WO2014029948A2

WO2014029948A2 - Flax extract and cosmetic composition comprising said extract for increasing the level of intracellular coenzyme q10

Info

Publication number: WO2014029948A2
Application number: PCT/FR2013/051954
Authority: WO
Inventors: Jean-Marie Botto; Nouha Domloge; Frédérique PORTOLAN
Original assignee: Isp Investments Inc.
Priority date: 2012-08-24
Filing date: 2013-08-21
Publication date: 2014-02-27
Also published as: WO2014029948A3; FR2994656B1; FR2994656A1

Abstract

The invention relates to an extract of flax (Linum) originating from the hydrolysis of flax proteins and to a cosmetic composition comprising same. The invention also relates to a synergic cosmetic composition comprising a flax extract as a first active principle and coenzyme Q10 as a second active principle. The invention further relates to the use of a cosmetic composition intended to protect the skin and appendages from external aggression and to delay the visible signs of cutaneous aging.

Description

FLAX EXTRACT AND COSMETIC COMPOSITION COMPRISING SAID EXTRACT FOR INCREASING THE LEVEL OF COENZYME Q10

INTRACELLULAR

The present invention is in the cosmetic field. The present invention relates to a flax extract (Linum) derived from the hydrolysis of proteins extracted from flax and a cosmetic composition comprising it. The invention also relates to a synergistic cosmetic composition, comprising a linseed extract as the first active ingredient, and coenzyme Q10 as the second active ingredient.

The invention further relates to the use of a cosmetic composition for increasing the level of transprenyl transferase enzyme expression and / or increasing the concentration of Coenzyme Q10 Binding protein and / or increasing the concentration of Coenzyme Q10 in cells. of the skin, intended to protect the skin and integuments of external aggressions and delay the appearance of the manifestations of skin aging.

The term "superficial body growths" according to the invention encompasses all the keratinous appendages present on the surface of the body, in particular the hairs, the eyelashes, the eyebrows, the nails and the hair.

Aging and the appearance of the skin are at the heart of research in the field of cosmetics and more particularly in the field of cosmetic skin care.

Indeed, the skin is the first protective barrier of the body and determines the external appearance of individuals. The skin is involved especially in maintaining body temperature, protecting tissues and organs from most external aggressions, perception, and immunity. The skin, like the other organs, is constantly subjected to the aggressions by various types of oxidative stress which, over time, amplify the phenomenon of chronological and intrinsic genetic aging. Oxidative damage is caused by free radicals, chemically unstable and highly reactive species, generated by intracellular metabolism or external aggression. These external aggressions include: UV radiation, toxins, air pollutants, food oxidants. In the skin, there is premature aging occurring in areas exposed to radiation, characterized by phenomena of alterations of macromolecules (lipid peroxidation, carbonylation of proteins) particularly affecting elastin, collagen or fibronectin.

The body has antioxidant defense mechanisms, capable of trapping or transforming free radicals such as enzymes, glutathione, vitamins A and E, coenzyme Q10, etc.

Coenzyme Q10 is a benzoquinone derivative flanked by a long isoprene side chain, most often composed of ten isoprenoid units (hence the name Coenzyme Q10). The biosynthesis of Coenzyme Q10 is made from tyrosine for the quinone nucleus, and from farnesyl pyrophosphate for the side chain. The enzyme responsible for this latter reaction, which is an essential step in the biosynthesis of coenzyme Q10, is transprenyl transferase (or polyprenyltransferase, hereinafter referred to as "PDSS1 / 2") composed of two subunits: PDSS1 and PDSS2.

Specifically, Coenzyme Q10 (or ubiquinone) is an endogenous, naturally occurring, lipophilic antioxidant found in all lipid membranes, such as the inner membrane of the mitochondria, where it can diffuse freely among membrane phospholipids. The mitochondrial respiratory chain is composed of four complexes that, from the metabolites generated by the Krebs cycle, catabolism of pyruvate, amino acids and fatty acids, can synthesize ΓΑΤΡ. ATP, or adenosine triphosphate, has for main role to provide the energy necessary for the chemical reactions of the cells, because it has links rich in energy. Lipophilic transporters allow the transport of electrons between different mitochondrial complexes. More particularly, coenzyme Q10 is the electron carrier between complexes I and II and complexes III of the mitochondrial respiratory chains, and therefore, it is involved in the oxidative phosphorylation leading to the production of TP.

A protein is particularly important for proper functioning of coenzyme Q10 in the mitochondrial respiratory chain: the Coenzyme Q10 binding protein (hereinafter referred to as "CoQ1OBP") located in the inner membrane of the mitochondria. This protein binds Coenzyme Q10 analogously to a chaperone protein, and is involved in the transport of Coenzyme Q10 from its synthetic site to the respiratory complexes. The localization of CoQlOBP is correlated with the presence of Coenzyme Q10 (Cui and Kawamukai, 2009).

Another fundamental property of Coenzyme Q10 is to be an antioxidant, neutralizing free radicals. Indeed, Coenzyme Q10 can exist in three states oxidation: a reduced form (CoQH2 or UQH2), an oxidized form (CoQ10), and an intermediate form the radical ubisemiquinone (Q °). Coenzyme Q10, present in the skin, protects the stability of cell membranes, protects DNA from oxidative damage induced by free radicals, and recycles and regenerates other antioxidants such as tocopherol or ascorbate (Skull, 1984, 2001). Therefore, coenzyme Q10 stimulates the natural functions of cells and acts as a defender against external aggressions.

In this context, the antioxidant and energizing properties of coenzyme Q10 appear to be particularly interesting for delaying the manifestations of aging of the skin.

One of the proposed solutions is coenzyme Q10 supplementation.

For example, the use of coenzyme Q10 as a dietary supplement, in beverages or food for its cellular energizing and antioxidant properties has been described.

Another solution proposed in the prior art is a direct supply of coenzyme Q10 to the skin, by application of cosmetic compositions comprising Coenzyme Q10 for its effects as an antioxidant, skin repairing and for its anti-aging and anti-wrinkle properties ( Hojerov and others, 2006).

However, the disadvantage of the known solutions is a low efficiency to protect the skin from external aggressions and to delay the appearance of the manifestations of aging of the skin. Indeed, coenzyme Q10 is a large molecule thermolabile and lipophilic so its bioavailability by a topical intake is low. In addition, the exogenous coenzyme Q10 is still in oxidized form, which prevents it from acting as an antioxidant (Katawan et al., 2007).

The present invention aims to solve all or part of the disadvantages mentioned above. The inventors have demonstrated a cosmetic activity of a particular linen extract described in the present invention. In particular, it has been demonstrated that this flaxseed extract, when applied to the skin, has a strong protective activity vis-à-vis the aggressions suffered by the skin, responsible for the appearance of the signs of aging.

This new active ingredient, capable of increasing cellular energy and protecting the skin from oxidative damage, significantly promotes the synthesis of ATP, as well as the expression of the enzyme PDSS1 / 2 and / or CoQlOBP and / or coenzyme Q10 and thus opens up new cosmetic perspectives. According to a first aspect, the present invention relates to a flax extract derived from the hydrolysis of proteins of flax (Linum), characterized in that it contains at least 0.1 to 5 g / l of peptide-like compounds by weight. of dry extract, 0.1 to 2 g / l of sugar by weight of dry extract and essentially comprises peptide compounds with a molecular weight of less than 5 kDa, preferably with a molecular weight of less than 2.5 kDa.

The invention and the advantages thereof will be better understood from reading the description.

The invention is directed to mammals in general, and more particularly to humans.

The term "peptide" refers to a sequence of two or more amino acids linked together by peptide bonds or modified peptide bonds; the term "polypeptide" designating a peptide of larger size; the term "peptide compounds" designating the protein fragments, peptides and free amino acids present in the mixture.

The term "hydrolyzate or derived from hydrolysis" refers to any substance or mixture of substances, or isolated preparation, obtained after hydrolysis of plant material.

To carry out the extraction, one can use the whole plant, or a specific part of the plant (leaf, seed, etc.).

More particularly according to the invention, one of the many plants of the linaceae family, of the genus Linum (flax), is used. The genus Linum has nearly 200 species growing throughout the northern hemisphere. They are herbaceous plants with fibrous stems, single leaves, flowers with 5 petals. Preferably, according to the invention, the cultivated species Linum usitatissimum L. is used. The linseed extract (Linum) according to the invention is preferably a peptide extract originating from the hydrolysis of the proteins extracted from flaxseeds and preferably the freed seed. of its envelope by a step of dehulling.

Any method of extraction or purification known to those skilled in the art can be used to prepare the flax extract according to the invention.

Preferably, the flax extract is obtained by a process which comprises:

a step of extracting proteins of plant origin,

a controlled hydrolysis step which releases biologically active peptide compounds. Many proteins found in plants are likely to contain logically active organic peptide compounds within their structures. The controlled hydrolysis makes it possible to release these peptide compounds. It is possible, but not necessary to carry out the invention, to extract either the proteins concerned first and then hydrolyze them, or to carry out the hydrolysis first on a crude extract and then to purify the peptide compounds. .

A particular embodiment of the process for obtaining the flax extract according to the invention is described below.

In a first step, the plant is ground using a plant grinder. The powder thus obtained may subsequently be "delipidated" with the aid of a conventional organic solvent (for example an alcohol, hexane or acetone).

In a second step, the proteins of the plant are extracted according to a conventional method. The plant meal is suspended in an alkaline solution containing an insoluble polyvinylpolypyrrolidone adsorbent material (PVPP) (0.01-20%); in fact, it has been observed that the hydrolysis and subsequent purification operations are facilitated by this means. The concentration of phenolic-type substances interacting with proteins is thus reduced.

The soluble fraction is collected after centrifugation and filtration steps, this crude solution then constituting a first form of the extract comprising proteins, carbohydrates and optionally lipids.

According to a particular mode of implementation of the process, the proteins can then be precipitated by varying the ionic strength by acidifying the medium, which makes it possible to eliminate the soluble components. The precipitate is then washed with an organic solvent such as, for example, ethanol or butanol and the solvent is evaporated by drying in vacuo. The protein-rich precipitate is dissolved in water or other solvent and is then a more purified form of the hydrolyzate.

The protein extraction step of the plant can also be carried out in neutral or acidic medium always in the presence of polyvinylpolypyrrolidone. After a filtration step, a precipitation step may be carried out in a particular mode of implementation, using a conventional precipitation agent such as salts (sodium chloride, ammonium sulfate) or a solvent organic (alcohol, acetone). The precipitate obtained can be separated from the precipitating agents by dialysis after redissolving in water or another solvent. The soluble fraction, comprising proteins, carbohydrates and optionally lipids, is collected after centrifugation and filtration steps. This crude solution is then hydrolyzed under mild conditions to generate peptidic compounds, polypeptides and soluble peptides. Hydrolysis is defined as a chemical reaction involving the cleavage of a molecule by water, this reaction being possible in a neutral, acidic or basic medium. According to the invention, the hydrolysis is carried out chemically and / or advantageously by proteolytic enzymes.

According to particular characteristics, the hydrolysis is carried out by a protease or a mixture of proteases and / or a cellulase or a mixture of cellulases.

According to preferred features, an enzyme mixture comprising:

endoproteases of plant origin (for example papain, bromelain, ficin, etc.) and / or derived from microorganisms (Aspergillus, Rhizopus, Bacillus, Alcalase®, etc.),

and / or a cellulase or a mixture of cellulases (for example Celluclast® CL).

Advantageously, cellulases allow a better hydrolysis of the cell wall of flax which increases the accessibility to proteins and facilitates filtration. Indeed, cellulases allow the hydrolysis of cell walls into oligosaccharides of small sizes. The joint action of cellulases and proteases then leads to the production of polypeptides of low molecular weight less than 5 kDa and more particularly a significant fraction less than 2.5 kDa. Once these different hydrolyses have been performed, a thermal deactivation step is necessary to inactivate these enzymes.

For the same reasons as above, that is to say the removal of the polyphenol substances, an amount of polyvinylpolypyrrolidone is added to the reaction medium during this mild hydrolysis step.

After filtration, the solution obtained constitutes a first advantageous form of peptide extract of flax according to the invention. The peptide extract of flax can be further purified in order to select the molecular weights and the nature of the peptides generated. The fractionation can advantageously be carried out by ultrafiltration and / or by a chromatographic type method.

The use of peptide extracts, and in particular peptide extracts of low molecular weight, has many advantages in cosmetics. In addition to generating peptide compounds that did not pre-exist in the starting protein mixture, hydrolysis and purification provide a mixture of more peptide compounds. stable, more easily reproducible composition and not causing allergic reactions in cosmetics.

According to particular features, the flax extract according to the invention essentially comprises peptide compounds of low molecular weight. Preferably, the peptide extract of flax according to the invention essentially comprises peptide compounds with a molecular weight of less than 2.5 kDa.

Any of the more or less purified forms of the hydrolyzate is then solubilized in water or in any mixture containing water, and then sterilized by ultrafiltration.

The flaxseed extract obtained according to the invention is analyzed qualitatively and quantitatively for its physico-chemical characteristics and its content of peptide compounds. The term "peptide compounds" means the protein fragments, the peptides and the free amino acids present in the mixture. The peptides, amino acids and protein fragments are determined according to conventional techniques, which are well known to those skilled in the art.

Thus, according to an advantageous embodiment of the invention, the flax extract has a pH of between 4 and 5, and preferably between 4 and 4.5. Advantageously, this acidic pH promotes dissolution of the proteins in the water and stabilizes the proteins. The flax extract according to the invention has a dry weight of between 0.1 and 8 g / l, and preferably between 0.1 and 5 g / l, and comprises:

between 0.1 and 5 g / l of peptide compounds by weight of dry extract,

and between 0.1 and 2 g / l of sugars by weight of dry extract.

The extract is then diluted in water or in any solvent mixture containing water and sterilized by sterilizing filtration (0.2 μιη).

According to particular characteristics, the extract is diluted in one or more physiologically adapted solvents, such as water, glycerol, ethanol, propanediol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, polyols cyclic or any mixture of these solvents. "Physiologically suitable" means that the chosen solvent is suitable for coming into contact with the skin without causing toxicity or intolerance reactions.

Preferably, after dilution, the flax extract according to comprises:

between 1.5 and 3.5 g / l of peptide compounds by weight of dry extract,

and about 0.3 g / l of sugar by weight of dry extract. The sugar content is even more preferably less than 0.3 g / l in the flax extract according to the invention.

After this dilution step, the extract may be encapsulated or included in a cosmetic vector such as liposomes or any other microcapsules used in the field of cosmetics or adsorbed on powdery organic polymers, mineral supports such as talcs and bentonites, and more generally solubilized in, or attached to, any physiologically adapted vector.

According to a second aspect, the invention relates to a cosmetic composition comprising a flax extract according to the invention.

The cosmetic composition according to the invention comprises a quantity of linseed extract necessary in order to obtain the desired result, namely: increasing the intracellular levels of coenzyme Q10 and / or coenzyme Q10 Binding Protein and / or the expression of the transprenyl transferase enzyme, and more generally, protect the skin and integuments of external aggressions and delay the onset of manifestations of skin aging.

According to an advantageous embodiment of the invention, the flax extract according to the invention is present in the composition at a concentration of between 0.0001% to 20% approximately, and preferably at a concentration of between 0.05%. and about 5%, even more preferably at a concentration of between about 1% and 3% relative to the total weight of the final composition.

The compositions according to the invention may be applied by any appropriate route, in particular oral, parenteral or external topical, and their formulations are adapted by those skilled in the art, in particular for cosmetic compositions.

Advantageously, the compositions according to the invention are intended for administration topically cutaneous, on at least a portion of the skin of the face or body. The cosmetic composition according to the invention can be used as a care product and / or as a makeup product for the skin. These compositions must therefore contain a physiologically adapted medium according to the invention, that is to say a medium which is suitable for use in contact with the skin or the human integuments, without risk for example of toxicity, incompatibility, d instability, or allergic response.

Preferably, the composition according to the invention intended to be applied topically to at least a part of the skin of the face or body is in the form of an aqueous, hydroalcoholic or oily solution, an oil-in-water emulsion, or a water-in-oil or multiple emulsion, solution, suspension, microemulsion, aqueous or anhydrous gel, serum, or dispersion of vesicles, colloid. These compositions may also be in the form of creams, suspensions or powders, suitable for application to the skin, mucous membranes, lips and / or integuments. These compositions may be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a cream, a paste, a an ointment, or foam. They can also be in solid form, such as a stick, a patch, or applied to the skin in the form of an aerosol or spray. They can be used as a care product and / or as a make-up product for the skin. These compositions may also be adapted to applications on the scalp and / or the hair, and in particular a shampoo, a conditioner, a styling lotion, a treatment lotion, a cream or a styling gel, a lotion for hair, a mask, etc. The cosmetic composition according to the invention can be used in particular in treatments using an application that is followed or not followed by rinsing, or in the form of shampoo. It can also be in the form of dye or mascara to be applied with a brush or a comb, in particular on eyelashes, eyebrows or hair.

These compositions additionally comprise any additive commonly used in the field of application envisaged as well as the adjuvants necessary for their formulation, such as co-solvents (ethanol, glycerol, benzyl alcohol, humectant, etc.), thickeners, diluents, emulsifiers, antioxidants, dyes, sunscreens, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, trace elements, essential fatty acids, surfactants, film-forming polymers, chemical or mineral filters, moisturizing agents or thermal waters, etc. It is possible, for example, to mention water-soluble polymers of the natural polymer type, such as polysaccharides, or polypeptides, cellulose derivatives of the methylcellulose or hydroxypropylcellulose type, or also synthetic polymers, polaxamers, carbomers, siloxanes, PVAs or PVPs, and in particular polymers sold by Ashland. In all cases, those skilled in the art will ensure that these adjuvants and their proportions are chosen so as not to adversely affect the desirable properties of the composition according to the invention. These adjuvants may, 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 may represent from 5 to 80% by weight and preferably from 5 to 30% by weight relative to the total weight of the composition. The emulsifiers and co-emulsifiers used in the composition will be chosen from those conventionally used in the field under consideration. For example, they can be used in a proportion ranging from 0.3 to 10% by weight, relative to the total weight of the composition.

It is understood that the active ingredient according to the invention can be used alone or in combination with at least one other active ingredient in a cosmetic composition.

Advantageously, the compositions that may be used according to the invention may furthermore comprise various active principles intended, in particular, for the prevention and / or treatment of disorders associated with photoaging. Thus, the composition according to the invention can associate, with the active principle according to the invention, other active ingredients promoting its action. For example, it may be added active ingredients having an anti-radical or antioxidant action, selected from vitamin C, vitamin E, coenzyme Q10 and polyphenolic extracts of plants.

There may be mentioned, in a non-limiting manner, the following classes of ingredients: other peptide active ingredients, plant extracts, cicatrizing, anti-aging, anti-wrinkle, soothing, anti-radical, anti-UV, agents stimulating the synthesis of dermal macromolecules or energy metabolism, moisturizing, antibacterial, antifungal, anti-inflammatory, anesthetic agents, modulating agents differentiation, pigmentation or skin depigmentation, stimulating agents the growth of nails or hair etc. Preferably, use will be made of an agent having an activity in the field of anti-wrinkles, such as an anti-radical agent or antioxidant, or an agent stimulating the synthesis of dermal macromolecules, or an agent stimulating energy metabolism. More particularly, the active ingredient is chosen from vitamins, phytosterols, flavonoids, DHEA and / or one of its precursors or one of its chemical or biological derivatives, a metalloproteinase inhibitor, or a retinoid.

In a particularly preferred embodiment, the invention relates to a synergistic cosmetic composition, comprising a peptide extract of flax as the first active ingredient, and coenzyme Q10 as the second active ingredient.

Advantageously, the inventors have demonstrated that the simultaneous use in a composition of coenzyme Q10 associated with the flax extract according to the invention allows a Significantly greater increase in the level of coenzyme Q10 in cells compared to the use of coenzyme Q10 alone or flaxseed extract according to the invention. This synergistic effect is particularly advantageous in a cosmetic composition.

According to an advantageous embodiment of the invention, the flax extract according to the invention is present in the synergistic composition according to the invention at a concentration of between about 0.0001% and about 20%, and preferably at a concentration of between between about 0.05% and 5% relative to the total weight of the final composition.

According to a particularly advantageous embodiment of the invention, the coenzyme Q10 is present in the synergistic composition according to the invention at a concentration of between 0.0001% and about 5% relative to the total weight of the final composition, and preferentially at a concentration of between 0.01% and 0.3% relative to the total weight of the final composition.

A third aspect of the invention relates to the use of a flax extract as described above, or a composition comprising it, for increasing the activity or synthesis of transprenyl transferase enzyme and / or increasing the concentration of Coenzyme Q10 Binding protein and / or increase the concentration of coenzyme Q10 in skin cells.

In a particular embodiment, the flax extract according to the invention may advantageously be used in a cosmetic composition intended to delay the onset of the manifestations of cutaneous aging and, more specifically, in order to delay the appearance of the manifestations of aging. photo-induced (photo-aging).

"Cutaneous manifestations of aging" means any changes in the external appearance of the skin and skin appendages due to aging, for example, wrinkles and fine lines, wilted skin, soft skin, thinned skin, lack of skin. elasticity and / or tone of the skin, dull and lackluster skin or skin pigmentation spots, discoloration of the hair or spots on the nails, but also any internal change in the skin that does not result not systematically by a modified external appearance such as, for example, any internal degradation of the skin following exposure to ultraviolet (UV) radiation. The flax extract according to the invention, or the composition comprising it, will make it possible, in particular, to combat the loss of elasticity and firmness of the skin. The flax extract according to the invention makes it possible to protect the skin and integuments against all types of external aggressions. The use of the flax extract, or a composition comprising it, will allow the skin and the integuments to be protected and to better withstand environmental stresses.

The expression "external aggression" means the aggressions that the environment can produce. By way of example, mention may be made of aggressions such as pollution, UV, or irritating products 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 "internal" pollution, which may be due in particular to the solvent emissions of paints, glues , or wallpaper (such as toluene, styrene, xylene or benzaldehyde), or even cigarette smoke.

The invention also relates to the use in a cosmetic composition of a flax extract as described above, the flax extract, or the composition comprising it, being intended to increase the synthesis of intracellular ATP of the cells. skin.

The subject of the invention is also the use in a cosmetic composition of a linen extract as described above, the flax extract, or the composition comprising it, being intended to prevent damage to the skin caused by Sun exposure.

The invention also relates to the use in a cosmetic composition of a linen extract as described above, the flax extract, or the composition comprising it, being intended to protect the skin from damage caused by the radicals. free.

According to a fourth aspect, the invention also consists of a cosmetic treatment method, intended to stimulate the defenses and to protect the skin and the integuments from external aggressions and to delay the appearance of the manifestations of skin aging, characterized by the application on the skin or integuments to be treated with a flax extract according to the invention, or a composition comprising it.

The invention also consists of a cosmetic treatment method intended to give a healthier appearance to the skin and to improve the radiance and radiance of the complexion, characterized by the application on the skin to be treated of a linen extract according to the invention, or a composition comprising it.

Particular embodiments of this cosmetic treatment method also result from the foregoing description. Other advantages and characteristics of the invention will appear better on reading the examples given by way of illustration and not limitation. Example 1 Preparation of active principle from flax (Linum usitatissimum L.)

In a first step, 1 kg of shelled linseed (Linum usitatissimum L.) seeds are crushed in a cereal grinder. The flour obtained (1 kg) is brought into contact with 10 liters of hexane. The mixture is then stirred for 2 hours at room temperature in order to proceed to delipidation of the raw material. After filtration and drying under vacuum, the powder obtained is suspended in 800 ml of an aqueous alkaline solution (dilution 1/10) pH 10 containing 1% of polyvinylpolypyrrolidone (Polyclar V ISP). This mixture is stirred for 2 hours at room temperature to allow the solubilization of the soluble fractions. After this extraction phase, the medium is clarified by centrifugation and then filtered through a plate filter. This filtrate which contains the soluble fractions of flax is then subjected to protein precipitation by varying the ionic strength in neutral or acidic medium, which eliminates soluble carbohydrate components, lipids and nucleic acids. at pH 3.5. The supernatant is removed and the precipitate is then washed with ethanol and the solvent is evaporated by drying in vacuo.

At this stage, about 50 grams of light yellow powder of crude protein extract containing:

- Protein: 78%

- Carbohydrates: 20%

- Lipids <2%

The precipitate rich in proteins is dissolved in 500 grams of water.

The crude protein extract is then subjected to a series of controlled and selective enzyme hydrolyses in the presence of 0.5% PVPP (Polyclar V) and cysteine endopeptidases (2 g / l bromelain and 2 g / 1 alkalase). . After 2 hours of reaction at 50 ° C and then deactivation of the enzyme cocktail for 2 hours at 80 ° C., the hydrolyzate is filtered on plates of decreasing porosity then on sterilizing cartridge (0.2 μιη).

A light-colored hydrolyzate, titrating with 15 to 30 g / l of dry extract, is then obtained, which is then diluted so that the concentration of peptide compounds determined by the Lowry method is between 0.1 and 5. g / 1 and preferably between 1.5 and 3.5 g / l. The physicochemical analysis of the vegetable hydrolyzate, which constitutes the active ingredient, shows that its pH is between 4 and 5, and preferably between 4 and 4.5. The flax extract according to the invention has a solids content of between 1 and 8 g / l, and preferably between 0.1 and 5 g / l, and comprises between 0.1 and 5 g / l of peptide compounds. by weight of dry extract and between 0.1 and 2 g / l of sugars by weight of dry extract, preferably the flax extract according to the invention is diluted to contain between 1.5 and 3.5 g / l of peptide compounds by weight of extract dry and between 0.1 and 0.3 g / 1 of sugar by weight of dry extract.

Example 2 Analysis of the Peptide Compounds of the Linseed Extract According to Example 1

The molecular weights of the peptide compounds of the extract obtained according to Example 1 were evaluated by Steric Exclusion Chromatography.

Protocol:

A linen extract obtained according to Example 1 is analyzed by Agilent HP 1200 liquid chromatography controlled by ChemStation.

The following table details the characteristics of this analysis:

TOSOH BIOSCIENCE TSKGEL SuperSW 2000 column

Stationary phase Silica gel grafted with hydrophilic groups

Column size 4.6 mm I.D x 30 cm

Particle size 4 μιη

Pore size 125 Â

Mobile phase 0.1 mol / L Na ₂ S0 ₄ +0.05% NaN ₃ in 0.1 M buffer

phosphate pH = 6.7

280 nm UV detection

Flow rate of the phase 0.175 mL / min

mobile

Wash solution 0.5 mol / L Na ₂ SO ₄ , pH = 2.7

Temperature 25 ° C

Injection volume Ι Ο μΙ, Results:

It is observed that the molecular weights are as follows:

75.1% of the peptide compounds of the flax extract have a molecular weight of between 1.5 and 2.5 kDa.

20% of the peptide compounds of the flax extract have a molecular weight of less than 1.5 kDa.

Conclusion:

The flaxseed extract according to Example 1 is composed of peptides of low molecular weight essentially less than 2.5 kDa.

EXAMPLE 3 Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression of Coenzyme Q10

The purpose of this study is to determine the influence of flaxseed according to Example 1 on the expression of Coenzyme Q10. For this, the intracellular level of Coenzyme Q10 was evaluated by High Performance Liquid Chromatography (HPLC).

Protocol:

HaCaT cells (Human adult low calcium calcium keratinocytes) are treated twice daily with the flax extract according to Example 1 at 1% for 24 hours. The cells are then washed three times with PBS and detached from the culture medium using 200 mg / L trypsin-EDTA for 10 minutes at 37 ° C and 5% CO 2. The trypsin is neutralized with culture medium and the cell suspension is centrifuged twice at 9168 RCF at 4 ° C for 10 minutes. The cell pellet is then dissolved in 700 of a hexane / isopropyl alcohol mixture (5: 2) and undergoes three cycles of: freezing / thawing, sonication (10 seconds, 130 Watt, 20 KHz) and centrifugation at 9168 RCF at 4 ° C for 10 minutes. The supernatant is recovered and then evaporated. After total evaporation, 200 g of isopropyl alcohol are added and the sample is analyzed by HPLC. Calibration is performed using commercial Coenzyme Q10 dissolved in isopropyl alcohol (Prolab).

Results:

A marked 40% increase in the level of Coenzyme Q10 was observed in the cells treated with the flax extract according to Example 1 for 24 hours. Conclusions:

The flaxseed extract according to Example 1 stimulates the increase of the level of coenzyme Q10 in the cells of the skin.

EXAMPLE 4 Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression of the Coenzyme Q10 Binding Protein

The purpose of this study is to determine the influence of flaxseed extract according to Example 1 on the expression of the Coenzyme Q10 binding protein. For this, the expression level of the Coenzyme Q10 binding protein was evaluated by immuno-cytochemistry on keratinocytes treated for 48 hours with the flax extract according to Example 1 at 1%, or without treatment (control ) and included in paraffin.

Protocol:

NHK cells (normal primary human keratinocytes) are fixed with 10% formalin and then embedded in paraffin. The cell block is then cut into sections of 4 μιη thick with a microtome which are then transferred to a slide.

The sections are deparaffed in 100% xylene and then rehydrated in successive alcohol baths: 2 100% ethanol (EtOH) baths for 1 minute, 1 95% EtOH bath for 1 minute, 1 EtOH bath 90% for 1 minute and 1 H ₂ 0 bath for 5 minutes. The slides are immersed in a 0.01 M pH 6 citric acid buffer and heated in a microwave oven at 600 Watts, until lightly boiling to facilitate access of the antibody to the protein of interest . The sections are incubated with 5% BSA for 30 minutes, and then with the primary antibody, a polyclonal anti-Coenzyme Q10 binding protein B antibody (Abcam ab41997, Cambridge, UK) diluted 1: 200 in PBS during 1.5 hours with stirring and at room temperature. After several washings with PBS, the sections are incubated with the fluorescent secondary antibody, an Alexa Fluor 488 anti-rabbit antibody (Invitrogen A21206, Fisher) diluted 1/1000 in PBS for 1 hour at room temperature. The cell nuclei are labeled with 0.3 μl of 4 ', 6'-diamidino-2-phenylindole (DAPI) (Molecular Probes). The sections are rinsed for 5 minutes in PBS. The expression of the Coenzyme Q10 binding protein is detected by means of a fluorescence microscope (x40 objective). Results:

The results show that there is a 68.4% increase in the expression of the Coenzyme Q10 binding protein in keratinocytes treated with flaxseed extract according to Example 1 at 1% after 48 hours, compared to untreated keratinocytes.

Conclusion:

The flaxseed extract according to Example 1 stimulates the expression of the amount of the Coenzyme Q10 binding protein.

EXAMPLE 5 Demonstration of the Activating Effect of the Linseed Extract According to Example 1 on the Expression Level of the Transprényltransferase Messenger RNAs

The purpose of this study is to determine the influence of linseed extract according to Example 1 on the expression of transprenyl transferase (PDSSl / 2), which is the main enzyme in the biosynthesis of Coenzyme Q10 (composed two subunits: PDSS1 and PDSS2). For this purpose, the expression level of the PDSS1 messenger RNA is evaluated by real-time PCR.

Protocol:

NHK cells are treated with 1% flaxseed extract according to Example 1, twice daily, for 24 hours or untreated (control). In order to evaluate the level of expression of the PDSS1 messenger RNA, the total RNAs of the keratinocytes in culture, treated or not treated with the flax extract, must be isolated according to Example 1. The total RNAs are then transformed into Complementary DNAs by the action of a reverse transcriptase enzyme. Quantification of the complementary DNAs is then performed by real-time PCR using a StepOnePlus ™ thermocycler (Applied Biosystems). This quantification makes it possible to determine the level of expression of the messenger RNAs of PDSS 1.

Results:

A 33% increase in PDSS1 messenger RNA expression level was observed in the cells after 24 hours of treatment with the linseed extract according to Example 1, compared with the untreated cells. Conclusion:

The flaxseed extract according to Example 1 stimulates the expression level of the messenger RNA coding for PDSS1 in normal human keratinocytes.

Example 6 Evaluation of the Protective Effect of Linseed Extract According to Example 1 with Respect to Oxidative Damage

The purpose of this study is to determine the protective effect of the flaxseed extract according to Example 1 with respect to NHK cells subjected to oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ). at 2 mM, by UVB radiation or by a chemical agent, Rotenone at ΙμΜ. To evaluate the oxidative damage to the cells, a quantification of the superoxide ion level is performed. For this, the Mito S OX ™ reagent mitochondrial superoxide indicator (Invitrogen) is used as cationic derivative of dihydroethidium designed for the detection of superoxide ions in the mitochondria of living cells. Protocol:

NHK in culture were brought into contact with the flaxseed extract according to Example 1 at 1% for 24 hours. The cells are then subjected to different oxidative stress:

The NHKs are contacted with a solution of 2 mM H ₂ 0 ₂ for 30 minutes.

Or NHKs are irradiated with UVB radiation at 75 mJ / cm ² in PBS.

- Or the NHK are brought into contact with a solution of Rotenone at 1 μΜ for 4 hours. After each stress, the cells are incubated at 37 ° C. and 5% CO ₂ for 1 hour with culture medium. Untreated and non-oxidative stress controls are performed. The MitoSOX ™ Red mitochondrial superoxide indicator solution at 5μΜ is applied to the cells for 10 minutes at 37 ° C. The cells are washed three times with PBS and fixed with 3.7% formaldehyde (Sigma Aldrich, USA) for 10 minutes at room temperature with gentle shaking and protected from light. The cell nuclei are labeled with 0.3 μl of 4 ', 6'-diamidino-2-phenylindole (DAPI) (Molecular Probes). The cells are rinsed for 5 minutes in PBS. The superoxide ion level is detected by immunofluorescence using a microscope (40x objective) and quantified. Results:

Pretreatment with 1% linseed extract according to the example reduces the production of superoxide ions after oxidative stress (decrease of 38.2% after stress ¾ (¾ 36.1% after UVB radiation stress). and 62.7% after Rotenone stress).

Conclusion:

The flaxseed extract according to Example 1 effectively protects skin cells against oxidative damage caused by hydrogen peroxide, UVB radiation or by the chemical Rotenone.

EXAMPLE 7 Evaluation of the Synergistic Effect of Linseed Extract According to Example 1 with Coenzyme Q10

The purpose of this study is to determine the synergistic effect of the linseed extract according to Example 1, with Coenzyme Q10 vis-à-vis the intracellular amount of Coenzyme Q 10 binding protein.

Protocol:

Human skin biopsies are maintained in culture ex vivo, and then treated, once a day for 24 hours, with 20 μl of a 1% solution of the flax extract according to Example 1 and / or 20 μl of 1 μΜ solution of purified Coenzyme Q10 (Sigma), or untreated.

Skin biopsies are then fixed and then paraffin-embedded after passage through a Shandon Hypercenter XP (Shandon, UK). The paraffin-embedded skin biopsies are then cut into sections of 4 μιη thick with a microtome and are then transferred to a slide. The sections are deparaffed in 100% xylene and then rehydrated in successive alcohol baths: two 100% ethanol (EtOH) baths for 1 minute, 1 95% EtOH bath for 1 minute, 1 EtOH bath 90% for 1 minute and 1 H ₂ 0 bath for 5 minutes. The slides are immersed in a 0.01M pH 6 citric acid buffer and heated until light boiling to facilitate access of the antibody to the protein of interest. The sections are incubated with 5% BSA for 30 minutes, and then with the primary antibody, a polyclonal rabbit anti-Coenzyme Q10 binding protein B (Abcam ab41997, Cambridge, UK) diluted 1/200 in PBS. for 1 hour and a half with stirring and at room temperature. After several washings with PBS, the sections are incubated with the fluorescent secondary antibody, an Alexa Fluor 488 anti-rabbit antibody (Invitrogen A21206, Fisher) diluted 1/1000 in PBS for 1 hour at room temperature. The cell nuclei are labeled with 0.3 μl of 4 ', 6'-diamidino-2-phenylindole (DAPI) (Molecular Probes). The slides are rinsed for 5 minutes in PBS. The expression of the Coenzyme Q10 binding protein is evaluated by means of a fluorescence microscope (20x objective). The resulting images are converted to grayscale and analyzed by the Image-Pro Analyzer software 6.3 (MediaCybemetics, Inc.) in terms of intensity and number of pixels. Finally, the analysis is completed by statistical analyzes.

Results:

Quantification of the fluorescence present on the skin sections shows that the skin biopsies treated with the combination of the flax extract according to Example 1 and Coenzyme Q10 have a higher fluorescence level (+77%), very statistically significant, compared with untreated skin biopsies.

In addition, the increase obtained with the combination of linseed extract and Coenzyme Q10 is greater than the sum of the increased expression of Coenzyme Q10 binding protein by treatment with the extract. flax according to Example 1 alone (+47%) or by purified Coenzyme Q10 alone (+ 9%).

Conclusion:

A synergistic effect on the expression of the Coenzyme Q10 binding protein is obtained by combining the flax extract according to Example 1 with the purified coenzyme Q10 on human skin biopsies.

Examples: Preparation of compositions

1 - Sun protection cream:

The constituents of phase A and phase B are heated separately between 70 ° C and 75 ° C. Phase B is emulsified in phase A with stirring. Phase C is added at 45 ° C, increasing stirring. Phase D is then added when the temperature is below 40 ° C. Cooling is continued up to 25 ° C with vigorous stirring. -After-sun milk

The constituents of phase A and phase B are heated separately between 70 ° C and 75 ° C. Phase A is emulsified in phase B with stirring. Phase C is added at 45 ° C, increasing stirring. Phases D and E are then added when the temperature is below 40 ° C. Cooling is continued up to 25 ° C with vigorous stirring.

3 -Anti-age cream:

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 + B in C. At about 45 ° C, the carbomer is neutralized by addition of phase D. Phase E is then added with gentle stirring and cooling is continued up to 25 ° C. Phase F is then added if desired.

4 -Protective protector of day:

Prepare phase A and heat to 75 ° C. Prepare phase B by dispersing the carbopol, then the xanthan gum with stirring. Let stand until perfectly homogeneous. Heat B at 75 ° C.

At 75 ° C., emulsify A in B with rotor-stator stirring. Neutralize with phase C with rapid stirring. After cooling to 40 ° C, add the clear D phase, then the E phase (preheated to 40 ° C and homogenized to perfect clarity). The cooling is continued with gentle stirring to 25 ° C and the F phase added.

Claims

1. Flax extract (Linum) originating from the hydrolysis of proteins of flax, characterized in that it contains at least 0.1 to 5 g / l of peptide compounds by weight of solids, 0.1 to

2 g / 1 of sugar by weight of dry extract and essentially comprises peptide compounds with a molecular weight of less than 5 kDa.

2. Flax extract according to claim 1, characterized in that it essentially comprises peptide compounds with a molecular weight of less than 2.5 kDa.

3. Flax extract according to one of claims 1 to 2, characterized in that it is obtained by hydrolysis of flax proteins in the presence of cellulases.

4. flax extract according to one of claims 1 to 3, characterized in that it is previously solubilized in one or more physiologically suitable solvents, such as water, glycerol, ethanol, propanediol, butylene glycol , dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols or any mixture of these solvents.

5. Cosmetic composition characterized in that it comprises a flax extract according to one of claims 1 to 4, as active ingredient in a physiologically suitable medium.

6. Composition according to claim 5, characterized in that the flax extract is used in an amount representing from 0.0001% to 20% of the total weight of the composition, and preferably in an amount representing from 0.05% to 5% of the total weight of the composition.

7. Synergistic cosmetic composition according to one of claims 5 to 6, characterized in that it comprises a flax extract according to one of claims 1 to 4 as the first active ingredient, and coenzyme Q10 as a second active ingredient.

8. synergistic cosmetic composition according to claim 7, characterized in that the coenzyme Q10 is used in an amount representing from 0.0001% to 5% of the total weight of the composition, and preferably in an amount representing from 0.01% to 0.3% of the total weight of the composition.

9. Cosmetic composition according to one of claims 5 to 8, characterized in that the composition is in a form suitable for topical application comprising a physiologically suitable medium.

10. Composition according to one of claims 5 to 9, characterized in that it further comprises, at least one other active ingredient promoting the action of said flax extract, selected from antioxidant active ingredients and / or active ingredients stimulating the synthesis of dermal macromolecules, and / or the active ingredients stimulating the energy metabolism.

11. Use of a composition according to one of claims 5 to 10 for increasing the level of expression of transprenyl transferase enzyme and / or increase the concentration of Coenzyme Q10 Binding protein and / or increase the concentration of coenzyme Q10. in the cells of the skin.

12. Use of a composition according to one of claims 5 to 10, for protecting the skin and integuments against all types of external aggression.

13. Use of a composition according to one of claims 5 to 10, for delaying the appearance of cutaneous manifestations of aging and / or photo-aging.

14. Cosmetic treatment method for stimulating the defenses and protecting the skin and integuments against all types of external aggressions, characterized in that it is applied topically on the skin or integuments to treat a composition comprising a quantity effective agent, as defined in any one of claims 1 to 4.

15. Cosmetic treatment process intended to protect the skin and superficial body growths, characterized in that a composition comprising an effective amount of active principle, as defined according to any one of Claims 1, is applied topically to the skin to be treated. at 4.