases <SEP> Materials <SEP> first <SEP> 0/0
<tb> Aqueous <SEP> Water <SEP> purified <SEP> 55.55
<tb> Blend <SEP> preservatives <SEP> 0.8
<tb> Pemulen <SEP> TR2 <SEP> NF <SEP> 0.2
<tb> Carbopol <SEP> 981 <SEP> NF <SEP> 0.05
<tb> NaOH <SEP> (10% <SEP> m / m) <SEP> 0.8
<tb> Peptide SEP Extract <SEP> of <SEP> Lupine <SEP> 7
<tb> (Structurine)
<tb> Troxerutin <SEP> 5
<tb> Oil <SEP> of <SEP> Rose <SEP> Muscat <SEP> 30
<tb> Grasse
<tb> Blend <SEP> preservatives <SEP> 0.2
<tb> EDTA <SEP> di <SEP> Nua
<tb> (Dissolvine <SEP> Na <SEP> 2P)
<tb> Aroma <SEP> yerbatone <SEP> 0.3
<tb> TOTAL <SEP> 100
<tb> Composition <SEP> mixture <SEP> preservatives <SEP>: <SEP> Phenoxyethanol <SEP> 73, <SEP> 3 <SEP>%
<Tb>

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<Tb>
<tb> PHB <SEP> Methyl <SEP> 20, <SEP> 0 <SEP>%
<tb> PHB <SEP> Propyl <SEP> 6, <SEP> 7 <SEP>%
<Tb>

Exemple 2 : Composition contenant de la Troxérutine (a) et de l'huile de Rosier Mucat (b)

EXAMPLE 2 Composition Containing Troxerutin (a) and Rosier Mucat Oil (b)

<Tb>
<tb> Phases <SEP> Materials <SEP> first%
<tb> Aqueous <SEP> Water <SEP> purified <SEP> 62.55
<tb> Blend <SEP> preservatives <SEP> 0.8
<tb> Pemulen <SEP> TR2 <SEP> NF <SEP> 0.2
<tb> Carbopol <SEP> 981 <SEP> NF <SEP> 0.05
<tb> NaOH <SEP> (10% <SEP> m / m) <SEP> 0.8
<tb> Troxerutin <SEP> 5
<tb> Grasse <SEP> Oil <SEP> of <SEP> Rose <SEP> Muscat <SEP> 30
<tb> Blend <SEP> preservatives02
<tb> EDTA <SEP><SEP> Na <SEP> 0, <SEP> 1
<tb> (Dissolvine <SEP> Na <SEP> 2P)
<tb> Aroma <SEP> yerbatone <SEP> 0, <SEP> 3
<tb> TOTAL <SEP> 100
<tb> Composition <SEP> mixture <SEP> preservatives <SEP>: <SEP> Phenoxyethanol <SEP> 73,3 <SEP>%
<tb> PHB <SEP> Methyl <SEP> 20, <SEP> 0%
<tb> PHB <SEP> Propyl <SEP> 6, <SEP> 7%
<Tb>

Exemple 3 : Composition contenant de la Troxérutine (a) et de l'huile de Rosier Mucat (b)

Example 3 Composition Containing Troxerutin (a) and Rosier Mucat Oil (b)

<Tb>
<tb> Phases <SEP> Materials <SEP> first <SEP>%
<tb> Aqueous <SEP> Water <SEP> purified <SEP> 62.2
<tb> Blend <SEP> preservatives <SEP> 0.8
<tb> Pemulen <SEP> TR2 <SEP> NF <SEP> 0.2
<tb> Carbopol <SEP> 981 <SEP> NF <SEP> 0.05
<tb> EDTA <SEP> di <SEP> Na <SEP> (Dissolvine <SEP> NA <SEP> 2 <SEP> P) <SEP> 0, <SEP> 1
<tb> Troxerutin <SEP> 5
<tb> Grasse <SEP> Oil <SEP> of <SEP> Rose <SEP> Muscat <SEP> 30
<tb> Blend <SEP> preservatives <SEP> 0.2
<tb> Butylhydroxyanisole <SEP> (BHA) <SEP> 0, <SEP> 015
<tb> Butylhydroxytoluene <SEP> (BHT) <SEP> 0.03
<tb> DL <SEP> alpha <SEP> tocopheryl <SEP> acetate <SEP> 0.3
<tb> Aroma <SEP> yerbatone <SEP> 0.3
<tb> After <SEP> Emulsification <SEP> NaOH <SEP> (Solution <SEP> to <SEP> 10 <SEP>% <SEP> m / m) <SEP> 0.8
<tb> TOTAL <SEP> 100
<Tb>

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Evaluation of trophic and hemodynamic changes induced by the application of compositions according to the present invention on an experimental model of human skin maintained in survival The object of this study is to evaluate the action of compositions according to the present invention on an experimental model of human skin maintained in survival. The compositions used are those of Examples 1 and 2, which are in the form of an emulsion for cutaneous application. These compositions will be designated hereinafter by the terms M109 / 1 (example 1) and Moo9 / 2 (example 2).

The action of products M109 / 1 and Ml 09/2 on the one hand on structural proteins and epidermal lipids, and on the other hand on the vascular tone leading to a reduction of edema by reducing the capillary permeability and thus allowing to contribute to the prevention of the occurrence of pressure ulcers was thus evaluated.

1. Experimental model
The first experimental model on human skin maintained in survival allowed to alter the protective qualities of the epithelium as they are presented at the initial stage of the eschar (stage 1), and to show the action of the products M109 / 1 and MI09 / 2. To do this, a model of alteration of the stratum corneum by application of acetone was used. The study was carried out by analyzing the modulation of transglutaminase type 1 (indicating terminal differentiation), the quality of the stratum corneum and the level of epidermal lipids. Lipids play an essential role in water retention and the limitation of water evaporation, as well as in the maintenance of NMF within corneocytes. The modulation of transglutaminase type 1 indicates good epithelial differentiation. These results were correlated with the histological analysis of the stratum corneum (SC) and more particularly the SC compactum zones.

 The second protocol used an experimental model of vasodilation on human skin maintained in survival using as substance neuromediator substance P (SP) which is a powerful cutaneous vasodilator agent. The evaluation of the effect was histological with evaluation of the capillary caliber

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Finally, the modulation of cell proliferation was also evaluated by immunohistochemical analysis of the Ki67 protein (control of proliferating cells).

 2. Material and methods 2.1. Survival of Human Skin Organ cultures (6 different donors) were made from skin fragments from plastic surgery operations. The skin fragments were placed in inserts themselves positioned on culture wells. The culture medium (antibiotics, FCS) was added to the bottom of the wells, a passage being carried out by slow diffusion between the two compartments via a porous membrane (12 μm). Two sets of skins were maintained in survival, one for a study at the epidermic level, the other for a study at the dermal level.

2.2. Experimental model for the evaluation of structural proteins, epidermal lipids, stratum corneum, and increased vascular tone The complex of 2 products (lots M109 / 1 and M109 / 2) was surface applied once per day for 48 hours. The skin fragments were subjected to the action of a neuromediator for vascular tone analysis: substance P (5 μM), which was added to the culture medium for an additional 24 hours. Finally, on the last day of culture, the skin fragments were also dehydrated in order to alter the barrier function by applying acetone to the surface of the skin for 15 minutes. The following comparisons were made: - control skin, - dehydrated skin and subjected to the action of the substance P, - dehydrated skin, subjected to the action of the substance P and treated with the complex M109 / 1, - dehydrated skin subjected to the action of the substance P and treated with the M109 / 2 complex.

The skins kept alive were stopped on day 3 (after 3 days) for histological analyzes (after fixation in Bouin's liquid and inclusion in paraffin) and lipid assays.

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 3. Analyzes 3.1. Immunohistochemical Analysis of Transglutaminase Type 1 From the paraffin embedded fragments, it is possible to detect by immunohistochemistry a terminal differentiation marker such as transglutaminase type 1 (antibody BT 621, Clinisciences). Immunodetection was performed using a 3-layer indirect immunoperoxidase technique (ABC Peroxidase kit, Vector Laboratories) and revealed in DAB (Diaminobenzidine). The intensity of immunohistochemical staining of transglutaminase type 1 was evaluated using different semi-quantitative histological scores: Score 1: + low intensity Score 2: + to ++ intensity slightly increased Score 3: ++ moderate intensity Score 4: ++ to +++: significant intensity 3.2. Histological analysis of the thickness of the stratum corneum The thickness of the stratum corneum was also measured on the histological sections.

The number of compactum stratum corneum sectors was counted (10 fields at the objective x 40). Morphometric analysis of the thickness of this stratum compactum was performed (.mu.m).

3.3. Lipid assay The lipids of sebum (cholesterol esters, squalene, triglycerides and diglycerides) and stratum corneum (free fatty acids, cholesterol and various lipids including ceramides type 1 to VI, glycerol, monoglycerides) were obtained by direct extraction using a chloroform / methanol type solvent (2/1). They were separated by thin layer chromatography. The spot size corresponding to each lipid class was measured by densitometry (scanner). The results are expressed in% of each of the lipid classes relative to the total amount of lipid by separating lipids from sebum and stratum corneum. The total amount of lipids per skin was also evaluated.

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3.4. Histological analysis of vasoconstriction After staining with haemalun-eosin, vascular dilatation was evaluated by counting the number of dilated vessels over the entire histological section (16 fields at 40 magnification). This number has been reported to the total number of vessels to calculate the percentage of dilated vessels. On the other hand, a morphometric analysis of the surface (.mu.m.sub.2) occupied by the lumen of the vessels was carried out in order to determine the average surface (.mu.sub.2) occupied by the vessels.

3.5. Evaluation of Cellular Proliferation by Immunolabeling Using an Anti-Ki67 Antibody The epithelial proliferation was analyzed by immunohistochemistry using an anti-Ki67 antibody (M, S, G 1 and M phase cell marker). G2 of the cell cycle). Immunodetection was performed using an amplified 3-layer indirect immunoperoxidase technique (DAKO kit) and revealed in AEC.

The number of labeled cells is evaluated relative to a total of 200 to 400 cells. The percentage of cells undergoing proliferation at the level of the basal layer has thus been calculated.

3.6. Statistics The statistical analysis will be performed by the Student's test said the reduced gap or test paired samples, with an alpha risk of 5%.

 4. Results 4.1. Evaluation of transglutaminase type 1 The results of immunohistochemical evaluation of transglutaminase type 1 (intensity of immunostaining using semi-quantitative scores) are shown in Table I.

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Table 1:

<Tb>
<tb> Skin <SEP> control <SEP> 2.25 <SEP> 1
<tb> Skin <SEP> + <SEP> acetone <SEP> 1.66 <SEP> 1
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 1 "3, <SEP> 3 <SEP> 0.5 <SEP>&num;
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 2 <SEP> 2, <SEP> 8 <SEP> 0, <SEP> 7 <SEP>&num;
<Tb>
&Num; : statistically significant difference compared to the skin treated with acetone (Student paired test, p <0.05) Thus, the application of acetone on the experimental model tends to decrease the intensity of this immunostaining. The preventive treatment of these skins with the M 109/1 and M109 / 2 products made it possible to increase the intensity of this immunostaining at a level statistically different from that of the dehydrated skin (p <0.05). These products thus make it possible to improve the terminal differentiation of the epithelium, with a slight superiority of Ml 09/1 compared to Ml 09/2, but without significance.

4.2. Evaluation of the thickness of the stratum corneum compactum The results concerning the stratum corneum are given in Tables II and III. The average thickness of the stratum comeum compactum is thus presented in Table II, while the percentage of stratum corneum compactum per section analyzed is presented in Table III.

Table II: Average thickness of stratum corneum compactum (hemalun-eosin, in μm):

<Tb>
<tb> Skin <SEP> control <SEP> 10.7 <SEP> 3
<tb> Skin <SEP> + <SEP> Acetone <SEP> 4 <SEP> 1, <SEP> 65 *
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> MI09 / 1 <SEP> 09/18, <SEP> 5 <SEP> 3, <SEP> 6 <SEP>&num;
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 27, <SEP> 65 <SEP> 1, <SEP> 9 <SEP> * &num;
<Tb>
*: statistically significant difference compared to control skin (Student paired test, p <0.05)

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&Num; : statistically significant difference compared with acetone-treated skin (Student's paired test, p <0.05) Table III: Percentage of stratum comeum compactum per section analyzed (10 fields examined at objective x 40, hemalun- eosin):

<Tb>
<tb> Skin <SEP> control <SEP> 69.55 <SEP> 1, <SEP> 73
<tb> Skin <SEP> + <SEP> Acetone <SEP> 33.13 <SEP> 14, <SEP> 6 *
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 1 <SEP> 49, <SEP> 9 <SEP>! <SEP> 2, <SEP> 2 * &num;
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 2 <SEP> 49, <SEP> 16 <SEP>: <SEP> 13, <SEP> 35 * &num;
<Tb>
*: statistically significant difference compared to control skin (Student paired test, p <0.05) &num; : statistically significant difference compared with acetone-treated skin (paired test of
Student, P <0.05)
Dehydration with acetone resulted in a significant reduction of the compactum stratum corneum (p <0.05). The application of products M109 / 1 and M 109/2 shows a significant increase in stratum corneum compactum and its thickness (p <0.05). Their efficiency is practically identical on this parameter.

4.3 Determination of lipids
Lipid measurement results are shown in Tables IV, V and
VI. The total lipid assay is thus presented in Table IV, the sebum lipid assay is presented in Table V, while the intracellular lipid assay of the stratum corneum is shown in Table VI.

Table IV: Total lipid assay (peak area):

<Tb>
<tb> average <SEP> SD
<tb> Skin <SEP> control <SEP> 10 <SEP> 604, <SEP> 5 <SEP> 4239
<Tb>

<Desc / Clms Page number 17>

<Tb>
<tb> Skin <SEP> + <SEP> Acetone <SEP> 6579.2 <SEP> 2748 *
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 1 <SEP> 14 <SEP> 035, <SEP> 3 <SEP> 3637 * &num;
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 2 <SEP> 14 <SEP> 344, <SEP> 5 <SEP> 5289 <SEP>&num;
<Tb>
*: statistically significant difference compared to control skin (Student paired test, p <0.05) &num; : statistically significant difference compared to acetone treated skin (Student's paired test, p <0.05) Table V: Sebum lipid assay (mean ET (%)):

<Tb>
<tb> diglycerides <SEP> triglycerides <SEP> squalens <SEP> Ester <SEP> from
<tb> cholesterol
<tb> Skin <SEP> Control <SEP> 10, <SEP> 2 <SEP> 27.2 <SEP> ~ <SEP> 16 <SEP> 30.75 <SEP> ~ <SEP> 10, <SEP> 5 <SEP> 0.35 <SEP> 0, <SEP> 3
<tb> Skin <SEP> + <SEP> Acetone <SEP> 1,16 <SEP>: <SEP> 1 <SEP>: <SEP> 0, <SEP> 6 <SEP> 31 <SEP> ~ <SEP> 20, <SEP> 5 <SEP> 27, <SEP> 7 <SEP> 8, <SEP> 8 <SEP> 0.55 <SEP> 0, <SEP> 6
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> 1.9 <SEP> 0, <SEP> 8 * <SEP> 47 <SEP> ~ <SEP> 21 * <SEP> 20, 54 <SEP> 6, <SEP> 8 <SEP> 0.47 <SEP> ~ <SEP> 0, <SEP> 3
<tb> M109 / 1
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> 1, <SEP> 7 <SEP> 0, <SEP> 84 <SEP> 52, <SEP> 56 <SEP> 20 * <SEP > 19, <SEP> 35 <SEP> 7, <SEP> 2 * <SEP> 0.42 <SEP>: <SEP> 0, <SEP> 24
<tb> M109 / 2
<Tb>

* : amerence statistiquemem signlcattve par rapport a la peau temon est apparie ae maenr, pu, u) Tableau VI : Dosage des lipides intercellulaires du stratum comeum (moyenne ET (%)) :

*: Statistically significant amerence compared to the skin temon is related to maenr, pu, u) Table VI: Determination of intercellular lipids of the stratum comeum (mean AND (%)):

<Tb>
<tb> cholesterol <SEP> Lipids <SEP> various, <SEP> Free <SEP> fatty acids <SEP>
<tb> ceramides
<tb> Skin <SEP> control <SEP> 19, <SEP> 5 <SEP> ~ <SEP> 4, <SEQ> 4 <SEP> 11.4 <SEP> ~ <SEP> 3 <SEP> 8, <SEP> 4 <SEP> ~ <SEP> 10, <SEP> 5
<tb> Skin <SEP> + <SEP> Acetone <SEP> 23, <SEP> 35 <SEP> ~ <SEP> 7 <SEP> 8 <SEP> ~ <SEP> 3, <SEP> 25 <SEP> 11 , <SEP> 9 <SEP>: <SEP> t <SEP> 14, <SEP> 7
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 1 <SEP> 12.6 <SEP> 4, <SEP> 4 * &num; 9, <SEP> 3 <SEP> t , <SEP> 1 <SEP> 8 <SEP> 10, <SEP> 2
<tb> Skin <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 2 <SEP> 8.9 <SEP>: <SEP> 3, <SEP> 5 * &num; 10, <SEP> 7 <SEP>: <SEP> 5, <SEP> 611, <SEP> 2 <SEP> 18
<Tb>

Lipides divers : céramides de type 1 à VI, glycérol, monoglycérides... c * : différence statistiquement significative par rapport à la peau témoin (test apparié de Student, p < O, 05)

Various lipids: ceramides of type 1 to VI, glycerol, monoglycerides ... c *: statistically significant difference compared to the control skin (Student paired test, p <0.05)

<Desc / Clms Page number 18>

&num;: différence statistiquement significative par rapport à la peau traitée par l'acétone (test apparié de Student, p < O, 05)
Une diminution statistiquement significative des lipides totaux, après application d'acétone, peut ainsi être observée dans le tableau IV (p < 0,05). L'application préventive des produits M109/1 et M109/2 induit une augmentation significative des lipides de façon similaire (p < 0, 05).

&num;: statistically significant difference from skin treated with acetone (Student paired test, p <0.05)
A statistically significant decrease in total lipids, after application of acetone, can thus be observed in Table IV (p <0.05). The preventive application of M109 / 1 and M109 / 2 products induced a significant increase in lipids in a similar manner (p <0.05).

 With regard to sebum lipids (Table V), the preventive treatment with the M109 / 1 product makes it possible to observe a significant increase in diglycerides and triglycerides significantly compared with the control skin (p <0.05). The preventive treatment with the M109 / 2 product makes it possible to observe an increase of squalenes and triglycerides significantly compared to control skin (p <0.05).

différent des peaux témoins, des peaux + acétone et des peaux + acétone + MI09/1). With regard to the lipids of the stratum corneum (Table VI), the preventive application of the products M109 / 1 and M109 / 2 does not imply a change in the percentage of various lipids, including ceramides. A different calculation method made it possible to express the results in ng of various lipids / mg of epidermis. The following results were obtained: - control skin: 4121.4 1903 ng / mg - skin + acetone: 2992 1368 ng / mg - skin + acetone + Ml 09/1: 3721, 5 1304, 8 ng / mg - skin + acetone + M109 / 2: 5379.45 1635 ng / mg (result significantly

different from control skin, skin + acetone and skins + acetone + MI09 / 1).

In addition, the application of the product Ml 09 did not change the level of free fatty acids. In the experimental model, the percentage of cholesterol is significantly decreased after application of the products M109 / 1 and M109 / 2 (p <0.05), either compared to the control skin or in relation to the skin dehydrated with acetone.

4.4. Vascular modulation evaluation a) Evaluation of the overall percentage of dilated capillaries The results concerning the overall percentage of dilated capillaries are shown in Table VII.

<Desc / Clms Page number 19>

Table VII: Histological evaluation of the caliber of the dermal capillaries (hemalunetosine),% of dilated vessels:

<Tb>
<tb>%
<tb> Skin <SEP> control <SEP> 58.25 <SEP> 8, <SEP> 4
<tb> Skin <SEP> + <SEP> SP <SEP> 88, <SEP> 9 <SEP> 6, <SEP> 2 *
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> M109 / 1 <SEP> 43 <SEP> 10, <SEP> 2 <SEP>&num;
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> M109 / 2 <SEP> 54.45 <SEP> ~ <SEP> 9.3 <SEP>&num;
<tb> * <SEP>: <SEP> difference <SEP> statistically <SEP> significant <SEP> by <SEP> report <SEP> to <SEP><SEP> water <SEP> control <SEP> (test <SEP> matched <SEP> of <SEP> Student. <SEP> p <0.05)
<Tb>

' : ainerence stansnquement signincauve par rapport a la peau temoin rest apparie ae ùtuaenr, p < u, uj) &num;: différence statistiquement significative par rapport à la peau traitée par la substance P (test apparié de Student, p < 0, 05) L'application de substance P induit une vasodilatation statistiquement significative par rapport à la peau témoin (p < 0,05). Après application des produits M109/1 et M109/2, le pourcentage de capillaires dilatés est significativement diminué, par rapport aux peaux traitées par l'acétone (p < 0,05). Le produit Ml 09/1 montre une supériorité d'action par rapport au produit MI 09/2, mais pas de façon significative. b) Mesure de la surface moyenne des capillaires Les résultats concernant la mesure de la surface moyenne des capillaires sont exposés dans le Tableau VIII.

A statistically significant difference between the skin and the skin was consistent with the difference between the skin treated with substance P (Student's paired test, p <0.05). Substance P application induced statistically significant vasodilation compared to control skin (p <0.05). After application of the products M109 / 1 and M109 / 2, the percentage of dilated capillaries is significantly decreased, compared with the acetone-treated skin (p <0.05). The product Ml 09/1 shows a superiority of action compared to the product MI 09/2, but not significantly. b) Measurement of the average surface of the capillaries The results concerning the measurement of the mean surface of the capillaries are set out in Table VIII.

Table VIII: Measurement of the surface of dilated capillaries:

<Tb>
<tb> Surface <SEP> in <SEP> pm2
<tb> Skin <SEP> control <SEP> 73.84 <SEP> 44, <SEP> 6
<tb> Skin <SEP> + <SEP> SP <SEP> 155, <SEP> 2 <SEP> 31, <SEP> 2 *
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> MI09 / 1 <SEP> 43, <SEP> 7 <SEP> 12, <SEP> 35 <SEP>&num;
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> MI0912 <SEP> 09/254, <SEP> 4 <SEP> 9, <SEP> 1 <SEP>&num;
<Tb>

* : différence statistiquement signiticative par rapport a la peau temom (test apparie de Student, p < u, u-))

*: statistically significant difference with respect to skin temom (Student's paired test, p <u, u-))

<Desc / Clms Page number 20>

&num;: différence statistiquement significative par rapport à la peau traitée par l'acétone (test apparié de Student, p < 0, 05) L'application de substance P induit une augmentation statistiquement significative de la surface moyenne des capillaires par rapport à la peau témoin (p < 0, 05). Après l'application des produits M109/l et Mol09/2 permet de diminuer la surface moyenne des capillaires par rapport aux peaux traitées par l'acétone (p < 0, 05). Là encore, le produit M109/2 montre une supériorité d'action par rapport au produit Ml09/l, mais pas de façon significative.

&num;: statistically significant difference from acetone-treated skin (Student's paired test, p <0.05) Substance P application induced a statistically significant increase in mean capillary surface area relative to skin control (p <0.05). After the application of the products M109 / l and Mol09 / 2 reduces the average surface of the capillaries compared to the skin treated with acetone (p <0.05). Again, the M109 / 2 product shows superiority of action over the Ml09 / 1 product, but not significantly.

4. Evaluation of the mitotic activity The results concerning the evaluation of the epithelial proliferation analyzed by immunohistochemistry are set out in Table IX.

Table IX: Analysis of the proliferation after immunolabeling using an anti-Ki67 antibody (% of basal cells positive):

<Tb>
<tb> Skin <SEP> control <SEP> 6.3 <SEP> 4, <SEP> 3
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> Acetone <SEP> 5.7 <SEP> 4, <SEP> 4
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 18, <SEP> 2 <SEP> 4, <SEP> 75 <SEP>&num;
<tb> Skin <SEP> + <SEP> SP <SEP> + <SEP> Acetone <SEP> + <SEP> M109 / 211, <SEP> 74 <SEP> 8, <SEP> 1 * <SEP>&num;
<Tb>

* : différence statistiquement significative par rapport à la peau témoin (test apparié de Student, p < 0, 05) &num;: différence statistiquement significative par rapport à la peau traitée par l'acétone (test apparié de Student, p < 0, 05)
La prolifération épithéliale analysée par immunohistochimie à l'aide d'un anticorps anti-Ki67 n'est pas modifiée dans le modèle expérimental (application de substance P, puis d'acétone) : il n'y a pas de différence significative entre les peaux témoins et les peaux expérimentales.

*: statistically significant difference compared to the control skin (Student paired test, p <0.05): statistically significant difference compared to the skin treated with acetone (Student paired test, p <0.05 )
The epithelial proliferation analyzed by immunohistochemistry using an anti-Ki67 antibody is not modified in the experimental model (application of substance P, then of acetone): there is no significant difference between the skins witnesses and experimental skins.

 On the other hand, the proliferation is increased statistically significantly after application of the M109 / 1 and MI09 / 2 products compared with the skins.

<Desc / Clms Page number 21>

 experimental (p <0.05). Again, the Moo9 / 2 product shows superiority of action over the Ml09 / 1 product, but not significantly.

5. Conclusions
The experimental model of dehydrated human skin has made it possible to demonstrate a positive modulation of the M109 / 1 and M109 / 2 products on the protection of a structural protein of the epithelium, transglutaminase type 1. In fact, the Preventive application of these products prior to the dehydration step has helped to improve epithelial differentiation. This result can be correlated with a significant increase in stratum corneum compactum and its thickness.

après application des produits M109/1 et Moo9/2 dans ce modèle de peau déshydratée. En ce qui concerne les céramides, l'efficacité de MI09/2 est significativement supérieure à celle de Ml 09/1. The amount of total epidermal lipids is significantly increased

after application of M109 / 1 and Moo9 / 2 products in this dehydrated skin model. With regard to ceramides, the efficiency of MI09 / 2 is significantly greater than that of Ml 09/1.

 The experimental model of vasodilatation with the substance P made it possible to demonstrate an efficacy of the products M 109/1 and M 109/2 on the vascular tone.

Finally, the Ml09 / 1 and Ml09 / 2 products made it possible to obtain a positive modulation of the epithelial proliferation, significantly increased compared to the experimental skin.

Thus, the MI09 / 2 product seems particularly effective on the ceramide, vasodilatation and proliferation parameters, whereas the MI09 / 1 product seems particularly effective as regards the epithelial differentiation.

Claims (20)

A composition comprising in combination: (a) at least one flavonoid, and (b) at least one vegetable oil containing at least 20% by weight of linoleic acid and / or at least 10% by weight of alpha-linolenic acid .  2. Composition according to claim 1, characterized in that the vegetable oil (b) contains 30 to 50% by weight of linoleic acid and / or 25 to 40% by weight of alpha-linolenic acid.  3. Composition according to claim 2, characterized in that the vegetable oil (b) is Rosehip Muscat oil.  4. Composition according to any one of claims 1 to 3, characterized in that the flavonoid (a) is a flavanone, preferably selected from the group consisting of naringenin, naringin, hesperetin, and hesperidin.  5. Composition according to any one of claims 1 to 3, characterized in that the flavonoid (a) is a flavone, advantageously chosen from the group consisting of rutin, troxerutin, diosmin, diosmetin, chrysin, quercetin and luteolin.  6. Composition according to any one of the preceding claims, characterized in that the flavonoid (a) and the vegetable oil (b) are the only active ingredients of the composition.  7. Composition according to any one of claims 1 to 5, characterized in that it further comprises: (c) at least one peptide extract of lupine.  8. Composition according to claim 7, characterized in that the peptide extract of lupine (c) comprises glutamate peptides of low molecular weight and oligosaccharides.  9. Composition according to claim 7 or 8, characterized in that the peptide extract of lupine (c) is obtained by hydrolysis of the protein fraction of lupine.  10. Composition according to any one of the preceding claims, characterized in that the flavonoid (a) is present at a concentration between <Desc / Clms Page number 23>  0, 1 and 20%, advantageously between 1 and 10%, relative to the total weight of the composition.  11. Composition according to any one of the preceding claims, characterized in that the vegetable oil (b) is present at a concentration of between 10 and 60%, advantageously between 20 and 40%, relative to the total weight of the composition. .  12. Composition according to any one of claims 7 to 11, characterized in that the peptide extract of lupine (c) is present at a concentration of between 0, 1 and 20%, advantageously between 1 and 10%, relative to to the total weight of the composition.  13. Composition according to any one of the preceding claims, characterized in that the composition is formulated to be administered topically.  14. Composition according to any one of the preceding claims, characterized in that the composition is in the form of an emulsion.  15. The composition of claim 14, characterized in that the composition further contains a emulsifier polymer Pemulen type.  16. Composition according to any one of claims 13 to 15, characterized in that the composition is packaged in the form of a spray.  17. Composition according to any one of claims 1 to 16 for use as a dermatological or dermato-cosmetic agent.  18. A composition according to claim 17 for use as an anti-inflammatory agent or as a regenerative agent for epidermal and dermal tissue.  19. Composition according to claim 17 for its use in the prevention or treatment of oedemas, pressure ulcers, disorders of the skin microcirculation, or ulcers.  20. Composition according to claim 17 for its use in the treatment of radiodermites.