# Patent

## Title
Use of an active principle originating from flax in a composition intended to reinforce the barrier function of the skin and to protect the skin and the appendages against external aggressions

## Abstract

The present invention concerns the use, in a cosmetic composition, of an effective quantity of active principle originating from flax (genus Linum) to reinforce the barrier function of the skin and to protect the skin and the appendages against external aggressions. The active principle originates from the hydrolysis of flax proteins and contains principally polypeptides or peptides. The invention likewise relates to the use, in a cosmetic composition, of a peptidic hydrolysate of flax as active principle capable of activating HMG-CoA reductase. The invention likewise relates to the use of a peptidic hydrolysate of flax as active principle capable of activating transglutaminase. The active principle can likewise be used to prepare pharmaceutical compositions intended to prevent or combat against the pathologies linked to alterations to the barrier function, such as certain hypersensitivies, irritations of the skin and reactive skins, or atopic dermatitis.

## Background
This application is a continuation-in-part of co-pending application Ser. No. 12/669,836 filed on Jan. 20, 2010, which is the 35 U.S.C. §371 national stage of International PCT/FR2008/001072 filed on Jul. 21, 2008, which claims priority to French Application No. 0705295 filed on Jul. 20, 2007. The entire contents of each of the above-identified applications are hereby incorporated by reference. Any disclaimer that may have occurred during prosecution of the above referenced applications is hereby expressly disclaimed.


The present invention lies in the cosmetic and pharmaceutical field, and more particularly in the field of dermatology. The present invention concerns the use, in a cosmetic composition, of an effective quantity of active principle originating from flax (genus Linum); to reinforce the barrier function of the skin and to protect the skin and the appendages against external aggressions.
Preferably, the active principle originates from the hydrolysis of flax proteins and principally contains polypeptides or peptides. It can be used alone or in association with at least one other active principle. The invention likewise relates to the use of a cosmetic composition to stimulate the functions of the mitochondria and to increase the cellular energy level. The invention further relates to a method of cosmetic treatment intended to protect the skin and the appendages from external aggressions and to combat against cutaneous ageing.
The invention likewise relates to the use, in a cosmetic composition, of a peptidic hydrolysate of flax as active principle capable of activating HMG-Co-A reductase.
The invention likewise relates to the use of a peptidic hydrolysate of flax as active principle capable of activating transglutaminase.
The active principle can likewise be used to prepare pharmaceutical compositions intended to prevent or combat against the pathologies linked to mitochondrial dysfunctions; for example, certain neuromuscular or cardiac degenerations, type II diabetes or also certain pathologies of ageing.
The active principle can likewise be used to prepare pharmaceutical compositions intended to prevent or combat against the pathologies linked to alterations to the barrier function, such as certain hypersensitivities, irritations of the skin and reactive skins, or atopic dermatitis.
The term “appendages” according to the invention encompasses all the keratinic appendages present on the surface of the body, in particular hairs, eyelashes, eyebrows, nails and head hair.
The skin is a vital organ which covers the entire surface of the body and ensures protective, sensitive, immune, metabolic or thermoregulatory functions. The skin, like the other organs, is subject to ageing. Now, one of the major mechanisms involved in the process of ageing is the accumulation of oxidative damage in essential molecules such as the membrane lipids, the proteins, the DNA and most particularly the mitochondrial DNA (mtDNA).
Oxidative damage is caused by the free radicals, chemically unstable and very reactive species generated by the intracellular metabolism or external aggressions. These external aggressions can include: UV radiation, toxins, atmospheric pollutants, alimentary oxidants.
In the skin, a premature ageing is observed, occurring in the areas exposed to radiation, characterised by phenomena of alteration to the macromolecules (lipidic peroxidation, carbonylation of proteins), affecting in particular elastin, collagen or fibronectin. A progressive decline of the mitochondrial functions with age has likewise been able to be shown, probably linked to the accumulation of mutations on mtDNA (K. Singh, Ann. N.Y. Acad. Sci. 1019, 2004).
One of the important consequences of the accumulation of oxidative damage is a reduction in the capacity of the cell to produce ATP (Porteous et al., Eur J Biochem 1998, 257(1):192-201). Thus, the phenomenon of cellular ageing is in relation to the oxidative damage which the cell undergoes, but also to the process of energy production necessary for the cell to survive.
The organism possesses defence mechanisms capable of trapping or transforming the free radicals (enzymes, glutathione, vitamins A and E, coenzyme Q10, etc.). However, these antioxidant defence systems often prove to be insufficient with respect to the numerous stresses and external aggressions to which the organisms and the skin in particular, are subjected.
In this context, the particular properties of cytochrome c appear as being particularly interesting:
Cytochrome c is a small soluble protein of 15 kDa which plays an essential role in mitochondrial function and in cellular survival. Cytochrome c is a highly conserved molecule in the majority of eucaryotes; it is found in the mitochondria of plants, animals and numerous unicellular organisms. Cytochrome c presents a proteic structure organised around a porphyrin, constituted by four pyrrole nuclei, themselves linked to an iron atom.
The principal role of cytochrome c is to ensure the transfer of electrons due to the change in valency of the iron atom. Cytochrome c which is soluble thus transports the electrons from complex III (coenzyme QH2 cytochrome c reductase) to complex IV (cytochrome oxydase). The electrons, which are the substrate of the cytochrome oxydase, are then transferred by the enzyme to the oxygen.
The search for compounds which are able to stimulate the mitochondria and to increase the cellular energy level so as to prevent or combat against the signs of cutaneous ageing or of damage caused by external aggressions, such as UV rays, radiations, or exposures to toxins or pollutants, is an important preoccupation in medical and cosmetics research. In this respect, solutions have been proposed such as the addition of substances involved in the energy metabolism, and more particularly of intermediaries or cofactors of the Krebs cycle, such as fumarate, L-malate, acetyl CoA (WO 02064129) or else the treatment of the skin by substances capable of reducing the free radicals, such as vitamin C (US 2004/0086526) or L-ergothioneine (WO 9836748). In addition, extracts of lignans extracted from flax have been described as beneficial for the skin (WO 2004/010965). However, to the knowledge of the applicant, no cosmetic or pharmaceutical composition has yet been described comprising compounds of peptidic nature, originating from flax, capable of activating cytochrome c.
One of the essential functions of the epidermis is to constitute a barrier between the exterior environment and the interior environment. It is the most external layer of the epidermis, the stratum corneum, which ensures this function. It is composed of keratinocytes at the final stage of their differentiation, the corneocytes, sealed to each other by a thick intercellular cement rich in lipids. Cholesterol is to be found among these lipids.
In fact, the epidermis is a very active site for the synthesis of cholesterol. The limiting stage of this synthesis is catalysed by a membrane enzyme named HMG-CoA-reductase (E.C.1.1.1.34). In physiological situation, the cholesterol is synthesized at a level necessary to maintain homeostasis. On the other hand, following a brutal alteration of the cutaneous barrier, a high and rapid increase in the synthesis of cholesterol is observed, associated with an increase in the expression and the activity of HMG-CoA reductase (Menon G. K. et al., J. Lipid. Res., 1985, (26), P. 418-427).
The importance of HMG-CoA reductase has made this a target of choice to combat against the ageing of the skin or to prevent the alteration or to re-establish or reinforce the barrier function of the epidermis.
Moreover, the transglutaminases (EC 2.3.2.13) are a family of calcium-dependent enzymes expressed in the skin, which catalyze the formation of peptidic bridges which are extremely resistant to deterioration (Lorand et al., Nat Rev Mol Cell Biol. February; 4(2), 2003). More particularly, in the epidermis, TG1, TG3 and TG5 are involved in the formation of the cornified envelope (Lorand et al., Nat Rev Mol Cell Biol. February; 4(2), 2003). Thus, the TGs are capable of cross-linking the keratins with each other and with the filaggrin, which leads to the stabilisation and coordination of the keratin-filaggrin network in the corneous cells. The key role of the transglutaminases in the formation of the stratum corneum, and more widely in the epidermal differentiation and, consequently, on the barrier function, is confirmed by certain pathological models. Thus, in humans, mutations carried by the TG1 are responsible for more or less severe forms of ichthyosis (Huber et al. Science 267, 1995).
In the course of cutaneous ageing, the integrity of the cutaneous barrier and also its capacities for repair alter. An overall deficiency in lipids is observed, leading to a reduction in the lipidic multi-layers of the extracellular compartment of the stratum corneum. These functional changes are in correlation with an increased susceptibility of aged skins to external aggressions (Ghadially R. et al., J Clin Invest., 1995 (95 (5), p. 2281-90).
Independently of intrinsic or photo-induced ageing, alterations to the cutaneous barrier can occur in the case of external aggressions.
The present invention has as its main objective the use of an effective quantity of an active principle of peptidic nature, originating from the hydrolysis of flax proteins (genus Linum), in a cosmetic composition to activate cytochrome c and to stimulate the mitochondria, with the aim of protecting the skin from external aggressions and of combating against cutaneous ageing. The said active principle will be able to be used alone or in association with at least one other active principle. The inventors have in fact demonstrated a biological activity, and more particularly a dermatological and cosmetic activity, of an active principle of peptidic nature originating from the hydrolysis of flax, capable of activating cytochrome c. It has been demonstrated in particular that these polypeptides or these peptides, which constitute the active principle, when they are applied on the skin, stimulate the mitochrondrial functions to a considerable extent. This has been demonstrated, inter alia, by an increase in the expression of cytochrome c, an increase in the activity of the cytochrome oxydase and an increase in the synthesis of ATP.
It has likewise been demonstrated that this new active principle activates the expression of transglutaminases, enzymes involved in the formation of the epidermal cornified envelope, improving therein the barrier function of the epidermis.
It has likewise been demonstrated that this new active principle activates the expression of HMG-CoA reductase, thus reinforcing the barrier function of the epidermis, and stimulates the epidermal differentiation.
The present invention has as an objective the use, in a cosmetic composition, of an effective quantity of active principle originating from flax (genus Linum) to reinforce the barrier function of the skin and to stimulate epidermal differentiation.
“To reinforce the cutaneous barrier function and to stimulate epidermal differentiation” is understood to mean the improvement of the structure of the horny layer, and the increase of the expression of markers of keratinocyte differentiation.
“Active principle capable of activating cytochrome c” is understood to mean a hydrolysate of peptidic nature originating from the hydrolysis of flax proteins (genus Linum), capable of increasing the expression of cytochrome c, either by the activation of the proteic synthesis (by direct or indirect modulation of the genic expression of cytochrome c), or by the increase of the biological activity of cytochrome c, or by other biological processes such as the stabilisation of the protein cytochrome c or else the stabilisation of the messenger RNA transcripts.
“Active principle capable of stimulating the functions of the mitochondria” is understood to mean entirely a hydrolysate of peptidic nature originating from the hydrolysis of flax proteins (genus Linum), capable of increasing the expression or the activity of the principal active molecules present in the mitochondria, and more generally capable of increasing the principal energetic functions of the mitochondria; namely, the chain of oxidative phosphorylation and the synthesis of ATP.
“Active principle capable of activating transglutaminase” is understood to mean a hydrolysate of peptidic nature originating from the hydrolysis of flax proteins (genus Linum), capable of increasing the expression of transglutaminase, either by the activation of the protein synthesis (by direct or indirect modulation of the gene expression of transglutaminase), or by the increase of the biological activity of cytochrome c, or by other biological processes such as the stabilisation of the protein cytochrome c or else the stabilisation of the messenger RNA transcripts.
“Active principle capable of activating HMG-CoA reductase” is understood to mean a hydrolysate of peptidic nature originating from the hydrolysis of flax proteins (genus Linum), capable of increasing the expression of HMG-CoA reductase, either by the activation of the protein synthesis (by direct or indirect modulation of the gene expression of transglutaminase), or by the increase of the enzymatic activity, or by other biological processes such as the stabilisation of the protein or else the stabilisation of the messenger RNA transcripts.

    “Of peptidic nature” is understood to mean a mixture of compounds, represented mainly by peptides or polypeptides.
    The term “peptide” designates a chain of two or more amino acids linked to each other by peptidic links or by modified peptidic links; the term “polypeptide” designating a peptide of larger size.
    The expression “biologically active” is understood to mean “which possesses an activity in vivo or in vitro which is characteristic of the activity of the active principle according to the invention”.


The term “hydrolysate or originating from hydrolysis” designates any substance or mixture of substances, or isolated preparation, obtained after hydrolysis of vegetal manner.
The active principle according to the invention can be obtained by extraction of proteins of vegetal origin, followed by a controlled hydrolysis which releases biologically active peptidic fragments.
Numerous proteins found in plants are likely to contain biologically active peptidic fragments in the core of their structure. The managed hydrolysis allows these peptidic fragments to be released. It is possible, but not necessary to realize the invention, to extract either firstly the proteins concerned and to then hydrolyse them, or to carry out the hydrolysis first on a crude extract and to then purify the peptidic fragments. It is likewise possible to use certain hydrolysed extracts without purifying the peptidic fragments thereof corresponding to the biologically active peptides according to the invention, but nevertheless ascertaining the presence of the said fragments by suitable analytical means.
To carry out the extraction, the entire plant can be used, or a specific part of the plant (leaf, seed, etc.).
More particularly, according to the invention one of numerous plants are used of the linaceae family, of the genus Linum (flax). The genus Linum numbers almost 200 species, growing particularly in the northern hemisphere. These are herbaceous plants with fibrous stems, with simple leaves, with flowers having 5 petals. Preferably, according to the invention, the cultivated species Linum usitatissimum L. is used. According to the invention, the vegetal material which is used will be the seed and preferably the seed having had its envelope removed by a decortication step.
In a first stage, the plant is crushed by means of a plant crusher. The powder which is thus obtained can be subsequently “delipided” by means of a conventional organic solvent (such as for example an alcohol, hexane or acetone).
The extraction of the proteins from the plant is then carried out according to the modified conventional process (Osborne, 1924); the crushed plant material is placed in suspension in an alkaline solution containing an adsorbent product of the insoluble polyvinylpolypyrrolidone (PVPP) type (0.01-20%); in fact, it has been observed that the subsequent operations of hydrolyses and purifications were facilitated by this means. The concentration of substances of phenolic type interacting with the proteins is thus reduced.
The soluble fraction is collected after centrifugation and filtration stages, this crude solution therefore constituting a first form of the extract containing the proteins, the carbohydrates and possibly lipids.
The proteins are then precipitated, varying the ionic force by acidifying the medium, which allow the soluble components and the nucleic acids to be eliminated.
The precipitate is then washed by means of an organic solvent such as, for example, ethanol or methanol, then the solvent is evaporated by drying under vacuum. The precipitate, which is rich in proteins, is returned to solution in water or another solvent, and therefore constitutes a more purified form of the hydrolysate.
The extraction can likewise be realized in neutral or acid medium, still in the presence of polyvinylpolypyrrolidone. After a filtration stage, the precipitation stage is then carried out by means of a conventional precipitation agent such as salts (sodium chloride, ammonium sulphate) or an organic solvent (alcohol, acetone). The precipitate which is obtained can be separated from the precipitation agents by dialysis after returning to solution in water or another solvent.
The isolated proteic fraction according to the invention is then hydrolysed in conditions arranged to generate soluble peptides and polypeptides. Hydrolysis is defined as being a chemical reaction involving the cleavage of a molecule by water, this reaction being able to be carried out in neutral, acid or basic medium. According to the invention, the hydrolysis is realized chemically and/or advantageously by proteolytic enzymes. The use can therefore be cited of endoproteases of vegetal origin (papain, bromelaine, ficin) and micro-organisms (Aspergillus, Rhizopus, Bacillus, etc.).
For the same reasons as previously, in this managed hydrolysis stage, a quantity of polyvinylpolypyrrolidone is added to the reaction medium. After filtration, the solution which is obtained constitutes the active hydrolysate. The active hydrolysate can be further purified so as to select the molecular weights and the nature of the generated peptides. The fractionation can be carried out advantageously by ultrafiltration and/or by a method of the chromatographic type.
Any of the more or less purified forms of the hydrolysate is then solubilised in water or in any mixture containing water, then sterilised by ultrafiltration.
The vegetal hydrolysate obtained according to the invention is analysed qualitatively and quantitatively for its physico-chemical characteristics and its content of compounds of proteic and peptidic nature. Compounds of peptidic nature are understood to mean the fragments of proteins, the peptides and the free amino acids present in the mixture. The peptides, amino acids and fragments of proteins are dosed according to conventional techniques which are well known to the man skilled in the art.
Thus, according to an advantageous embodiment of the invention, the active vegetal hydrolysate has a pH comprised between 4 and 7, and preferably between 5 and 6, a dry extract titrating between 1 and 8 g/l, and preferably between 2 and 5 g/l, its content of compounds of peptidic nature is comprised between 0.1 and 5 g/l, and preferably between 0.5 and 2 g/l and its content of sugars is from 0.5 to 2.5 g/l.
According to an advantageous embodiment of the invention, the active principle according to the invention is previously solubilised in one or more cosmetically or pharmaceutically acceptable solvents, conventionally used by the man skilled in the art, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols or any mixture of these solvents.
According to another further advantageous embodiment of the invention, the active principle according to the invention is previously solubilised in a cosmetic or pharmaceutical vector such as liposomes, or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilised in, or fixed on, any cosmetically or pharmaceutically acceptable vector.
The composition which is able to be used according to the invention can consist in particular of a composition for capillary care, and in particular a shampoo, a conditioner, a setting lotion, a treatment lotion, a hairdressing cream or gel, a restructuring lotion for the hair, a mask, etc. The cosmetic composition according to the invention can be used in particular in treatments implementing an application which is followed, or not followed, by a rinsing, or else in the form of a shampoo.
It can likewise present itself in the form of a dye or mascara to be applied by a brush or by a comb, in particular on the eyelashes, the eyebrows or the hair.
It is readily understood that the active principle according to the invention can be used alone or else in association with at least one other active principle, in a cosmetic composition or for the preparation of a pharmaceutical and/or dermatological composition.
The compositions according to the invention will be able to be applied by any suitable manner, in particular orally, parenterally or externally topically, and their formulation will be adapted by the man skilled in the art, in particular for cosmetic or dermatological compositions. Advantageously, the compositions according to the invention are intended for an administration in a topical cutaneous manner. These compositions must therefore contain a cosmetically and/or dermatologically acceptable medium, i.e. compatible with the skin and the appendages, and cover all the cosmetic or dermatological forms. In particular, these compositions will be able to be in the form of creams, oil-in-water emulsions, or water-in-oil or multiple emulsions, solutions, suspensions, gels, milks, lotions, sticks or else powders, suited to an application on the skin, the lips and/or the appendages.
These compositions comprise the necessary excipients for their formulation, such as solvents, thickeners, thinners, surfactants, antioxidants, colouring agents, preservatives, perfumes.
Advantageously, the compositions which are able to be used according to the invention further contain other active principles intended to promote its action. Among these other active principles, the active principles can be named which have an anti-radical or antioxidant action, selected from vitamin C, vitamin E, the coenzyme Q10 and polyphenolic plant extracts.
“Anti-radical active principles” are understood to mean any compound capable of trapping the free radicals. These active principles are capable of blocking the chain reactions of the free radicals before the final degradation stages of the biological constituents of the skin and thus have an antioxidant activity. Among these other active principles, one can likewise name the active principles stimulating the syntheses of the dermic macromolecules (laminin, fibronectin, collagen), for example collagen peptide sold under the name “Collaxyl®” by the company Vincience.
Among these other active principles, one can finally name the active principles stimulating the energetic metabolism, like the active principle sold under the name “GP4G®” by the company Vincience.
According to another aspect, the composition according to the invention can be a sun composition, i.e. a composition assisting in the protection against solar radiation. Thus, actives assisting in solar protection, such as for example, solar filters, can be advantageously added to the composition according to the invention.
It is readily evident that the invention is addressed to mammals in general and more particularly to human beings.
The effective quantity of active principle corresponds to the quantity necessary so as to obtain the result which is sought, namely: to activate the cytochrome c, to stimulate the mitochondria and to increase the cellular energy level, and more generally to protect the skin and the appendages from external aggressions and to combat against cutaneous ageing.
The effective quantity of active principle corresponds to the quantity necessary to obtain the result which is sought, namely to active HMG-CoA reductase and/or transglutaminase, and at the skin level to improve the barrier function of the epidermis and to stimulate epidermal differentiation.
According to an advantageous embodiment of the invention, the active principle is present in the compositions of the invention at a concentration comprised between 0.0001% to 20% approximately, and preferably at a concentration comprised between 0.05% and 5% approximately with respect to the total weight of the final composition.
These compositions will be able to present themselves, in particular, in the form of an aqueous, hydroalcoholic or oily solution; an oil-in-water, water-in-oil emulsion or multiple emulsions; they can also present themselves in the form of creams, suspensions, or else powders, suited to an application on the skin, the mucosa, the lips and/or the appendages. These compositions can be more or less fluid and can have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or a foam. They can also present themselves in solid form, as a stick or can be applied on the skin in the form of an aerosol. They can be used as a care product and/or as a make-up product for the skin.
These compositions further comprise any additive commonly used in the envisaged field of application and also the adjuvants necessary for their formulation, such as solvents, thickeners, thinners, antioxidants, colouring agents, solar filters, suntan simulating agents, pigments, charges, preservatives, perfumes, odour absorbers, cosmetic or pharmaceutical actives, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc.
In all cases, the man skilled in the art will ensure that the adjuvants and their proportions are selected in such a way as to not be prejudicial to the sought advantageous properties of the composition according to the invention. These adjuvants can, for example, correspond to 0.01 to 20% of the total weight of the composition. When the composition of the invention is an emulsion, the fatty phase can represent from 5 to 80% by weight and preferably from 5 to 50% by weight with respect to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be selected from those conventionally used in the field concerned. For example, they can be used in a proportion from 0.3 to 30% by weight, with respect to the total weight of the composition.
By its particular activities, the active principle according to the invention will be able to be used advantageously in a cosmetic composition or for the preparation of a pharmaceutical composition.
In particular, the active principle according to the invention will be able to be used advantageously in a cosmetic preparation intended to combat in a preventive and/or curative manner against the manifestations of cutaneous ageing and, more specifically, so as to combat against and/or to prevent photo-induced ageing (photo-ageing). Cutaneous manifestations of ageing are understood to mean all modifications of the exterior appearance of the skin and the appendages due to ageing, such as, for example, wrinkles and fine lines, withered skin, flabby skin, thinned skin, the lack of elasticity and/or of tonus of the skin, dull skin without brightness, or pigmentation blemishes of the skin, discolouration of the hair or blemishes on the nails, but likewise any internal modification of the skin which is not ultimately resulting systematically in a modified exterior appearance, such as for example any internal degradation of the skin following an exposure to ultraviolet radiation (UV). The active principle according to the invention, or the composition containing it, will allow one to combat, in particular, against the loss of elasticity and of firmness of the skin.
The active principle according to the invention allows the skin and the appendages to be protected against all types of external aggressions.
According to a particular embodiment of the invention, the active principle is capable of preventing damages caused to the skin and the appendages caused by external aggressions
According to another particular embodiment of the invention, the active principle is capable of improving the repair of the skin and the appendages after an external aggression.
The inventors have in fact demonstrated that a hydrolysate of peptidic nature originating from the hydrolysis of flax proteins (genus Linum) presented protective properties allowing a reduction of the cellular and histological damage in tissues subjected to a stress of physico-chemical or environmental origin, and allowed improvement to the repair of the skin after stress.
The expression “external aggression” is understood to mean the aggressions which can be produced by the environment. By way of example, one can name aggressions such as pollution, UV rays (ultraviolet radiations), or else products of an irritant character such as surfactants, preservatives or perfumes. Pollution is understood to mean both “external” pollution, due for example to diesel particles, ozone or heavy metals, and “interior” pollution which can be due in particular to the emissions of solvents of paints, glues or wallpapers (such as toluene, styrene, xylene or benzaldehyde), or else cigarette smoke.
The active principle according to the invention can be advantageously used in a cosmetic composition or for the preparation of a pharmaceutical composition, as a photo-protective agent and, more particularly, as a photo-protective agent which is designated “secondary”. A distinction is in fact made between primary photo-protective agents and secondary photo-protective agents. The primary photo-protective agents are substances which exert a physical power: they are able to absorb UV radiation and to restore it in the form of heat so as to protect the skin. The secondary photo-protective agents are substances which generally have a biological effect; they are, for example, agents capable of limiting the damage caused to the DNA and to the membranes by the penetration of UV radiation on the skin.
The invention further relates to use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective quantity of active principle according to the invention, the active principle or the composition containing it, being intended to increase the intracellular ATP synthesis of the cells of the skin.
The invention likewise has as an object the use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective quantity of active principle according to the invention, the active principle, or the composition containing it, being intended to prevent damage caused to the skin by an exposure to the sun or an exposure to ionising radiation during radiotherapies.
The invention likewise has as an object the use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective quantity of active principle according to the invention, the active principle, or the composition containing it, being intended to stimulate the mitochondria, in particular on the areas of the body which are exposed to UV radiation.
The invention further relates to the use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective quantity of active principle as previously described, the active principle, or the composition containing it, being intended to protect the skin from damage caused by free radicals.
The invention further consists in the use of an effective quantity of active principle to prepare a pharmaceutical composition intended to prevent or to combat against certain pathologies linked to mitochondrial dysfunctions, for example certain neuromuscular or cardiac degenerations, type II diabetes, certain pathologies of ageing.
It is possible, by targeting the enzymes involved in the formation of the stratum corneum (transglutaminase and HMG-CoA reductase) to improve certain pathological dysfunctions linked to the barrier function (hypersensitive skins, irritated or reactive skins, atopic eczema).
Thus, the invention further consists in the use of an effective quantity of active principle to prepare a pharmaceutical composition intended to prevent or treat the pathologies characterized by an alteration to the barrier function, such as hypersensitive, irritated or reactive skins and atopic dermatitis.
The invention further consists of a method of cosmetic treatment intended to stimulate the defences and to protect the skin and the appendages from external aggressions and to combat against cutaneous ageing, characterized by the application on the skin or the appendages which are to be treated of a composition containing an effective quantity of active principle according to the invention.
The invention further consists of a method of cosmetic treatment intended to give a healthier appearance to the skin and to improve the brightness and radiance of the complexion, characterized by the application on the skin which is to be treated of a composition containing an effective quantity of active principle according to the invention.
The invention further relates to the use, in a cosmetic composition, of a peptidic hydrolysate of flax as active principle capable of activating HMG-CoA.
The invention likewise relates to the use, in a cosmetic composition, of a peptidic hydrolysate of flax as active principle capable of activating transglutaminase.
Particular embodiments of this method of cosmetic treatment likewise result from the preceding description. Other advantages and characteristics of the invention will be better apparent from reading examples, given by way of illustration and not restrictively.


EXAMPLE 1
Preparation of Active Principle from Flax (Linum usitatissimum L.)
The active principle is obtained from plants of the species Linum usitatissimum L. Of course, the extract can be prepared from plants from at least any one of the numerous varieties and species belonging to the genus Linum. 
In a first stage, 1 kg of decorticated flax seeds are crushed in a cereal crusher. The flour which is obtained is delipided by the action of an organic solvent, hexane. After filtration and drying under vacuum, the powder which is obtained is placed in suspension in an alkaline aqueous solution (dilution at 1/10) pH 10 containing 1% of polyvinylpolypyrrolidone (Polyclar V ISP). This mixture is kept under stirring for a sufficiently long time to allow the solubilisation of the soluble fractions. The extraction temperature is variable (comprised between 4 and 80° C.); preferably the operation will be carried out cold. After this extraction phase, the medium is clarified by centrifuging, then filtered on a plate filter. This filtrate which contains the soluble fractions of the flax is then subjected to a precipitation of the proteins, varying the ionic force in neutral or acid medium, which allows the soluble glucidic components to be eliminated, the lipids and the nucleic acids, the medium is brought to pH 3.5. The supernatant is eliminated and the precipitate is then washed by means of a solvent such as, for example, ethanol or methanol then the solvent is evaporated by drying under vacuum.
At this stage, one obtains approximately 50 grams of powder of light yellow colour of crude bruteic extract containing:

    
    
        Proteins: 75%
        Carbohydrates: 20%
        Lipids: 5%
    
    


The precipitate, rich in proteins, is returned to solution in water or another solvent.
The crude proteic extract is then subjected to a series of managed and selective hydrolyses consisting of chemical and enzymatic hydrolyses in the presence of 0.5% of PVPP (Polyclar V) and of cysteine endopeptidases (papain, ficin). After reaction, the hydrolysate is filtered on a plate then on sterilising cartridge (0.2 μm).
A light-coloured hydrolysate is then obtained, titrating from 15 to 30 g/l of dry extract, which is then diluted such that the concentration of compounds of peptidic nature determined by the Lowry method, is comprised between 0.1 and 5 g/l and preferably between 0.5 and 2 g/l. The physico-chemical analysis of the vegetal hydrolysate, which constitutes the active principle, shows that its pH is comprised between 4 and 7, and preferably between 5 and 6, the dry extract titrates from 1 to 8 g/l and preferably between 2 and 5 g/l, its content of compounds of peptidic nature is comprised between 0.1 and 5 g/l, and preferably between 0.5 to 2 g/l and its content of sugars between 0.5 to 2.5 g/l.
EXAMPLE 2
Preparation of Active Principle from Flax (Linum usitatissimum L.)
A variant of the protocol of Example 1 consists in carrying out the same sequence of managed and selective enzymatic hydrolyses, but in the presence of 0.5% of PVPP.
One then obtains a light-coloured hydrolysate titrating from 15 to 30 g/l of dry extract after sterilising filtration.
One then proceeds to an ultrafiltration of the solution on a Millipore Helicon filtration cartridge (cutoff threshold; 1 kDa). The high molecular weights contained in the retentate are eliminated, the filtrate is retained. The analysis by electrophoresis of the peptides thus purified shows that their molecular weight is less than 10 kDa.
The concentration of compounds of peptidic nature is determined by the Lowry method, either comprised between 0.1 and 5 g/l and preferably between 0.5 and 2 g/l. The physico-chemical analysis of the vegetal hydrolysate, which constitutes the active principle, shows that its pH is comprised between 4 and 7, and preferably between 5 and 6, the dry extract titrates between 1 to 8 g/l, and preferably between 2 and 5 g/l, its content of compounds of peptidic nature is comprised between 0.1 and 5 g/l and preferably between 0.5 to 2 g/l and its content of sugars between 0.5 to 2.5 g/l.
Another variant consists in carrying out a purification of the active principle, obtained according to Example 1 or 2, by ion exchange chromatography, on a gel TSK column (TosoHaas) with a pH7 phosphate buffer.
EXAMPLE 3
Demonstration of the Stimulating Effect of the Active Principle According to Example 1 on the Synthesis of Intracellular ATP
The aim of this study is to determine the influence of the active principle according to Example 1 on the synthesis of ATP, produced by the mitochondria.
Protocol: This study is carried out by means of a kit “ATP Bioluminescence Assay Kit HS II” (Roche Applied Science). Dermal fibroblasts are treated with a 1% solution of active principle according to Example 1, for a period of from 1 to 3 hours. At the end of the incubation, the wells are rinsed with 2 ml of cold PBS before adding 250 μl of a lysis buffer provided by the kit. The cells of each well are then scraped, then collected in 14 ml tubes. Each well is rinsed with 2×500 μl cold PBS and the whole is collected again in the respective tubes. From these samples, a dilution is carried out at 1/12000th in cold PBS before each reading. The ATP dosage is carried out on these samples: 50 μL of this dilution are deposited in a luma cuvette and 50 μL of luminol are added. After 10 seconds, the reading of the luminescence is started. The values are standardised with respect to the quantity of proteins for each sample. The measurements are carried out by means of an apparatus: the Biocounter M2010A LUMAC®/3M.
Results: The dosages of ATP show that there is a great increase in the quantity of intracellular ATP after 1 hour and 3 hours, in cells treated by the active principle according to Example 1, in comparison with the non-treated cells.
Conclusion: The active principle according to Example 1 increases appreciably the energetic level of cutaneous cells such as fibroblasts, and more generally stimulates the mitochondrial functions.
EXAMPLE 4
Demonstration of the Activating Effect of the Active Principle According to Example 1 on the Expression of Cytochrome c
The aim of this study is to determine the influence of the active principle according to Example 1 on the expression of cytochrome c. For this, the quantity of cytochrome c was evaluated by the technique of immunoblot (or Western blot).
Protocol: Human dermal fibroblasts are treated with a 1% solution of active principle according to Example 1, for 72 hours. The cells are then lysed and homogenised by sonication, then centrifuged for 10 minutes at 1000 g. The samples, which are standardised for their protein concentration (dosage kit BCA, Pierce), are subjected to an electrophoresis on gel Bis-TRIS 4-12% (InvitroGen). After electrotransfert, the membranes are incubated for one night with an anti-cytochrome c antibody, diluted at 1/500 (Mouse monoclonal anti cytochrome c, TEBU). A secondary antibody, coupled to peroxydase and diluted at 1/5000 is then used (peroxydase conjugated F(ab′)2, Goat antimouse, Immunotech). The chemiluminescent signal is then quantified with the aid of the kit Supersignal West Femto Trial kit and read in a reading chamber (Multilmage light Cabinet, Alpha Immunotech Corporation).
Results: A clear increase is observed of the expression of cytochrome c in the fibroblasts treated by the active principle according to Example 1.
Conclusions: The active principle according to Example 1 greatly stimulates the expression of cytochrome c in the cutaneous cells, and more generally the mitochondrial functions.
EXAMPLE 5
Demonstration of the Activating Effect of the Active Principle According to Example 1 on the Enzymatic Activity of the Cytochrome Oxydase
The aim of this study is to determine the influence of the active principle according to Example 1 on the mitochondrial activity. For this, the total enzymatic (mitochondrial) activity of the cytochrome oxydase was measured.
Protocol: Normal human fibroblasts are treated with a 1% solution of active principle according to Example 1 for 3 hours and 24 hours. The cells are collected, rinsed, then lysed by sonication. A first centrifuging is carried out to eliminate the principal cellular debris and the supernatant is then centrifuged again at 10 000 g for 10 min. The enzymatic activity is dosed in the supernatant and/or in the 2nd cell pellet by a biochemical method, with the aid of the kit Cytocox 1 (Sigma), then standardised for the content of proteins (dosed by the kit PCA, Pierce).
Results: The dosages show that there is a very great increase in the enzymatic activity of the cytochrome oxydase after 3 hours and 24 hours of application of the active principle according to Example 1, in comparison with the non-treated cells.
Conclusion: The active principle according to Example 1 stimulates very greatly the enzymatic activity of the cytochrome oxydase, and more generally the mitochondrial activity, in the cutaneous cells.
EXAMPLE 6
Demonstration of the Protective Effect of the Active Principle According to Example 1 on the Mitochondrial Membrane Potential
The aim of this study is to determine the protective effect of the active principle according to Example 1, with respect to mitochondria of dermal fibroblasts subjected to an oxidative stress, caused by oxygenated water (H2O2) or a UVB irradiation. For this, one uses a marker of the membrane potential of the mitochondria (JC-1). JC-1 is a marker which emits a different fluorescence according to the level of polarisation of the mitochondrial membrane.
Protocol: The human dermal fibroblasts are treated with a 1% solution of active principle according to Example 1 for 96 hours, then subjected to an oxidative stress caused by H2O2 at 2 mM for 30 minutes, or to an irradiation by UVB at 50 mJ/cm2. Controls which are not treated by the peptide or by H2O2 or are not irradiated are realized under the same conditions. At the end of the experiment, the cells are washed, fixed and subjected to a marking by a solution of JC-1 (Molecular Probes) at 0.2 μg/ml, so as to reveal the membrane potential of the mitochondria.
Results: In the fibroblasts treated by the active principle according to Example 1, the mitochondria present a red fluorescence (JC-1 aggregate), the sign of a high membrane potential, greater than in the control cells. The fibroblasts which were irradiated or subjected to an oxidative stress have mitochondria which fluoresce little in the red range, and principally in the green range (monomeric JC-1), the sign of an alteration of the mitochondrial membrane potential. Under these latter conditions, the application of the active principle according to Example 1 allows a more marked red fluorescence to be observed.
Conclusions: The application of the active principle according to Example 1 causes an increase in the membrane potential of the mitochondria. Moreover, the active principle according to Example 1 efficiently protects the mitochondria of the cutaneous cells subjected to an oxidative stress or to an irradiation by UVB.
EXAMPLE 7
Demonstration of the Activating Effect of the Hydrolysate According to Example 2 on the Expression of Transglutaminases TG1, TG2, TG3 and TG5
The aim of this study is to determine the influence of the hydrolysate according to Example 2 on the expression of the different transglutaminases expressed in the human skin. For this, specific labellings by immunofluorescence were made on culture of normal human keratinocytes (NHK) and on skin biopsy. Labelling by immunofluorescence were likewise made on cultures of normal human fibroblasts, specifically for TG2, expressed in this type of cells.
Protocol of Immunolabellings on Normal Human Keratinocytes in Culture:
NHKs in culture are treated with a 1% solution of hydrolysate according to Example 2 for 24 hours. For immunolabelling by the antibody anti TG1, the cells are washed and fixed in paraformaldehyde at 3.7% for 10 minutes. The cells are then incubated in the presence of a specific antibody anti TG1 (Clinisciences BT-621, monoclonal mouse), then an adapted secondary antibody, coupled to a fluorescent marker. For the other immunolabellings, the cells are washed and fixed in cold methanol for 1 minute. The cells are then incubated in the presence of a specific antibody; anti-TG2 (Abcam ab2972, polyclonal rabbit), anti TG3 (Abcam ab53236, monoclonal mouse) or anti TG5 (Abcam ab26992, polyclonal rabbit). The slides are then observed under an epifluorescence microscope (Nikon Eclipse E 80i microscope), after mounting in an ad hoc medium.
Protocol of immunolabellings on normal human fibroblasts in culture: Human dermic fibroblasts are treated and immuno-marked by means of an antibody anti TG2 according to the same protocol as for the NHKs.
Protocol of the immunolabellings on skin biopsies: Human skin biopsies are placed in culture at the air/liquid interface. A 1% solution of hydrolysate according to Example 2 is applied topically for 24 hours.
For the labellings of TG1 and TG2, the skin biopsies are then enclosed in resin and frozen in nitrogen. Sections of approximately 6 μm are then made in the cryostat. The immunolabelling is carried out by means of a specific antibody: anti TG1 (Clinsciences BT-621, monoclonal mouse) or anti-TG2 (Abcam ab2972, polyclonal rabbit) then an adapted secondary antibody, coupled with a fluorescent marker. The sections of skin are then examined under the Epi-fluorescence microscope (Nikon Eclipse E 80i microscope).
For the labellings of TG3, the skin biopsies are enclosed in paraffin and histological sections of 3 μm thickness are carried out. The slides are deparaffinated, hydrated then subjected to immunlabelling by an antibody directed against TG3 (Abcam ab53236 monoclonal mouse) then an adapted secondary antibody, coupled to a fluorescent marker. The sections of skin are then examined under the Epi-fluorescence microscope (Nikon Eclipse E 80i microscope).
Results: In all the conditions tested, a more intense fluorescence is observed in the cultures and on the sections of skin treated by the hydrolysate according to example 2 at 1%, than in the untreated control conditions.
Conclusions: The hydrolysate according to Example 2 stimulates the expression of TG1, TG2, TG3 and TG5 in normal human keratinocytes in culture, and also the expression of TG2 in human fibroblasts.
The hydrolysate according to Example 2 stimulates the expression of TG1, TG2 and TG3 in skin biopsies cultivated ex vivo.
EXAMPLE 8
Study of the Expression of Hmg-CoA Reductase in Biopsies of Skin, in the Presence of the Hydrolysate According to Example 2
The aim of this study is to determine the influence of the hydrolysate according to Example 2 at 0.5% on the expression of HMG-CoA reductase.
Protocol: Samples of human skin are placed in culture at the air/liquid interface. The hydrolysate according to Example 2 at 0.5% is applied topically, then the samples are incubated for 24 hours or 48 hours.
These samples of skin are then fixed with formaldehyde then enclosed in paraffin. Sections of 2 to 3 μm are then made. The immunolabelling is carried out after demasking of the specific sites by microwave treatment then incubation in trypsin. The immunolabelling is carried out by means of a specific polyclonal rabbit antibody of HMG-CoA reductase (Millipore, Upstate), then a secondary antibody, coupled to a fluorescent marker. The sections of skin are then examined under the Epi-fluorescence microscope (Nikon Eclipse E600 microscope).
Results: The microscopic observations show a stronger fluorescence in the upper layers of the epidermis of the skins treated by the hydrolysate according to Example 2 at 0.5% compared with the non-treated control.
Conclusion: The hydrolysate according to Example 2 stimulates the expression of HMG-CoA reductase in the upper layers of the epidermis.
EXAMPLE 9
Demonstration of the Activating Effect of the Hydrolysate According to Example 2 on Epidermal Differentiation
The aim of this study is to determine the influence of the hydrolysate according to Example 2 on epidermal differentiation. For this, the expression of the principal markers of epidermal differentiation, expressed specifically in the keratinocytes of the suprabasal layers was studied. The markers tested are transglutaminase 1, pankeratins, filaggrin, involucrin and loricrin. Moreover, filaggrin, involucrin and loricrin are precursors of the cornified envelope and substrates of transglutaminases.
Protocol of the immunolabellings on normal human keratinocytes in culture: NHKs in culture are treated with a 1% solution of a solution of hydrolysate according to Example 2 for 24 hours. The cells are then washed and fixed in paraformaldehyde at 3.7% for 10 minutes. After demasking of the specific sites, the cells are incubated in the presence of a specific antibody directed against TG1 (Clinisciences BT-621, monoclonal mouse), or against loricrin (Abcam ab24722, polyclonal rabbit), or involucrin (Novocastra NCL-INV, monoclonal mouse, clone SY5), then incubated in the presence of an adapted secondary antibody, coupled to a fluorescent marker. For greater ease of observation, the nuclei of the cells can be counterstained by DAPI (4′,6′ Di Amidino-2-Phenyl Indole), a blue fluorescent molecule capable of binding strongly to DNA). The slides are then observed under the epifluorescence microscope (Nikon Eclipse E 80i microscope), after mounting in an ad hoc medium.
Protocol of the immunolabellings on skin biopsies: Human skin biopsies are placed in culture at the air/liquid interface. A 1% solution of hydrolysate according to Example 2 is applied topically for 24 hours. The skin biopsies are then enclosed in paraffin and histological sections of 3 μm thickness are carried out. The slides are deparaffinated, hydrated, then subjected to an immunolabelling by an antibody directed against TG1 (Clinisciences BT-621), monoclonal mouse or cytopankeratins (Novocastra (NCL-CK10, monoclonal mouse), or loricrin (Abcam ab24722, polyclonal rabbit), or involucrin (Novocastra NCL-INV, clone SY5, monoclonal mouse) or filaggrin (Tebu Santa Cruz sc-58761, monoclonal mouse). For a greater ease of observation, the nuclei of the cells can be counterstained by DAPI (4′,6′ Di Amidino-2-Phenyl Indole, a blue fluorescent molecule capable of binding strongly to DNA). An adapted secondary antibody, coupled to a fluorescent marker is then used. After mounting in an ad hoc medium, the slides are observed under the epifluorescence microscope (Nikon Eclipse E 80i microscope).
Protocol of the immunoblots: NHKs in culture are treated with a 1% solution of hydrolysate according to Example 2 for 24 hours. The cells are then rinsed then detached from the support by scraping in a RIPA buffer in the presence of a cocktail of protease inhibitors (Thermo Scientific, Rockford, USA). The lysed cells are centrifuged at 4° C. at 10000 rpm for 20 minutes and the supernatants are collected. The samples are then standardized by dosage of the proteins by the kit BCA (Pierce, France). The samples are mixed then subjected to an electrophoresis on gel NuPAGE 3-8% tris-acetate, in a migration buffer NuPAGE tris-acetate, then blotted on membrane of nitrocellulose by means of a blot device (Invitrogen, Paisley, UK). The membranes are saturated in 5% TBS milk for 2 hours at ambient temperature, then incubated at 4° C. overnight with a primary antibody directed against TG1 (Clinisciences BT-621, monoclonal mouse) or against involucrin (Novocastra NCL-INV, clone SY5, monoclonal mouse) or against loricrin (Abcam ab24722, polyclonal rabbit). After washing by buffer TBS-Tween 0.05%, the membrane is incubated with an adapted secondary antibody, coupled to peroxydase. The blots are then developed by means of a chemiluminescent substrate (SuperSignal West Dura Extended Duration Substrate, Pierce, Brebiere, France). The specific bands of proteins which are thus revealed are quantified by means of a Chemi-Imager technology image analyzer (Alpha Innotech Corporation).
Results: In all the conditions tested by immunofluorescence, a more intense fluorescence is observed in the cultures and on the sections of skin treated by the hydrolysate according to Example 2 at 1% than in the untreated control conditions. On the sections of skin ex vivo, an epidermal differentiation is noted with presence of a thicker horny layer.
The quantitative analysis of the immunoblots permits an evaluation of the increase of the expression of the tested markers. The increase of TG1 is in the order of 20%, for involucrin is greater than 25% and for loricrin is in the order to 15%.
Conclusions: The hydrolysate according to Example 2 stimulates the expression of pankeratins, involucrin, filaggrin, loricrin, and also TG1 is the normal human keratinocytes. The hydrolysate according to Example 2 likewise improves the epidermal differentiation and in particular the morphology of the horny layer.
EXAMPLE 10
Demonstration of the Protective Effect with Respect to External Aggressions of the Hydrolysate According to Example 2
The aim of this study is to determine the protective effect on the skin of the hydrolysate according to Example 2 with respect to external aggressions. For this, a model ex vivo of severe aggression of the cutaneous barrier was used.
Protocol: The biopsies of human skin are subjected to an aggression caused by successive tearing of strata of the horny layer by means of adhesive tape (technique known as “tape stripping”). The tearing stage is repeated 20 times in succession on the same zone. The “stripped” biopsies of human skin are then placed in culture and treated by the hydrolysate according to Example 2 at 1% and 3% according to the protocol of Example 3, for 24 hours and 48 hours. The skin biopsies are then enclosed in paraffin and histological sections of 3 μm thickness are made. The sections are deposited on Superfrost slides (Menzel Gläser, Thermo Scientific), then deparaffined in xylene and rehydrated in a series of alcohol-water solutions. The sections are then stained by hematoxylin at 50% for 3 minutes, rinsed, then stained with eosin 60% for 3 minutes, and rinsed in water. The sections are dehydrated, mounted in the Eukitt and examined by optical microscopy.
Results: The histological sections of skin treated by the hydrolysate according to Example 2 at the concentration 1% and 3%, show a greater neo-synthesis of the horny layers. The whole of the epidermal layers presents fewer vacuolized cells and a greater cellular density.
Conclusion: The hydrolysate according to Example 2 improves the reconstruction of the aggressed epidermis.
EXAMPLE 11
Study of the Protective Effect of the Hydrolysate According to Example 2 on the Cutaneous Cells Subjected to Ultraviolet Radiation (UVB)
The aim of this study is to determine the protective effect of the hydrolysate according to Example 2 with respect to normal human keratinocytes subjected to a stress by UVB radiation. For this, cellular viability tests were carried out by the MTT technique.
Protocol: The normal human keratinocytes are treated with the hydrolysate according to Example 2 at 0.5% for 24 hours, irradiated by UVB (50 mJ/cm2) then cultivated for a further 24 hours in the presence of the same concentration of hydrolysate according to Example 2. Non-treated and irradiated controls are made under the same conditions. At the end of the experiment, the cells are incubated in a solution containing 0.1 mg/ml MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium, bromide). This compound is absorbed by the living cells, then metabolized by the mitochondrial enzymes in a blue violet compound, formazan, which will be dosed by spectrophotometry at 540 nm. The optical density (O.D.) is then directly proportional to the mitochondrial enzymatic activity and also to the number of living cells.
Results: The evaluation of the cellular viability by the MTT technique shows that the hydrolysate according to Example 2 increases the cellular viability after irradiation by UVB of 16%.
Conclusion: The hydrolysate according to Example 2 at 0.5% increases the cellular viability and efficiently protects the cutaneous cells against cytotoxic effects and UVB radiation.
EXAMPLE 12
Clinical Study of the Effect of the Hydrolysate According to Example 2 on the Improvement of the Barrier Function of the Stratum Corneum and the Reduction of Damage to the Skin
The aim of this study is to evaluate clinically the protective effect on the skin of the hydrolysate according to Example 2 with respect to external aggressions. The selected model of aggression is a stress by SDS (sodium dodecyl sulphate).
Protocol:
The test was carried out as a double blind test.
12 volunteers applied twice a day the cream formulated at 1.5% of hydrolysate according to Example 2 for 21 days onto their forearms. At D21, the volunteers were subjected to a slight stress, by application of a patch of SDS (from 0.25% to 2% according to the volunteers) on the treated area. The concentration of applied SDS was selected to cause a moderate redness of score 2 in all the volunteers, according to the system for evaluation of erythema based on the Frosch and Kligman evaluation:
0=no erythema
1=slight redness, by small spots or diffused
2=moderate redness
3=intense redness
4=inflammatory redness and oedema
The SDS patch was kept for 24 hours under occlusion.
After the stress, the volunteers continued to apply the cream for 1 week.
Measurements were carried out at T0, T21 days (before the stress) then at T24 H after the stress, at T48 H and T1 week and T2 weeks after the stress.
The analyses of the skin were carried out by VivaScope® and the study concerned the stratum corneum and the non-corneous epidermis.
Apparatus:
The Lucid Vivascope (Rochester) is a confocal imager allowing images of the skin to be obtained “layer by layer”, in vivo, in a non-invasive manner with a cellular resolution.
Results:

    
    
        Results on the Stratum Corneum after 21 Days of Treatment:
    
    


In 90% of the volunteers, a highly significant reduction (p=0.00195) is observed of the thickness of the stratum corneum in the treated area compared with the control area treated by the placebo. The observation shows a better organisation of the cells, a greater compacting of the corneocytes, which is the sign of an improvement to the barrier function of the stratum corneum.

    
    
        Results on the Stratum Corneum at 24 H, 48 H and 1 Week after the SDS Stress:
    
    


The SDS stress causes on the microscopic scale the degradation by dissolution of the lipids of the stratum corneum, leading to a reduction in the thickness of the stratum corneum, the appearance of a parakeratosis (persistence of the nuclei in the stratum corneum) and the appearance of isolated corneocytes. (Astner S et al., Non-invasive evaluation of the kinetics of allergic and irritant contact dermatitis. J Invest Dermatol. 2005 February; 124(2):351-9; Astner S et al., Noninvasive evaluation of allergic and irritant contact dermatitis by in vivo reflectance confocal microscopy. Dermatitis, 2006 December; 17(4):182-91).
In accordance with the results of the literature, at 24 H, 48 H and 1 week after the stress, the observation shows a reduction in the thickness of the stratum corneum and a parakeratosis in the control treated areas treated by the placebo.
On the contrary, in the areas treated with the hydrolysate according to Example 2, there is no change in the thickness of the stratum corneum, the cells have not changed in morphology and parakeratosis is not noted.

    
    
        Results on the Epidermis after 21 Days of Treatment:
    
    


The observation shows a reduction in the thickness of the epidermis, due to a greater compacting and better organisation of the different layers of non-corneified cells, which is a sign of good health in the epidermis.
After the SDS stress, it can be noted that the reduction in the thickness of the epidermis in the control area, treated by the placebo, is greater than in the area treated by the hydrolysate according to Example 2.
In the treated area, the granulocytes present a homogeneous morphology, the cells are cohesive and smaller than in the placebo area, a sign of a greater cellular renewal, and sign of better health of the skin than in the control area.
The measurements of the thickness of the epidermis carried out at 24 H, 48 H and 1 week after the stress show that the thickness of the epidermis is reconstituted more rapidly in the treated area.
Conclusions:
This study demonstrates the protective effect with respect to a SDS stress. The skin treated by the hydrolysate according to Example 2 appears better prepared for the stress, due to a reinforcement of the cutaneous barrier and a better cohesion of the corneocytes.
Moreover, the hydrolysate according to Example 2 allowed the entire epidermis to maintain its integrity and to better realize its barrier function with respect to external aggressions. The hydrolysate according to Example 2 allowed a more rapid restoration of the epidermis after the stress.
EXAMPLE 12
Preparation of Compositions










1 - Sun protection cream:









Commercial names
INCI names
% mass










PHASE A









Demineralised water
Aqua (Water)
qs


Pemulen TR1
Acrylates/C10-30 Alkyl
0.40



Acrylate Crosspolymer


Glycerine
Glycerin
3.00


Nipastat Sodium
Sodium Methylparaben (and)
0.15



Sodium Ethylparaben (and)



Sodium Butyl paraben (and)



Sodium Propylparaben (and)



Sodium Isobutylparaben







PHASE B









Parsol MCX
Ethylhexyl Methoxycinnamate
7.50


Eusolex 4360
Benzophenone-3
3.00


Parsol 1789
Butyl Methoxydibenzoyl-
2.00



methane


Myritol 318
Caprylic/Capric Triglyceride
4.00


Emulgade SEV
Hydrogenated Palm Glycerides
5.00



(and) Ceteareth-20 (and)



Ceteareth-12 (and) Cetearyl



Alcohol


Propylparaben
Propylparaben
0.15


Nacol 16-98
Cetyl Alcohol
1.00







PHASE C









TEA
Triethanolamine
0.20







PHASE D









Active principle

3


of Example 2


Perfume
Parfum (Fragrance)
qs


Colouring agent

qs









The constituents of Phase A and Phase B are heated separately between 70° C. and 75° C. Phase B is emulsified in Phase A under stirring. Phase C is added, at 45° C., increasing the stirring. Phase D is then added when the temperature is below 40° C. The cooling is continued to 25° C. under strong stirring.










2 -After-sun milk:











Commercial names
INCI names
% mass











PHASE A











Montanov L
C14-22 Alcohols (and) C12-
3.00




20 Alkyl Glucoside



Waglinol 2559
Cetearyl Isononanoate
4.00



Tegosoft TN
C12-15 Alkyl Benzoate
3.00



Apricot kernel oil
Prunus Armeniaca
2.00




(Apricot) Kernel Oil



Avocado oil
Persea Gratissima
1.00




(Avocado) Oil



Abil 350
Dimethicone
1.00







PHASE B











Demineralised water
Aqua (Water)
qs







PHASE C











Simulgel EG
Sodium Acrylate/Acryloyl-
0.4




dimethyl Taurate Copolymer




(and) Isohexadecane (and)




Polysorbate 80 Copolymer




(and) Polysorbate 80







PHASE D











Phenonip
Phenoxyethanol (and)
0.30




Methylparaben (and)




Ethylparaben (and)




Butylparaben (and)




Propylparaben (and)




Isobutylparaben




Ethylparaben and




Propylparaben and




Buthylparaben



Germall 115
Imidazolidinyl Urea
0.20







PHASE E











Active principle

0.1



of Example 2










Prepare Phase A under stirring. Incorporate the xanthan gum progressively, under deflocculating stirring. Phases C and D will be incorporated once the gel is finished. Phase E, prepared previously up to perfect dissolving of the DHA, will then be added. Adjust the pH if necessary to 4-4.5. Colour and perfume.










3 -Anti-ageing cream:









Commercial names
INCI names
% mass










Phase A









Montanov 68
Cetearyl Alcohol (and)
6.00



Cetearyl Glucoside


Squalane
Squalane
3.00


Cetiol SB 45
Butyrospermum Parkii (Shea
2.00



Butter)


Waglinol 250
Cetearyl Ethylhexanoate
3.00


Amerchol L- 101
Mineral Oil (and) Lanolin
2.00



Alcohol


Abil 350
Dimethicone
1.50


BHT
BHT
0.01


Coenzyme Q10
Ubiquinone
0.10







Phase B









Avocado oil
Persea Gratissima (Avocado)
1.25



Oil


Phenonip
Phenoxyethanol (and)
0.75



Methylparaben (and)



Ethylparaben (and)



Butylparaben (and)



Propylparaben (and)



Isobutylparaben







Phase C









Demineralised water
Aqua (Water)
qs


Butylene Glycol
Butylene Glycol
2.00


Glucam TM E10
Methyl Gluceth-10
1.00


Allantoin
Allantoin
0.15


Carbopol ® Ultrez 10
Carbomer
0.20







Phase D









TEA
Triethanolamine
0.18







Phase E









Active principle

0.5


of Example 2


GP4G
Water (and) Artemia Extract
1.50


Collaxyl
Water (and) Butylene Glycol
3.00



(and) Hexapeptide-9







Phase F









Perfume
Parfum (Fragrance)
qs


Colouring agent

qs









Prepare and melt Phase A at 65-70° C. Heat Phase C at 65-70° C. Phase B is added to Phase A just before emulsifying A in B. At approximately 45° C., the carbomer is neutralised by addition of Phase D. Phase E is then added under light stirring and the cooling is continued to 25° C. Phase F is then added, if desired.










4 -Protective day cream:









Commercial names
INCI names
% mass










Phase A









Emulium Delta
Cetyl alcohol (and) Glyceryl
4.00



Stearate (and) PEG-75



Stearate (and) Ceteth-20



(and) Steareth-20


Lanette O
Cetearyl Alcohol
1.50


D C 200 Fluid/100 cs
Dimethicone
1.00


DUB 810C
Coco Caprylate/Caprate
1.00


DPPG
Propylene Glycol
3.00



Dipelargonate


DUB DPHCC
Dipentaerythrityl
1.50



Hexacaprylate/Hexacaprate


Cegesoft PS6
Vegetable Oil
1.00


Vitamin E
Tocopherol
0.30


Phenonip
Phenoxyethanol (and)
0.70



Methylparaben (and)



Ethylparaben (and)



Butylparaben (and)



Propylparaben (and)



Isobutylparaben







Phase B









Demineralised water
Aqua
qsp 100


Glycerine
Glycerin
2.00


Carbopol ETD 2020
Acrylates/C10-30 Alkyl
0.15



Acrylate Crosspolymer


Keltrol BT
Xanthan Gum
0.30







Phase C









Sodium Hydroxide
Sodium Hydroxide
0.30


(sol. 10%)







Phase D









Demineralised water
Aqua
5.00


Stay-C 50
Sodium Ascorbyl Phosphate
0.50







Phase E









Butylene Glycol
Butylene Glycol
2.00


Dekaben CP
Chlorphenesin
0.20







Phase F









GP4G
Water (and) Artemia Extract
1.00


Active principle

5


of Example 2









Prepare Phase A and heat at 75° C. under stirring. Prepare Phase B, dispersing the carbopol, then the xanthan gum under stirring. Allow to rest. Heat at 75° C.
At temperature, emulsify A in B under rotor-stator stirring. Neutralise with Phase C under rapid stirring. After cooling at 40° C., add Phase D, then Phase E. The cooling is continued under light stirring and Phase F is added.

## Claims

1. A method of reinforcing barrier function of the skin and stimulating epidermal differentiation of a patient's skin in need thereof, consisting essentially of administering to said patient's skin in need thereof, a cosmetic composition consisting essentially of a therapeutically effective quantity of a hydrolysate from flax seeds that have been hydrolyzed in the presence of polyvinylpolypyrrolidone.

2. The method of claim 1, wherein said flax seeds have been previously solubilised in one or more solvents selected from the group consisting of water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated diglycols, propoxylated diglycols, and cyclic polyols.

3. The method of claim 1, wherein the hydrolysate from flax seeds are 0.0001% to 20% of the total weight of the composition.

4. The method of claim 1, wherein the hydrolysate from flax seeds are 0.05% to 5% of the total weight of the composition.

5. A method of reinforcing barrier function of the skin and stimulating epidermal differentiation of a patient's skin in need thereof, consisting essentially of administering to said patient's skin in need thereof, a cosmetic composition consisting essentially of a therapeutically effective quantity of an hydrolysate from flax seeds that have been hydrolyzed in the presence of polyvinylpolypyrrolidone and an active principle selected from the group consisting of vitamin C, vitamin E, the coenzyme Q10, collagen peptide, artemia extract, and combinations thereof.

6. The method of claim 1, wherein said cosmetic composition is intended to protect the skin and the appendages against all types of external aggressions.

7. The method of claim 6, wherein said external aggressions are UV radiations.

8. The method of claim 6, wherein said cosmetic composition improves and repairs skin and the appendages after all types of external aggressions.

9. The method of claim 1, wherein the hydrolysate from flax seeds has a pH between 5 and 6.

10. The method of claim 1, wherein the hydrolysate from flax seeds has a dry extract titrated between 2 to 5 g/l.

11. The method of claim 1, wherein the hydrolysate from flax seeds has between 0.5 and 2 g/l of compounds of peptidic nature.

12. The method of claim 1, wherein the hydrolysate from flax seeds has a pH between 4 and 7, a dry extract titrated between 1 to 8 g/l, between 0.1 and 5 g/l of compounds of peptidic nature, and between 0.5 to 2.5 g/l of sugars.

13. The method of claim 5, wherein the cosmetic composition is a sun protection cream.