FR2903305A1 - USE OF PLANT EXTRACTS AS SLIMMING ACTIVE INGREDIENTS - Google Patents

Abstract

The present invention relates to novel compositions containing plant extracts comprising peptides homologous in sequence to protein fragments of the UCP family. The invention also relates to the use of these same vegetable extracts as slimming active agents, in or for the preparation of a pharmaceutical and / or dermatological and / or cosmetic composition.

Description

The present invention is in the field of pharmaceuticals, and

  more particularly in the field of dermatology and cosmetology. The present invention relates to the use of plant extracts comprising peptides homologous in sequence to protein fragments of the UCP family as slimming active agents, alone or in combination with at least one other active agent, in or for the preparation of a pharmaceutical and / or dermatological and / or cosmetic composition. The invention also relates to novel compositions containing at least one plant extract comprising peptides derived from proteins of the UCP family.

  The term UCP refers to the family of Uncoupling proteins which are proteins responsible for the heat dissipation of the energy produced by mitochondrial respiration (Nicholls et al., Physiol Rev., 64: 1-64, 1984). Mitochondrial respiration generates a proton gradient between the internal compartment of the mitochondria and the system of external double membranes. The return of the protons to the internal compartment is through the channels of ATPsynthase. The energy produced by this proton-motor force then allows the synthesis of ATP. The UCP-1 protein (Lin et al., FEBS Lett., 113: 299-303, 1980) is a proton transporter inserted into the inner membrane of mitochondria of brown adipose tissue cells (Cassard et al., Journal of Cell Biochemistry, 43, 1990), which bypasses the passage of protons by ATPsynthase. UCPs catalyze in this way the decoupling between the proton-motor force and the synthesis of ATP. Energy, which is no longer used to produce ATP, is released as heat. On the other hand, the decoupling of respiration stimulates the oxidation of fat. To date, three proteins of this family have been identified: UCP-1, UCP-2 and UCP-3. The less well-known UCP-2 and UCP-3 proteins probably play an important role in energy homeostasis (Klingenbeg, J. Bioenerg Biomembr 25: 447, 1993), particularly in the control of food metabolic efficiency. . Patent FR 2,866,233 has recently described the use of proteins or peptide fragments of UCP as active cosmetic agents.

  The technical problem to be solved was, for the inventors, to find a vegetable substitute, cosmetically and pharmaceutically acceptable, which contains an effective amount of peptides derived from proteins of the UCP family.  These plant extracts must have a therapeutic activity and, more particularly, dermatological and cosmetic slimming when applied to the skin.  The studies conducted by the inventors have demonstrated that the extracts according to the invention were capable of reducing, eliminating or preventing subcutaneous fat overloads.  According to a first aspect, the present invention relates to the use of plant extracts rich in at least one peptide derived from UCP proteins, as active agents, alone or in combination with at least one other active agent, in or for the preparation of a pharmaceutical and / or dermatological and / or cosmetic composition.  The term peptide refers to a sequence of two or more amino acids linked together by peptide bonds or modified peptide bonds; a polypeptide designating a larger peptide.  Peptides derived from proteins of the UCP family are understood to mean peptides homologous, by their amino acid sequence, to fragments of proteins of the UCP family.  Many proteins found in plants are likely to contain these sequences 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 to hydrolyze it, or to carry out the hydrolysis first on a crude extract and to purify the peptide fragments. then.  It is also possible to use certain hydrolysed extracts without purifying the peptide fragments according to the invention, but nevertheless ensuring the presence of said fragments by appropriate analytical means.  The term "extract" refers to any isolated substance or preparation obtained from plant material.  It is obtained, for example, by dissolving the active components by means of solvents (such as water, alcohol, etc.). ) then by concentration and purification of these assets, for example, by evaporation of the solvents.  Any method of extraction or purification known to those skilled in the art can be used to prepare the extract according to the invention.  To carry out the extraction, it is possible to use the entire plant, or a specific part of the plant (leaf, grain, etc.). ).  More particularly according to the invention is used one of the many plants of the family of poaceae, of the genus oryza (rice) and preferentially the species Oryza sativa L. .  According to the invention, the plant material used will be the grain and preferentially the grain freed from its envelope by a dehulling step.  According to another form of the invention, plants of the Fabaceae family are used; soybean or soybean meal and preferentially of the species Glycine max L. In a first step, the plant is ground using a plant mill.  The powder thus obtained may subsequently be "delipidated" with the aid of a conventional organic solvent (such as, for example, an alcohol, hexane or acetone).  The extraction of proteins from the plant is then conventionally carried out (Osborne, 1924); the crushed plant being suspended in an alkaline solution.  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.  Proteins are then precipitated by varying the ionic strength and acidifying the medium to remove soluble components and nucleic acids.  The precipitate is then washed with a solvent such as, for example, ethanol or methanol and the solvent is evaporated by drying in vacuo.  The protein-rich precipitate is redissolved in water or other solvent and then constitutes a more purified form of the extract.  The extraction can also be carried out in neutral or acidic medium.  The precipitation step is then carried out using a conventional precipitation agent such as salts (sodium chloride, ammonium sulfate).  The precipitate obtained can be separated from the precipitating agents by dialysis after redissolving in water or another solvent.  At this stage, the extract obtained contains at least 70% of compounds of protein and peptide nature.  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 and exopeptidases of plant origin (papain, bromelain, ficine) and microorganisms (Aspergillus, Rhizopus, Bacillus, etc.). ).  The solution obtained constitutes the active extract.  The active extract can be further purified by fractionation, especially by a chromatographic or purified method and concentrated by a dialysis method.  Any of the more or less purified forms of the extract is then solubilized in water or in any mixture containing water, and then sterilized by ultrafiltration.  The plant extract obtained according to the invention is analyzed qualitatively and quantitatively for its content of protein and peptide compounds.  Protein and peptide compounds are understood to mean protein fragments, peptides and free amino acids present in the mixture.  Peptides, amino acids and protein fragments are assayed according to standard techniques, well known to those skilled in the art.  Thus, according to an advantageous embodiment of the invention, the active plant extract contains a quantity of protein and peptide compounds representing between 30 and 90% of the total weight of the dry matter, more particularly this amount is between 60 and 90% of the total weight of the dry matter. and 80% of the total weight of the dry matter.  The plant extract according to the invention also has the remarkable property of containing 10 and 200 mg / l of biologically active protein and peptide compounds.  Preferably, according to the invention, the peptides present in the extract are derived from proteins of the UCP family of formula (I): ## STR2 ## Lys - X 1 Arg - X4 - X5 - (AA) n in which: X1 = Leu, Thr, Val; X 2 = Asp, Glu; X3 = Ala, Val; X4 = Leu, Phe, Tyr; X 5 = Gln, Ile, Met; and wherein (AA) is any amino acid, or a derivative thereof, and n is an integer of 0 to 2.  According to a particularly advantageous method of carrying out the invention, the extract contains at least one peptide corresponding to the sequences ID N: Pro Leu Asp Thr Ala Lys Val Arg Leu Gln Pro Thr Glu Val Ala Lys Val Arg Phe Gln Pro Thr Asp Val Ala Lys Val Arg Leu Gln Pro Thr Glu Val Ala Lys Val Arg Gln Pro Gln Asp Asp Val Ala Lys Val Arg Phe Gln (6) Pro Asp Asp Val Val Val Lys Thr Arg Phe Pro Val Val Asp Val Val Lys Thr Arg Tyr Met Pro Val Asp Val Val Lys Thr Arg Phe Met (5) 2903305 -5- (9) Pro Val Asp Val Val Lys Thr Arg Tyr Ile (10) Val Lys Arg Leu Gln Moreover, according to a method of carrying out the invention most preferably, the plant extract contains at least the peptide derived from UCP proteins of sequence ID N (10), that is to say the sequence Lys-Val-Arg-Leu-Gln.  Thus, one of the objects of the present invention is the use of a plant extract, as defined above, in or for the preparation of a cosmetic and / or dermatological slimming composition containing an effective amount of peptide of ID 10 N sequence. (10), Lys-Val-Arg-Leu-Gln.  The invention also relates to variant forms of these peptides and / or fragments.  The term "variant" herein refers to a polypeptide or peptide that differs from, for example, the sequence of a reference peptide while retaining its essential properties.  Generally, the differences are limited so that the sequences of the reference peptide and those of the variant are quite similar and, in some regions, identical.  Preferentially, the variant forms are those which vary from the reference sequences, by the substitution of chemically equivalent amino acids (or homologues), that is to say by the substitution of one residue for another having the same characteristics.  Thus, the classical substitutions are between Ala, Val, Leu and Ile; between Ser and Thr; between Asp and Glu acid residues; between Asn and Gln; and between the basic residues Lys and Arg; or between the aromatic residues Phe and Tyr.  The term "variant" thus refers to a peptide that differs, for example, from the sequence of a reference peptide while retaining these essential properties.  Generally, the differences are limited so that the sequences of the reference peptide and those of the variant are quite similar and, in some regions, identical.  A variant peptide and a reference peptide may thus differ from the amino acid sequence by one or more substitutions, additions, deletions in all combinations.  The use of the extract according to the invention also includes the use of any biologically active fragments, or one of its analogs or variants.  By the term all biologically active fragments is meant fragments which possess in vivo or in vitro activity characteristic of the activity of the active agent according to the invention, such as, for example, the slimming activities described above.  In the invention, the term "amino acid" refers herein to any natural or unnatural organic acid having the formula (II): embedded image where each -R -RNR-CR-C (O) -O- (II) is independently selected from hydrogen and an alkyl group having from 1 to 12 carbon atoms.  Preferably, at least one -R group of each amino acid is a hydrogen.  By the term "alkyl" herein is meant a carbon chain which may be linear or branched, substituted (mono- or poly-) or unsubstituted; saturated, mono-saturated (a double or triple bond in the chain) or polyunsaturated (two or more double bonds, two or more triple bonds, one or more double bonds and one or more triple bonds in the chain).  The effective amount of plant extract is the amount needed to achieve the desired result.  Thus, according to an advantageous embodiment of the invention, the abovementioned plant extract is used, in the compositions, at a concentration of between 0.00001% and 20% approximately, and preferably at a concentration of between 0.001% and 10%. % approximately by weight relative to the total weight of the final composition.  According to an advantageous embodiment of the invention, the extracts according to the invention are previously solubilized in one or more cosmetically or pharmaceutically acceptable solvents, conventionally used by a person skilled in the art, such as water, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petrolatum, glycerol (glycerine), a vegetable oil or any mixture of these solvents.  The use of the plant extract according to the invention will preferably be in the form of a composition suitable for external topical administration, mainly on the skin, the mucous membranes and / or the integuments, comprising a medium that is cosmetically or dermatologically acceptable.  It is understood that the extract according to the invention may be used alone or in combination with at least one other active agent.  According to a second aspect, the invention relates to a cosmetic and / or dermatological composition containing an effective amount of plant extract comprising at least one peptide derived from proteins of the UCP family, adapted for topical administration by cutaneous route comprising a cosmetically or pharmaceutically acceptable medium.  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 too can be in the form of suspensions, or powders, suitable for application on the skin, mucous membranes, lips and / or hair.  These compositions may be more or less fluid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste or of a foam.  They can also be in solid form, as a stick or be applied to the skin in aerosol form.  These compositions additionally comprise any additive usually used in the intended field of application and the adjuvants necessary for their formulation, such as solvents, thickeners, silicones, diluents, antioxidants, dyes, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, cosmetic or pharmaceutical active ingredients, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, etc. . . .  For example, in said compositions, the extract according to the invention may advantageously be combined with any other skin exfoliation stimulating, lipogenesis inhibiting or lipolysis stimulating ingredient such as xanthine derivatives.  For example caffeine, theophylline, theobromine, acefylline, xanthinol nicotinate, diniprophylline, diprophylline, etamiphylline and its derivatives, etophylline, proxyphyline, pentophylline, propentophylline, pyridophylline and bamiphylline.  In all cases, those skilled in the art will ensure that these active or non-active adjuvants and their proportions are chosen in such a way as not to adversely affect the desirable advantageous 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 selected 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.  According to a third aspect of the invention, the extract according to the invention can be used in or for the manufacture of a composition for topical use, intended for the treatment of cellulite and / or the treatment of orange peel. .  The extract or the composition containing it is advantageously used in order to reduce, eliminate or prevent subcutaneous fat overloads.  The hypoderm consists of large vacuolated cells, the adipocytes, almost entirely filled with triglycerides.  This adipose tissue also contains connective tissue in which, among other things, particular fibroblasts and preadipocytes are found.  This adipose tissue is the largest energy reservoir in the body.  It is capable of storing lipids as triglycerides or releasing them as fatty acids and glycerol.  The lipid reserves of the body are constantly renewed and there is a balance constantly adapted to the energy needs of the body, between the phenomenon of lipolysis, which releases fatty acids, and the lipogenesis phenomenon that stores them.  If an imbalance settles in the body between these two phenomena, the fatty acids are stored in the adipocytes, the volume and the number of which increases: it is called hypertrophy and hyperplasia of the adipocytes.  The excessive development of fat mass can then result in changes in the appearance of the skin, or even progress to overweight the individual, or progress to a real obesity.  Cellulite is a particular configuration of adipose tissue, considered today as unsightly.  It refers to a quilted and padded appearance of the skin that corresponds, schematically, to the accentuation of adipose tissue in certain areas of the body, in particular, in the woman, at the hips, thighs, buttocks, knees and forearms.  At an advanced stage of cellulite formation, the skin spontaneously has the appearance of orange peel or capiton, which is characterized by a succession of small bumps and depressions due to traction of the epidermis to the deep tissues.  It has been found that the extract according to the invention, or the composition containing it, has a very effective action at the level of the adipocytes.  Indeed, it helps to significantly reduce the amount of triglycerides contained in the adipocytes of the hypoderm.  This phenomenon is probably due to a blocking of the phenomenon of lipogenesis, that is to say the blocking of the triglyceride storage process which results in hypertrophy of the adipocytes.  Control of lipogenesis, that is to say of the synthesis reaction of triglycerides in adipocytes, will prevent hypertrophy of adipocytes and subsequent hyperplasia.  Thus, when, during adipocyte differentiation, the amount of triglycerides present in the vacuoles do not increase, the volume of the adipocytes and their number do not increase either.  The skin thus resumes, little by little, its normal appearance: the cellulite tissue no longer develops, the orange peel effect is attenuated.  The unsightly appearance of the body fades away.  The extract according to the invention thus makes it possible to prevent the appearance of cellulite as well as to fight against its aggravation.  It is obvious that the invention is directed to mammals in general and more particularly to humans.  According to a fourth aspect of the invention, the extract according to the invention can be used in 5 or for the manufacture of a pharmaceutical composition for topical use, intended for the treatment of cellulite and / or for the treatment of subcutaneous fat overloads. skin.  According to a fifth aspect, the present invention relates to a cosmetic care method for obtaining a slimming action, and a cosmetic care method for reducing, eliminating and / or preventing subcutaneous fat overload, and / or intended to combat cellulite and / or to fight against the phenomenon of orange peel, consisting in applying, topically, to the areas of the skin, an effective amount of at least one of the extracts according to the invention or a composition containing them.  The cosmetic treatment process of the invention may be carried out in particular by regularly applying the cosmetic compositions as defined above, according to the technique of usual use of these compositions, for example: application of creams, gels, serums, lotions, milks, shampoos or sunscreens on the skin.  Particular embodiments of this cosmetic treatment method also result from the foregoing description.  The advantage of the present invention is to have a topical treatment effective against adiposity while employing gentle methods.  Other advantages and characteristics of the invention will appear better on reading the examples given for illustrative and non-limiting purposes. EXAMPLE 1 Preparation of extracts from rice (Oryza sativa L. ) The active agent is obtained from an extract of plants of the species Oryza sativa L.  Of course the extract can be prepared from plants of at least one of the many varieties and species belonging to the genus Oryza.  In a first step, 1 kg of husked rice grains are delipidated by the action of an organic solvent: hexane.  The rice grains are then milled in a knife mill and then suspended in an aqueous alkaline solution (1/10 dilution).  This mixture is stirred for a time long enough to allow the solubilization of the soluble fractions.  The extraction temperature is variable (between 2903305 -10- 4 and 80 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 the rice, is then subjected to protein precipitation by varying the ionic strength in a neutral or acidic medium to remove soluble carbohydrate components, lipids and nucleic acids.  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 are obtained containing: 10 -Proteins: 75% - Carbohydrates: 20% - Lipids: 5% The precipitate rich in proteins is dissolved in the solution water or other solvent.  The extraction can also be carried out in neutral or acidic medium.  The crude protein extract is then subjected to a series of gentle and selective hydrolyses consisting of chemical and enzymatic hydrolyses.  By way of example, mention may be made of hydrolysis in the presence of exopeptidases (papain, ficin).  The hydrolyzate is then purified and dialyzed on a membrane to remove the high molecular weight compounds.  A light-colored hydrolyzate titrating 15 to 30 g / l of dry extract and containing a quantity of protein and peptide compounds representing approximately 70% of the total weight of the dry matter is then obtained.  The hydrolyzate is then diluted so that the protein concentration determined by the Lowry method is between 0.5 and 2 g / l.  On the other hand, HPLC analysis shows that the plant extract according to the invention contains at least 20 mg / l of biologically active protein and peptide compounds containing the sequence-like ID n (10) sequence. of UCP protein.  Example 2: Preparation of extracts from soybean meal (Glycine max L. The oilcakes are the solid residues obtained after oil extraction from the soybeans.  They represent 50 to 75% of the seed mass.  The cakes are suspended in an aqueous alkaline solution (1/10 dilution).  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 C); Preferably, the operation will be carried out 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 the rice, is then subjected to protein precipitation by varying the ionic strength in a neutral or acidic medium to remove soluble carbohydrate components, lipids, and nucleic acids.  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 are obtained containing: 10-Proteins: 75% Carbohydrates: 20% - Lipids: 5% The precipitate rich in proteins is dissolved in water or another solvent.  The extraction can also be carried out in neutral or acidic medium.  The crude protein extract is then subjected to a series of gentle and selective hydrolyses consisting of chemical and enzymatic hydrolyses.  By way of example, mention may be made of hydrolysis in the presence of exopeptidases (papain, ficin).  The hydrolyzate is then purified and dialyzed on a membrane to remove the high molecular weight compounds.  A light-colored hydrolyzate titrating 15 to 30 g / l of dry extract and containing a quantity of protein and peptide compounds representing approximately 70% of the total weight of the dry matter is then obtained.  The hydrolyzate is then diluted so that the protein concentration determined by the Lowry method is between 0.5 and 2 g / l.  On the other hand, HPLC analysis shows that the plant extract according to the invention contains at least 20 mg / l of biologically active protein and peptide compounds containing the sequence-like ID n (10) sequence. of UCP protein. Example 3: Demonstration of the activity of the extracts according to the invention on adipocytes.  I. I.  Cell culture: Undifferentiated 3T3-L1 preadipocyte cell line cells are seeded in LabTek 8 wells (for Oil Red staining), in 12-well plates (for ATP assay), in plates 24 wells (for cAMP assay) and in 6-well plates (for glycerol assay) as well as with DMEM culture medium (4.5 2903305 -12- g / l) containing antibiotics.  These 3T3-L1 preadipocytes are able to return, under certain conditions, in terminal differentiation phase.  II.  Differentiation of Adipocytes: Once 3T3-L1 cells have reached 100% confluence, these cells are differentiated by culture in different media over a period of 6 to 8 days.  During the first 2 days, the cells are cultured in the presence of IBMX, dexamethazone and insulin, diluted in 4.5 g / l DMEM culture medium.  Then they are cultured for two days in the presence only of insulin diluted in DMEM 4.5 g / l culture medium and finally the cells are cultured for 2 to 3 days in culture medium containing exclusively DMEM 4.5 g / l.  The differentiation of cells into mature adipocytes is visualized by the appearance of lipid droplets in the cytoplasm of the cells.  The effect of the plant extracts according to Examples 1 and 2 as well as their impact on the differentiated or differentiating 3T3-L1 cells was evaluated through different techniques: the oil red coloration, the dosage of the amount of intracellular ATP, the dosage of the amount of intracellular cyclic AMP, the determination of glycerol released by the cells.  III.  The "Oil Red" Coloring: The principle of this test for evaluating the effect of the extracts according to Examples 1 and 2 is based on a microscopic observation of the number and size of the lipid vacuoles of the adipocytes after staining.  The adipocytes being incubated or not, in the presence of the substance to be tested.  The 3T3-L1 cells, seeded in 8-well Lab-teks, are treated with the extract according to Example 1 or 2, rich in peptide of sequence ID N (10), that is to say of sequence Lys-Val-Arg-Leu-Gln, representative of the peptides derived from proteins of the UCP family according to the invention, dissolved in 0.5%.  The active ingredient was added at different stages of cultivation: - From seeding and until the end of adipocyte differentiation (72 hours of culture to 100% confluence and then during the 6 days of differentiation); 6 hours after seeding and until the end of cell differentiation (66 hours of culture to 100% confluence and then 6 days of differentiation); Once the cells have been differentiated into adipocytes, the cells are treated with the active agent for 6, 24, or 48 hours.  After these different incubation periods, in the presence or absence of the active agent to be tested, the 3T3-L1 are stained by the Oil Red technique.  The Oil Red solution (Sigma, 0-0625) is prepared by adding 0.5 g of product in 100 ml of isopropanol and by dilution to 4/10 in distilled water, followed by filtration.  The cells are fixed for 10 minutes in a 4% formalin solution and NaCl, the Red Oil solution is applied for 15 minutes.  Counterstaining with Hematoxylin for 30 seconds is possible.  The cells are then rinsed with warm water and mounted on slides in a hydrophilic medium (Aquatex).  The observation is carried out under an optical microscope so as to distinguish the fatty vacuoles stained in red.  The results of the observation of the cells demonstrate that the cells which were treated with the extract according to Example 1 or 2 before the beginning of the differentiation (that is to say from their sowing, or 6 hours later their seeding) have a less rounded morphology and an intra-adipocyte lipid vesicle content markedly decreased compared to the control cells (on which the active was not applied).  This same observation is also made by comparison with the mature adipocytes, that is to say differentiated, on which the active has been applied, which have a large spherical shape and a large accumulation of intra-cytoplasmic lipid vesicles.  The solution containing the extract according to the invention is therefore particularly effective at the level of differentiating adipocytes and not at the level of mature adipocytes which have already accumulated triglycerides in their intra-adipocyte vesicles.  Indeed, no decrease was observed when the asset was applied once the differentiation ended.  IV.  Intracellular cyclic AMP assay: The objective of this test is to measure the variation of the intracellular cAMP concentration in the adipocytes, in order to deduce a possible activation of the lipolysis phenomenon.  The test was performed on differentiated 3T3-L1 cells.  These adipocytes are treated with the extract according to Example 1 or 2, rich in peptide of sequence ID N (10), that is to say of sequence Lys-Val-Arg-Leu-Gln, representative of the peptides UCP protein derivatives according to the invention, dissolved in 0.5%, for periods of 15 minutes, 30 minutes or 1 hour.  In parallel, the differentiated 3T3-L1 cells are also treated with isoproterenol (a lipolysis inducing agent) at 1 M, thus constituting a positive control.  After the different incubation periods, in the presence of the active agent to be tested or in the presence of isoproterenol, the amount of cAMP contained in the adipocytes is measured by the Biotrak EIA System CAMP Kit from Amersham Biosciences as well as by a colorimetric principle of the ELISA type.  The kit allows the lysis of the cells with a solution thus allowing the hydrolysis of the cell membranes and the release of the intracellular cAMP into the medium.  Then binding to the capture antibody of the intracellular cAMP, extracted from the samples, is put into competition with cAMP coupled to an enzyme, peroxidase.  Thus, after revelation with the TMB substrate, the more cAMP will be in the samples to be assayed, the weaker the signal will be.  The results obtained demonstrate that in the presence of active plant extracts, there is no increase in the amount of intracellular cAMP on differentiated cells, compared with the control condition, that is to say with respect to the cells. treated with isoproterenol.  V.  Assay for Intracellular ATP: The purpose of this study is to determine the influence of the active plant extracts according to Example 1 or 2 on the amount of intracellular ATP.  This study is carried out using a kit "ATP Bioluminescence Assay Kit HS II".  The differentiated 3T3-L1 cells are treated with a 0.5% solution, containing the extract according to Example 1 or 2, rich in peptide of ID N sequence (10), representative of the peptides derived from UCP proteins according to US Pat. invention, for a period of up to 96 hours.  At the end of the incubation time, the wells are emptied of their medium and rinsed with 2 ml of cold PBS before adding 250 l of lysis buffer provided by the kit.  The cells are then mechanically detached and then harvested in 14 ml tubes.  Each well is rinsed with 2 x 500 gl PBS cold and all is harvested again in the respective tubes.  Each tube is then passed to the polytron for 10 seconds at 18,000 rpm.  From these samples, dilution is carried out at 1 / 12000th in cold PBS before each reading.  The ATP assay is performed on these samples: 50 L of this dilution are deposited in a luma-cuvette and 50 L of luminol are added.  After 10 seconds, the reading of the luminescence is triggered.  The values are standardized with respect to the amount of protein for each sample.  The measurements are made using a device: the Biocounter M2010A LUMAC / 3M.  The results obtained demonstrate that there is no increase in the amount of intracellular ATP.  The measurements obtained even show a decrease in the amount of intracellular ATP after 96 hours of culture, on differentiated cells treated by the active agent, in comparison with the control.  VI.  Determination of glycerol: The purpose of this study is to determine the influence of the extract according to the invention on the release of glycerol by the adipocytes.  This glycerol is the result of lipolysis.  The differentiated 3T3-L1 cells are treated at a dose of 0.5% containing the extract according to Example 1 or 2, rich in peptide of ID N sequence (10), representative of the peptides derived from UCP proteins according to the invention. invention during periods of 1h30, 3h, 5h, 7h and 24 hours.  The determination of glycerol is carried out by means of a cascade enzymatic reaction leading to the formation of NADH whose quantity produced is evaluated by spectrophometric reading at 340 nm, the formation of NADH being proportional to the quantity of glycerol released into the culture centre.  An internal control of glycerol at 1 mm was also used.  The values are standardized with respect to the amount of protein for each sample.  The results obtained allow us to observe that at the different times studied, the glycerol release is identical for the control wells and the wells treated with the active at 0.5% on differentiated cells.  VII.  CONCLUSIONS: The results of the "Oit Red" staining test allow us to conclude that the active plant extracts according to the invention have a particularly effective effect in reducing the formation of intra-adipocyte lipid droplets.  However, this effect is only observed when the active is applied before the onset and / or during cell differentiation, no decrease being observed when the active is applied to mature adipocytes.  These results thus suggest that the plant extracts according to Examples 1 and 2 act at the level of lipogenesis and not at the level of lipolysis.  The increases in the amounts of cAMP and intracellular ATP are indicators of the activation of the lipolysis pathway, the level of intracellular cAMP regulating the activity of Triglyceride-lipase, the enzyme allowing hydrolysis. triglycerides.  Thus, a non-increase in the amounts of cAMP and intracellular ATP suggests that the active agent according to the invention does not act by increasing the phenomenon of lipolysis.  These results are, moreover, confirmed by the non-increase of glycerol release in the extracellular medium, glycerol being a product of lipolysis.  These results thus demonstrate that the extracts according to the invention have a truly effective action on adipocytes, and that they are particularly effective in the process of limiting adipocyte hypertrophy.  The active plant extract according to the invention thus prevents the accumulation of lipid droplets in the adipocytes, and thus allows, more or less directly, a reduction of the adipose mass and an inhibition of its development.  This decrease in lipid vacuoles in non-mature adipocytes, combined with the results of the cAMP, ATP and glycerol assays, makes it possible to conclude that the plant extracts according to the invention act, at the level of the adipocytes, on the mechanism of the Lipogenesis and not on the mechanism of lipolysis.  The plant extracts according to the invention therefore promote the non-accumulation of triglycerides contained in intra-adipocyte vesicles.  Example 4 Preparation of compositions  15 1.  Slimming cream Trade names I INCI Names I% by mass PHASE A Montanov 68 Cetearyl Alcohol (and) Cetearyl Glucoside 5. 00 Squalane Squalane 2. 50 DUB IPP Isopropyl Palmitate 3. 50 Eutanol G Octyldodecanol 1. Phenonip Phenoxyethanol (and) Methylparaben 0. 50 (and) Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben PHASE B Demineralized Water Aqua (Water) Qsp Glycerine Glycerin 3. Butylene Glycol Butylene Glycol 3. 00 PHASE C Simulgel EG Sodium Acrylate / Acryloyldimethyl 0. Taurate Copolymer (and) Isohexadecane (and) Polysorbate 80 PHASE D Extract according to Example 1 or 2 2. 0 Perfume Fragrance Qsp 2903305 -17- Trade names INCI names% by mass Qsp dye The constituents of phase A are melted at 75 ° C. and the constituents of phase B heated at 75 ° C.  Phase A is emulsified at B, then the mixture is cooled below 40 C.  Phases C and D are then added with constant stirring.  2.  Slimming Firming Spray Trade Names I INCI Names I% Mass PHASE A Emulgate SEV Glyceryl Stearate (and) Ceteareth-20 4. 60 (and) Ceteareth-12 (and) Cetearyl Alcohol Eumulgin B2 Ceteareth-20 1. 40 Cetiol OE Dicaprylyl Ether 3. 00 DUB B1215 C12-C15 Alkyl Benzoate 5. Phenonip Phenoxyethanol (and) Methylparaben 0. 50 (and) Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben DUB ININ Isononyl Isononanoate 5. Phenonip Phenoxyethanol (and) Methylparaben 0. 50 (and) Ethylparaben (and) Butylparaben (and) Propylparaben (and) Isobutylparaben PHASE B Demineralized water Aqua (Water) 15. 00 Glycerin Glycerin 3. 00 PHASE C Demineralized water Aqua (Water) Qsp PHASE D Extract according to example 1 or 2 1. 0 Perfume Fragrance qsp Colorant qsp The constituents of phase A and phase B are separately heated to 65 C; phase B is incorporated in phase A with stirring.  The temperature of the mixture is raised to 10 83 C and then cooled to a phase inversion temperature.  Phase C is then added.  The asset is incorporated when the temperature reaches less than 40 C.  It is then possible to add perfumes and / or dyes.  3.  Firming Gel ù Slimming ù anti-cellulite Trade names INCI names% by weight Carbopol Ultrez 10 (2%) Carbomer 25. 00 Demineralized water Aqua (Water) Qsp DUB DIOL Methyl Propanediol 3. 00 EDTA Tetrasodium EDTA 0. 10 Glydant Plus Liquid DMDM Hydantoin (and) 0. Iodopropynyl butylcarbamate Extract according to Example 1 or 2 0. TEA Triethanolamine 0. 50 Fragrance Fragrance Qsp Water-Soluble Dye Qsp The Carbopol gel is prepared at 2%.  The

  ingredients are added in the order listed above, with stirring. The mixture is then neutralized with the TEA. The perfume and the dyes are added if necessary.

Claims (13)

claims   1. Cosmetic and / or pharmaceutical composition, characterized in that it contains, in a cosmetically or pharmaceutically acceptable medium, an effective amount of plant extract comprising at least one peptide derived from proteins of the UCP family.   2. Composition according to Claim 1, characterized in that the plant extract contains at least one peptide of formula (I) (AA) n-Pro-X1-X2-X1-X3-Lys-X1-Arg-X4-X5. (AA) n in which: X1 = Leu, Thr, Val; X2 = Asp, Glu; X3 = Ala, Val; X4 = Leu, Phe, Tyr; X 5 = Gln, Ile, Met; (AA) is any amino acid, or a derivative thereof, and n is an integer from 0 to 2.   3. Composition according to claims 1 or 2, characterized in that the peptide is of sequence ID N: (1) Pro Leu Asp Thr Ala Lys Val Arg Leu Gln (2) Pro Thr Glu Val Ala Lys Val Arg Phe Gln (3) Pro Thr Asp Val Ala Lys Val Arg Leu Gln (4) Pro Thr Glu Val Ala Lys Val Arg Leu Gln (5) Pro Thr Asp Val Ala Lys Val Arg Phe Gln (6) Pro Val Asp Val Val Lys Thr Arg Phe 7) Pro Val Asp Val Val Lys Thr Arg Tyr Met (8) Pro Val Asp Val Val Lys Thr Arg Phe Met (9) Pro Val Asp Val Val Lys Thr Arg Tyr Ile (10) Lys Val Arg Leu Gln 30 35 2903305 - 20 -   4. Composition according to any one of the preceding claims, characterized in that the peptide has the sequence ID N (10), that is to say the sequence Lys Val Arg Leu Gln. 5   5. Composition according to any one of the preceding claims, characterized in that the plant extract is obtained from plants of the family Poaceae, preferably of the species Oryza sativa L. and more particularly the grain of said plants. 10   6. Composition according to any one of claims 1 to 4 characterized in that the plant extract is obtained from plants of the fabaceae family, preferably of the species Glycine max L. and more particularly the seeds of said plants. 15   7. Composition according to any one of the preceding claims, characterized in that the plant extract contains a quantity of compounds of protein and peptide nature representing between 30 and 90% of the total weight of the solids and more particularly between 60%. and 80% of the total weight of the dry extract. 20   8. Composition according to any one of the preceding claims, characterized in that the plant extract is present in the composition at a concentration of between 0.00001% to 20% by weight or preferably 0.001% to 10% by weight relative to total weight of the final preparation. 25   9. Composition according to any one of the preceding claims, characterized in that it is in the form of a cosmetic or pharmaceutical composition suitable for topical administration to the skin.   10. Composition according to any one of the preceding claims, characterized in that the plant extract is previously solubilized in one or more cosmetically or pharmaceutically acceptable solvents such as water, ethanol, propylene glycol, butylene glycol, dipropylene. glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetable oil or any mixture of these solvents. -21- 2903305   11. Composition according to the preceding claim, characterized in that it is in the form of an aqueous solution, hydroalcoholic or oily or in the form of an emulsion oil-in-water, water-in-oil or multiple emulsions or in the form of creams, suspensions, or powders; these compositions may be more or less fluid or solid and have the appearance of a cream, a lotion, a milk, a serum, an ointment, a gel, a paste, a foam or a stick. 10   12. Use of an effective amount of at least one plant extract as defined in any one of the preceding claims, in or for the preparation of a composition; the compound or composition being intended for the treatment of cellulite and / or orange peel; and / or to reduce, eliminate or prevent subcutaneous fat overloads. 15   13. A cosmetic care method for reducing, eliminating and / or preventing subcutaneous fat overload, and / or for fighting against cellulite, and / or for combating the phenomenon of orange peel, characterized in that an effective amount of a composition as defined in any one of claims 1 to 11 is applied to the surface of the skin.