WO2008145850A2

WO2008145850A2 - Use of an active ingredient derived from amaranth (amaranthus hypochondriacus) for preparing a composition for protecting mitochondria

Info

Publication number: WO2008145850A2
Application number: PCT/FR2008/000573
Authority: WO
Inventors: Claude Dal Farra; Nouha Domloge; Jean-Marie Botto
Original assignee: Societe D'extraction Des Principes Actifs S.A. (Vincience)
Priority date: 2007-04-27
Filing date: 2008-04-23
Publication date: 2008-12-04
Also published as: FR2915377B1; WO2008145850A3; FR2915377A1

Abstract

The present invention relates to the use, in a cosmetic composition or for preparing a pharmaceutical composition, of an effective amount of an active ingredient derived from amaranth; wherein the active ingredient, or the composition containing the latter, is for protecting the mitochondria and for activating aconitase. The active ingredient comes from the hydrolysis of amaranth (Amaranthus hypochondriacus) proteins and contains mainly polypeptides and peptides. It may be used alone or in combination with at least one other active ingredient. The invention also relates to a cosmetic treatment process for protecting the skin and the appendages against external attacks and for combating skin ageing.

Description

USE OF AN ACTIVE INGREDIENT DERIVED FROM AMARANTE (AMARANTHUS HYPOCHONDRIACUS) FOR PREPARING A COMPOSITION INTENDED FOR

PROTECT MITOCHONDRIES

The present invention is in the cosmetic and pharmaceutical field, and more particularly in the field of dermatology. The present invention relates to the use in a cosmetic composition or for the preparation of a pharmaceutical composition, of an effective amount of active ingredient derived from amaranth; the active ingredient, or the composition containing it, being intended to protect mitochondria and to activate aconitase. The active substance is derived from the hydrolysis of amaranth (Amaranthus hypochondriacus) proteins and contains mainly polypeptides and peptides. It can be used alone or in combination with at least one other active ingredient. The invention also relates to a cosmetic treatment method intended to protect the skin and the integuments from external aggressions and to fight against skin aging. Said active ingredient, mitochondrial protector and activator of aconitase, can also be used to prepare pharmaceutical compositions intended to prevent or fight against pathologies related to mitochondrial dysfunctions; for example certain neuromuscular or cardiac degenerations, type II diabetes or certain pathologies of 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.

Mitochondria are the organelles of eukaryotic cells specialized in the production of energy. They have their own DNA (mtDNA). The energy produced by the respiratory chain is stored as ATP. Under certain cellular stress conditions, electrons, released by the oxidative phosphorylation respiratory chain, produce free radicals that can damage mitochondrial structures. These stressors can be intrinsic or external. Among these are: UV radiation, toxins, radiation, food oxidants. Thus, it has been possible to show a progressive decline in mitochondrial function with age, probably related to the accumulation of mutations on mtDNA (Singh, K., Ann Acad Sci 1019, 2004). These mutations in mtDNA can be induced by repeated UV radiation exposures, and would even be a control of photo-aging (M. Bernburg et al., J. of Invest Dermatol 122 (5), 2004). Mitochondria also play a central role in apoptosis. Mitochondrial degradations can thus be a triggering factor of the reaction cascade leading to apoptosis in many cell types (G. Kroemer et al., FASEB J. 9: 1277-87, 1995).

In mitochondria, natural mechanisms of repair have been identified. Aconitase (aconitate hydratase or Iron Regulatory Protein, EC 4.2.1.3) has thus been shown to be essential for maintaining the integrity of mtDNA, regardless of its catalytic activity. Aconitase was first identified as a mitochondrial enzyme involved in the Krebs cycle; it catalyzes the stereospecific conversion of citrate to isocitrate and thus participates in the energy production of the cell. It has an active site with an iron-sulfur (4Fe-4S) cluster with pseudocubic geometry. Recently, ^Dr. Butow's team (XJ Chen et al., Science 307, 2005) has shown that aconitase plays a key role in preserving the integrity of mitochondrial DNA. It would interact with the DNA within regulatory regions suggesting a role in compaction of the mitochondrial genome in the manner of a protein of the HSP 70 family. The transition from the DNA binding form to the active form of the enzyme would be based on the assembly or disassembly of the iron-sulfur cluster.

Due to its dual functionality, aconitase establishes a direct link between the mitochondrial energy-generating activity and the conservation of mtDNA. Aconitase could thus constitute a therapeutic target to prevent or fight against pathologies related to mitochondrial dysfunctions, for example, type II diabetes, certain neuromuscular disorders, certain pathologies of aging.

The search for compounds that can stimulate or protect mitochondria is a concern of medical research and cosmetics. With regard to the skin, these new compounds could be useful to prevent or fight against the signs of skin aging, but also to protect or repair the skin after burns, exposure to UV radiation or radiation, or exposure to toxins or to pollutants.

In order to protect the mitochondria and to fight against the associated pathologies or the effects of skin aging, solutions have been proposed such as the supply of substances involved in energy metabolism, and more particularly intermediates or cofactors of the skin cycle. Krebs such as fumarate, L-malate, acetyl CoA (WO 02064129) or the treatment of the skin with substances capable of reducing free radicals, such as vitamin C (US 2004/0086526) or L-ergothioneine (WO 9836748). But, to the knowledge of the applicant, the use of active principles derived from the hydrolysis of amaranth, capable of activating aconitase, in order to protect mitochondria and fight against associated pathologies and / or the effects of skin aging, has never been described.

The main object of the present invention is the use of an effective amount of an active principle of a peptide nature, originating from the hydrolysis of Amaranthus hypochondriacus amaranth proteins, in a cosmetic composition for activating aconitase. , protect mitochondria and fight against skin aging. Said active ingredient may be used alone or in combination with at least one other active ingredient. The inventors have indeed demonstrated a therapeutic activity, and more particularly a dermatological and cosmetic activity, of an active principle of a peptide nature, resulting from the hydrolysis of amaranth, capable of activating aconitase. In particular, it has been demonstrated that this active ingredient, when applied to the skin, significantly promotes the mitochondrial activity, demonstrated by an increased activity of the enzyme. The newly identified properties of this mitochondrial protective active ingredient thus open up new therapeutic and cosmetic perspectives.

"Protective active principle of mitochondria or capable of protecting mitochondria" is understood to mean a peptide hydrolyzate originating from the hydrolysis of amaranth proteins (Amaranthus hypochondriacus), capable of limiting the functional or structural alterations of cell mitochondria or tissues subject to physico-chemical or environmental stress.

The term "active ingredient capable of activating aconitase" means a peptide hydrolyzate originating from the hydrolysis of amaranth (Amaranthus hypochondriacus) proteins, capable of increasing cytoplasmic or mitochondrial aconitase, or by activation of protein synthesis of aconitase (by direct or indirect modulation of the gene expression of aconitase), either by increasing the enzymatic activity of aconitase, or by other biological processes such as that the stabilization of the protein aconitase or the stabilization of messenger RNA transcripts.

"Peptide" is understood to mean a mixture of compounds predominantly represented by peptides or polypeptides. 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" denotes a peptide of larger size.

By the term "biologically active" is meant "which has an activity in vivo or in vitro characteristic of the activity of the active ingredient according to the invention".

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

The active ingredient according to the invention can be obtained by extraction of proteins of plant origin, followed by controlled hydrolysis which releases biologically active peptide fragments.

Many proteins found in plants are likely to contain biologically active peptide fragments within their structure. The controlled hydrolysis makes it possible to release these peptide fragments. 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 fragments. . It is also possible to use certain hydrolysed extracts without purifying the peptide fragments corresponding to the biologically active peptides according to the invention, but nevertheless ensuring the presence of said fragments by appropriate analytical means.

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

Amaranths are annuals of the family Amaranthaceae belonging to the genus Amaranthus, some of which are grown as ornamental plants for their spectacular flowering spikes, and sometimes as vegetables, for their spinach-like edible leaves or for their seeds. . The Amaranthus genus includes many species, mainly from the tropical and temperate regions of America and Asia, including Amaranthus hypochondriacus, Amaranthus cruentus, Amaranthus edulis, Amaranthus ticolor, Amaranthus caudatus, Amaranthus hybridus.

According to the invention the species used will be Amaranthus hypochondriacus, the plant material used will be the seed and preferably the seed removed from its envelope by a dehulling step. 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).

The proteins of the plant are then extracted according to the conventional method (Osborne, 1924) modified; the plant meal is suspended in an alkaline solution containing an insoluble polyvinylpolypyrrolidone (PVPP) adsorbent product (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 the 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 containing the proteins, carbohydrates and optionally lipids.

The proteins are then precipitated by varying the ionic strength by acidifying the medium, which eliminates soluble components and nucleic acids.

The precipitate is then washed with an organic solvent such as, for example, ethanol or methanol 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 extraction can also be carried out in neutral or acidic medium always in the presence of polyvinylpolypyrrolidone. After a filtration step, the precipitation step is then carried out using a conventional precipitation agent such as salts (sodium chloride, ammonium sulfate) or an organic solvent (alcohol, acetone). The precipitate obtained can be separated from the precipitating agents by dialysis after redissolving in water or another solvent.

The isolated protein fraction according to the invention is then hydrolyzed under mild conditions to generate 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. We can then mention the use of endoproteases of plant origin (papain, bromelain, ficin) and microorganisms (Aspergillus, Rhizopus, Bacillus, etc.).

For the same reasons as above during this hydrolysis step, a quantity of polyvinylpolypyrrolidone is added to the reaction medium. After filtration solution obtained constitutes the active hydrolyzate. The active hydrolyzate 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.

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 vegetable hydrolyzate obtained according to the invention is analyzed qualitatively and quantitatively for its physico-chemical characteristics and its content of protein and peptide compounds. Peptide-like compounds are understood to mean protein fragments, peptides and 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 active plant hydrolyzate has a pH of between 4 and 7, and preferably between 5 and 6, a solids content of between 1 and 8 g / l and preferably between 2 and 5 g / 1, its content of peptide compounds is between 0.1 and 5 g / l and preferably between 0.5 and 2 g / 1 and its sugar content is 0.5 to 2.5 g / 1.

According to an advantageous embodiment of the invention, the active principle according to the invention is solubilized beforehand in one or more cosmetically or pharmaceutically acceptable solvents, conventionally used by those skilled in the art, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents.

According to yet another advantageous embodiment of the invention, the active principle according to the invention is solubilized beforehand 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 solubilized in, or attached to, any cosmetically or pharmaceutically acceptable carrier.

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 or for the preparation of a pharmaceutical and / or dermatological composition. The compositions according to the invention may be applied by any appropriate route, in particular oral, parenteral or external topical, and their formulation will be adapted by those skilled in the art, in particular for cosmetic or dermatological compositions. Advantageously, the compositions according to the invention are intended for topical administration to the skin. These compositions must therefore contain a cosmetically and / or dermatologically acceptable medium, that is to say compatible with the skin and superficial body growths, and cover all cosmetic or dermatological forms. These compositions may especially be in the form of creams, oil-in-water or water-in-oil emulsions, multiple emulsions, solutions, suspensions, gels, milks, lotions, sticks or else powders, suitable for application on the skin, lips and / or integuments.

These compositions comprise the excipients necessary for their formulation, such as solvents, thickeners, diluents, surfactants, antioxidants, dyes, preservatives, perfumes.

Of course, those skilled in the art will take care to choose any additional compounds, active or non-active, and / or their quantity, so that the advantageous properties of the mixture are not impaired by the addition envisaged.

The composition that may be used according to the invention may in particular consist of a composition for hair care, and in particular a shampoo, a conditioner, a setting lotion, a treatment lotion, a cream or a styling gel, a restructuring 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.

Advantageously, the compositions that can be used according to the invention also contain other active ingredients intended to promote the action of the active principle according to the invention. Among these other active ingredients, there may be mentioned the active ingredients having an anti-radical or antioxidant action, chosen from vitamin C, vitamin E or coenzyme Q10 or polyphenol extracts of plants.

By "anti-radical active ingredients" is meant any compound capable of trapping free radicals. These active principles are capable of blocking free radical chain reactions before the ultimate stages of degradation of the biological constituents of the skin and thus have an antioxidant activity. Among these other active ingredients, mention may also be made of the active ingredients stimulating the synthesis of dermal macromolecules (laminin, fibronectin, collagen), for example the collagen peptide sold under the name "Collaxyl®" by the company Vincience. Among these other active ingredients, we can finally mention the active ingredients stimulating energy metabolism, such as the active ingredient marketed under the name "GP4G®" by Vincience.

According to another aspect, the composition according to the invention may be a solar composition, that is to say a composition helping to protect against solar radiation. Thus, it can be advantageously added, to the composition according to the invention, active assisting solar protection such as, for example, sunscreens.

It is obvious that the invention is directed to mammals in general, and more particularly to humans.

The effective amount of active ingredient according to the invention corresponds to the amount necessary to obtain the desired result, namely: activate aconitase, protect mitochondria and thus protect the skin from external aggression and fight against skin aging.

According to an advantageous embodiment of the invention, the active principle derived from amaranth is present in the compositions of the invention at a concentration of from 0.0001% to about 20%, and preferentially at a concentration of between 0, Approximately 5% and 5% relative to the total weight of the final composition.

These compositions may especially be in the form of an aqueous solution, hydroalcoholic or oily; an oil-in-water, water-in-oil emulsion or multiple emulsions; they 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, lotion, milk, serum, ointment, gel, paste or paste. a foam. They can also be in solid form, as a stick, or be applied to 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 additionally comprise any additive commonly used in the intended field of application and the adjuvants necessary for their formulation, such as solvents, thickeners, diluents, antioxidants, dyes, filters solar, self-tanning principles, pigments, fillers, preservatives, perfumes, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers etc.

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 50% 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 30% by weight, relative to the total weight of the composition.

By its particular activities, the active ingredient according to the invention can be used advantageously in a cosmetic composition or for the preparation of a pharmaceutical composition.

In particular, the active ingredient according to the invention may advantageously be used in a cosmetic composition intended to fight in a preventive and / or curative manner against the manifestations of skin aging and, more specifically, in order to fight against and / or prevent aging. photo-induced (photo-aging). Skin manifestations of aging means any changes in the external appearance of the skin due to aging such as, for example, wrinkles and fine lines, wilted skin, soft skin, thinned skin, lack of elasticity and / or or skin tone, dull and lackluster skin or skin pigmentation spots, but also any internal changes in the skin that do not systematically result in a modified external appearance such as, for example, any internal degradation of the skin. skin following exposure to ultraviolet (UV) radiation. The active ingredient according to the invention, or the composition containing it, will make it possible, in particular, to combat the loss of elasticity and firmness of the skin.

The active ingredient according to the invention makes it possible to protect the skin and integuments against all types of external aggressions. The use of the active ingredient, or a composition containing it, will allow the skin and 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 active ingredient according to the invention may advantageously be used in a cosmetic composition or for the preparation of a pharmaceutical composition, as a photo-protective active ingredient and, more particularly, as a so-called "secondary" photo-protective active ingredient. . In fact, the primary photoprotective active principles are distinguished from the secondary photoprotective active ingredients. Primary photoprotective active ingredients are substances that exercise physical power: they are able to absorb UV radiation and return it as heat to protect the skin. Secondary photoprotective active ingredients are substances that usually have a biological effect; these are, for example, the active ingredients capable of limiting damage to DNA and membranes by the penetration of UV radiation into the skin.

The subject of the invention is also the use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective quantity of active principle as described above, the active ingredient, or the composition containing it, being intended to to prevent damage to the skin caused by exposure to the sun or exposure to ionizing radiation during radiation therapy.

The invention also relates to the use in a cosmetic composition, or for the preparation of a pharmaceutical composition, of an effective amount of active ingredient as described above, the active ingredient, or the composition containing it, being intended to to protect the skin from damage caused by free radicals.

The invention also relates to the use of an effective amount of active principle according to the invention, for preparing a pharmaceutical composition intended to prevent or to fight against certain pathologies related to mitochondrial dysfunctions, for example, certain neuromuscular degenerations or cardiac, type II diabetes, certain pathologies of aging. The invention finally relates to a cosmetic treatment method for stimulating the defenses and protecting the skin and the integuments of external aggressions, characterized in that it is applied topically to the skin or integuments to treat a composition containing a effective amount of active ingredient according to the invention.

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 Ingredient from Amaranth (Amaranthus hypochondriacus)

The active agent is obtained from an extract of plants of the species Amaranthus hypochondriacus.

In a first step, 1 kg of shelled amaranth seeds are crushed in a cereal mill. The flour obtained is delipidated by the action of an organic solvent, hexane. After filtration and drying under vacuum the powder obtained is suspended in an aqueous alkaline solution (1/10 dilution) pH 10, containing 1% of polyvinylpolypyrrolidone (Polyclar V ISP). This mixture is stirred for a time long enough to allow the solubilization of the soluble fractions. The extraction temperature is variable (between 4 and 80 ⁰ C); preferably the operation will be performed cold. 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 amaranth, is then subjected to precipitation of the proteins by varying the ionic strength in neutral or acidic medium, which eliminates soluble carbohydrate components, lipids and nucleic acids. is brought to pH 3.5. The supernatant is removed and the precipitate is then washed with a solvent such as, for example, ethanol or methanol 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: 75%

- Carbohydrates: 20%

- Fat: 5%

The protein-rich precipitate is dissolved in water or another solvent. The crude protein extract is then subjected to a series of gentle and selective hydrolyses consisting of chemical and enzymatic hydrolyses in the presence of 0.5% PVPP (Polyclar V) and cysteine endopeptidases (papain, ficin). After reaction, the hydrolyzate is filtered on a plate and then on a sterilizing cartridge (0.2 μm).

A light-colored hydrolyzate titrant of 15 to 30 g / l of dry extract is then obtained, which is then diluted so that the concentration of peptides determined by the method of Lowry is between 0.1 and 5 g. and preferably between 0.5 and 2 g / l. The physicochemical analysis of the vegetable hydrolyzate, which constitutes the active ingredient, shows that its pH is between 4 and 7, and preferably between 5 and 6, the dry extract is between 1 to 8 g / l and preferred manner between 2 and 5 g / 1, its content of peptide compounds is between 0.1 and 5 g / l and preferably between 0.5 to 2 g / 1 and its sugar content between 0.5 to 2, 5 g / 1.

Example 2 Preparation of Active Ingredient from Amaranth (Amaranthus hypochondriacus)

A variant of the protocol of Example 1 consists in carrying out the same sequence of controlled and selective enzymatic hydrolyses but in the presence of 0.5% of PVPP.

A light-colored hydrolyzate of 15 to 30 g / l of dry extract is obtained after sterilizing filtration.

The solution is then ultrafiltered on a Millipore Helicon filtration cartridge (eut off 1 kDa). The high molecular weights contained in the retentate are removed and the filtrate is preserved.

The concentration of compounds of peptide nature is determined by the method of Lowry, between 0.1 and 5 g / l and preferably between 0.5 and 2 g / l. The physicochemical analysis of the vegetable hydrolyzate, which constitutes the active ingredient, shows that its pH is between 4 and 7, and preferably between 5 and 6, the dry extract is between 1 to 8 g / l and preferably between 2 and 5 g / 1, its content of peptide compounds is between 0.1 and 5 g / l and preferably between 0.5 to 2 g / 1 and its sugar content between 0.5 to 2, 5 g / 1.

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 TSK gel column (TosoHaas) with a pH 7 phosphate buffer. EXAMPLE 3 Demonstration of the Activating Effect of the Active Principle According to Example 1 on the Enzymatic Activity of Aconitase

The purpose of this study is to determine the influence of the active ingredient according to Example 1 on the enzymatic activity of aconitase. For this, the total enzymatic activity (cytoplasmic and mitochondrial) and the mitochondrial enzymatic activity of aconitase were measured.

Protocol: Normal human fibroblasts are treated with a 1% solution, of the active principle according to Example 1, for 48 hours, then subjected to irradiation with UVB at 100 mJ / cm ² . After irradiation, the cells are treated under the same conditions for another 24 hours. Negative controls are carried out under the same conditions but in the absence of active principle according to Example 1.

To evaluate the enzymatic activity of aconitase, the fibroblasts are then harvested and centrifuged in PBS buffer. Part of the cells is then treated to perform the assay of the total enzyme activity, another part is used to isolate the mitochondrial fraction according to the following protocol:

The cells are lysed using a Dounce type homogenizer and the homogenate is centrifuged cold at 10,000 g for 10 minutes. The pellet, rich in mitochondria, is washed with TES buffer (10 mM Tris, 1 mM EGTA, 0.25 M sucrose) and resuspended in 0.2 mM trisodium citrate buffer before being used for enzymatic assay.

The determination of the enzymatic activity of aconitase is carried out using the "OXIS Bioxytech Aconitase-340 assay" kit (Oxys International), according to the following principle: aconitase causes the isomerization of citrate to isocitrate. The isocitrate is then converted to alpha-ketoglutarate by the enzyme isocitrate dehydrogenase. This second reaction is accompanied by the transformation of NADP + into NADPH. It is the formation of NADPH which is quantified by spectrophotometry at 340 nm. Under the chosen experimental conditions, the rate of formation of NADPH is proportional to the amount of aconitase. Analysis of the appearance kinetics of NADPH makes it possible to calculate the aconitase concentration of the sample (in mU / ml).

Results The assays of the total enzymatic activity of aconitase show an increase of more than 55.3% of the activity in the cells treated with the active principle according to Example 1 compared to the untreated control cells. When the cells are irradiated with UVB, a decrease of more than 30% of this activity. The treatment, before and after the UVB irradiation, with the active principle according to Example 1, makes it possible to limit the decrease observed in the untreated control cells.

Assays of enzymatic activity of mitochondrial aconitase show comparable results.

Conclusions: The active ingredient according to Example 1 strongly stimulates the enzymatic activity of cytoplasmic and mitochondrial aconitases of cutaneous cells. The action of the active ingredient according to Example 1 protects cells and mitochondria against stress by UVB.

EXAMPLE 4 Evaluation of the Protective Effect of the Active Principle According to Example 1 on Skin Cells Subjected to Ultraviolet Radiation (UVB)

The purpose of this study is to determine the protective effect of the active ingredient according to Example 1 vis-à-vis dermal fibroblasts subjected to stress by UVB radiation. For this, cell viability tests were performed by the MTT technique.

Protocol: Human dermal fibroblasts are treated with a solution containing 1% of the active principle according to Example 1, for 24 hours, irradiated with UVB (from 25 to 100 mJ / cm ² ) and then cultured for a further 24 hours in the presence of the same concentration of active principle according to Example 1. Untreated and non-irradiated controls and untreated controls but irradiated are carried out under the same conditions. At the end of the experiment, the cells are incubated in a solution containing 0.1 mg / ml of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide). This compound is absorbed by living cells and then metabolized by the mitochondrial enzymes into a blue violet compound, formazan, which will be assayed spectrophotometrically at 540 nm. The optical density (OD) is then directly proportional to the mitochondrial enzymatic activity as well as to the number of living cells.

Results: The evaluation of the cell viability by the MTT technique shows, on the one hand, that the active ingredient according to Example 1, independently of any UVB radiation, increases the cell viability and, on the other hand, that the irradiations by UVB have had a dose-dependent cytotoxic effect on human fibroblasts, but this cytotoxic effect is not manifested in the presence of 1% of active ingredient according to Example 1. Conclusions: The active ingredient according to Example 1 increases the cell viability and on the other hand, effectively protects the skin cells against the cytotoxic effects of UVB radiation.

EXAMPLE 5 Evaluation of the Protective Effect of the Active Principle According to Example 1 on Cutaneous Cells Under Oxidative Stress

The purpose of this study is to determine the protective effect of the active ingredient according to Example 1 vis-à-vis dermal fibroblasts subjected to oxidative stress caused by hydrogen peroxide (H ₂ O ₂ ) at 4 mM or 5 mM. For this, cell viability tests were performed by the MTT technique.

Protocol: The human dermal fibroblasts are treated with a 1% solution of the active principle according to Example 1, for 24 hours, subjected to oxidative stress caused by H ₂ O ₂ at 4 mM or 5 mM, for 30 hours. minutes and then cultivated another 24 hours in the presence of the same concentration of active principle according to Example 1. Controls not treated with the active ingredient or with H ₂ O ₂ and treatment controls with the active principle alone are carried out under the same conditions. At the end of the experiment, the cells are incubated in a solution containing 0.1 mg / ml of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium, bromide), according to protocol described in Example 4.

Results: The evaluation of cell viability by the MTT technique shows, on the one hand, that the active ingredient according to Example 1, independently of oxidative stress, increases cell viability, and on the other hand, that oxidative stress caused by 4 mM or 5 mM H ₂ O ₂ for 30 minutes had a dose-dependent cytotoxic effect on human fibroblasts, but this cytotoxic effect does not occur in the presence of 1% of active ingredient according to Example 1.

Conclusions: The active ingredient according to Example 1 increases cell viability and effectively protects the skin cells against the cytotoxic effects of oxidative stress.

Example 6 Evaluation of the cytoprotective effect of the active principle according to Example 1 on cutaneous cells subjected to ultraviolet radiation (UVB)

A study of the cytoprotective effect of the active principle according to Example 1 was carried out by evaluating the damage caused at the DNA level. This study was carried out using the comet test (or "Single CeIl Gel Electrophoresis"), a microelectrophoretic technique short and sensitive that can visualize and quantify DNA breaks on individual cells.

Protocol: Primary human fibroblasts are seeded in boxes of diameters 100. When the cells reached 70% confluence, they are treated with a 1% solution of active principle according to Example 1 for 24 hours. The cells are then subjected to UVB irradiation at 100 mJ / cm ² and then cultured in the presence of the active ingredient for a further 24 hours. Boxes not treated with the active ingredient serve as control. A cell suspension at 100,000 cells / ml is then performed, 500 .mu.l is taken, included in an agarose solution and cast between slide and coverslip. The cells are lysed cold. The DNA is then denatured with an alkaline buffer and then subjected to a short electrophoresis (250 mA for 30 minutes) and evidenced by propidium iodide staining. The slides are then observed under a fluorescence microscope. An altered cell sees its DNA stretch toward the anode in proportion to the number of breaks in the DNA and form a "comet". The highly degraded DNA is in the "tail" of the comet. An intact cell remains round and its DNA is then compacted at the level of the "head" of the comet.

The evaluation of the DNA breaks is carried out using an image analysis software which makes it possible to determine the percentage of degradation of the DNA, by the quantitative evaluation of the "Tail Moment", a parameter defining itself as the product of the length of the comet by the percentage of DNA in its distal part.

Results: The results obtained show that the cells subjected to UVB irradiation, in the absence of the active ingredient according to Example 1 (UVB control), have the most important "Tail Moment", that is to say the condition where the DNA is the most damaged. On the contrary, the cells treated with the active ingredient according to Example 1, have a "Tail Moment", that is to say, damage to the DNA, decreased by 25%.

Conclusion: The active ingredient according to Example 1 plays an important role in protecting the DNA of cutaneous cells subjected to irradiation with UVB.

Example 7 Evaluation of the Action of the Active Principle According to Example 1 on Mitochondria: Ultrastructural Analysis

The purpose of this study is to determine the action of the active principle according to Example 1 on the ultrastructure of mitochondria by observation in electron microscopy. Protocol: Normal human keratinocytes are treated with a 1% solution of active principle according to Example 1 for 72 hours. Negative controls are carried out under the same conditions but in the absence of active principle according to Example 1.

The cells are then fixed with Karnosky's fixative (20 ml of 16% paraformaldehyde / 8 ml of 50% glutaraldehyde / 25 ml of 0.2M sodium phosphate buffer / 25 ml of water), 1 h at room temperature and then left overnight. at 4 ° C. The culture supports are scraped into the Karnosky fixative and the cells are then centrifuged for 5 minutes at 5000 rpm. The supernatant is discarded and the pellet resuspended in 1 ml of 0.1M cacodylate. The samples are then stored at 4 ° C. until they are treated for observation by electron microscopy.

Results: The organelles of the cells treated with the active principle according to Example 1 have ultrastructural characteristics suggesting an increase in metabolic activity. In particular, the specificities of the mitochondrial population, and the abundance of elements of the GoI gi apparatus are consistent with stimulation of metabolic synthesis.

Conclusion: The active ingredient according to Example 1 has a stimulating action on the mitochondrial activity.

Example 8 Preparation of Compositions

1 - Sun protection cream:

The constituents of phase A and phase B are heated separately between 7O ⁰ 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.

2 -After-sun milk:

Prepare phase A with stirring. Incorporate the xanthan gum gradually, with deflocculating stirring. Phases C and D will be incorporated once the gel is complete. Phase E, prepared before perfect dissolution of the DHA, will be added later. Adjust the pH if necessary to 4 - 4.5. Color and perfume.

-Crème anti-aging:

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 neutralized by addition of phase D. Phase E is then added with gentle stirring and cooling is continued to 25 ^° C. Phase F is then added if desired.

4 -Protective protector of day:

Prepare phase A and heat to 75 ° C with stirring. Prepare phase B by dispersing the carbopol, then the xanthan gum with stirring. Let rest. Heat at 75 ^° C. At temperature, emulsify A in B with rotor-stator stirring. Neutralize with phase C with rapid stirring. After cooling to 40 ^° C., add phase D and then phase E. Cooling is continued with gentle stirring and phase F added.

Claims

1. Use of an effective amount of an active ingredient of a peptide nature, resulting from the hydrolysis of Amaranthus hypochondriacus amaranth proteins, used alone or in combination with at least one other active principle, in a composition cosmetic, to activate aconitase and protect mitochondria.

2. Use of an effective amount of at least one active principle of peptide nature, originating from the hydrolysis of Amaranthus hypochondriacus amaranth proteins, used alone or in combination with at least one other active principle, for the preparation of a pharmaceutical composition for activating aconitase and protecting mitochondria.

3. Use according to one of the preceding claims, characterized in that said active ingredient contains from 0.1 to 5 g / l, and preferably from 0.5 to 2 g / l of peptide-like compounds.

4. Use according to any one of claims 1 to 3, characterized in that said active ingredient is used in an amount representing from 0.0001% to 20% of the total weight of the composition, and preferably in an amount of 0 , 5% to 5% of the total weight of the composition.

5. Use according to any one of the preceding claims, characterized in that said active ingredient is first solubilized in one or more cosmetically or pharmaceutically acceptable solvents, such as water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents.

6. Use according to any one of the preceding claims, characterized in that the composition is in a form suitable for topical application comprising a cosmetically or dermatologically acceptable medium.

7. Use according to any one of the preceding claims, characterized in that the composition further contains at least one other active ingredient promoting the action of said active ingredient.

8. Use according to claim 7, characterized in that the other active ingredient is selected from the antioxidant active ingredients and / or the active ingredients stimulating the synthesis of the extracellular matrix, and / or the active ingredients stimulating the energy cellular metabolism.

9. Use of an effective amount of active ingredient, as defined in one of claims 1 or 3, in a cosmetic composition for protecting the skin and integuments against all types of external aggression.

10. Use according to claim 9, characterized in that the external aggressions are UV radiation.

11. Use of an effective amount of active ingredient, as defined in one of claims 1 or 3, in a cosmetic composition for preventing or treating damage to the skin and integuments by free radicals.

12. Use of an effective amount of active ingredient, as defined in one of claims 1 or 3, in a cosmetic composition for preventing or treating the cutaneous signs of aging and / or photo-aging.

13. Use of an effective amount of active principle, as defined in one of claims 1 or 2, for preparing pharmaceutical compositions intended to prevent or fight against mitochondrial dysfunctions such as neuromuscular or cardiac degeneration, diabetes Type II or certain pathologies of aging.

14. Cosmetic treatment method for stimulating the defenses and protecting the skin and integuments of external aggressions, characterized in that it is applied topically to the skin or integuments to treat a composition containing an effective amount of active ingredient such as defined in one of claims 1 or 3.