FR2973381A1 - New oligosaccharide compounds useful e.g. for maintaining the integrity of the constituent molecules in the extracellular matrix and for reducing the glycation and/or hyperkeratinization process - Google Patents

Abstract

Oligosaccharide compounds (I) are new. Oligosaccharide compounds of formula (I) and their salts, are new. R 1>-R 1> 2> : OH (preferred), alkyloxy, carboxyl, alkoxycarbonyl, acyloxy, sulfate, phosphate or OCH 2R1a ; R1a : OH, alkyloxy, acyloxy, sulfate or phosphate; and n : = 100, where n is selected in such a way that the molecular weight of (I) is = 100000 D, preferably 5000-10000 D. An independent claim is included for the preparation of (I). [Image] ACTIVITY : Dermatological. MECHANISM OF ACTION : None given.

Description

The present invention relates to novel oligosaccharide compounds, a cosmetic composition comprising one or more of these compounds and their pharmaceutically acceptable salts, and the use of these compounds or this composition to maintain the integrity of the constituent molecules of the matrix. extracellular and / or decrease the processes of glycation and / or hyperkeratinization.

Fenugreek (or Trigonella foenum-graecum L.) is a herbaceous plant, native to Asia Minor, belonging to the fabaceae family.

Yellowish-white fenugreek flowers produce pod-shaped pods with angular, light-brown seeds. Because of their characteristic aroma, these seeds are often used in different food products (such as for example Viandox®) or as spices in traditional dishes.

Various extraction processes carried out on fenugreek have been described in the prior art.

Some extraction processes carried out on fenugreek, in particular on its seeds, have allowed the extraction of polyphenol type compounds with which various cosmetic properties are associated.

Thus, patent application JP 2010-0229924 describes an extraction process carried out on fenugreek seeds by soaking in ethanol for one week. The resulting extract, which contains compounds of the polyphenol type but no sugars, is used as a promoter of the production of collagen and hyaluronic acid.

Other extraction processes carried out on fenugreek, in particular on its seeds, have allowed the extraction of polysaccharides of galactomannan type with which various properties are associated. Thus, in general, the polysaccharides are obtained from fenugreek seed by aqueous extraction at temperatures ranging from 50 to 100 ° C, followed by alcoholic precipitation. Such operating conditions allow the extraction of certain polysaccharides of galactomannan type contained in fenugreek, but do not allow their degradation in oligosaccharides of low molecular weight.

Thus, patent application WO-A-95/21199 describes polysaccharides of general formula: ## STR1 ## where n is greater than 100; as well as their use as a nutraceutical agent. These galactomannans are prepared by aqueous extraction at temperatures ranging from 80 to 90 ° C conducted on fenugreek, followed by alcohol precipitation. The patent application FR-A-2661833 describes an extraction process carried out on fenugreek seeds by soaking in water at 45 ° C followed by filtration aimed at keeping only the polysaccharides having a molecular weight Less than 300 kDa. The use of the polysaccharides thus obtained in cosmetic or pharmacological compositions slowing dermal aging or nourishing the dermis is also mentioned. However, in addition to the fact that no experimental data support this statement, the expected anti-aging effect can only correspond to a tensor effect on the superficial cutaneous layer, since the polysaccharides can not penetrate the dermis to act. at the cellular level. As a result, the expected aging effect can only be short-lived.

However, the skin is constantly subject to many intrinsic and / or extrinsic stresses such as UV, pollution, heat, cold, various pathologies, wounds, inflammation, aging or activity. hormonal. Despite the presence of various intrinsic protection systems, the skin ends up being damaged by the succession of these aggressions, which results in particular in an alteration of the cutaneous architecture leading to an alteration of structures and cutaneous functions. These structural changes may be mainly attributed to disorganization of the extracellular matrix, itself induced by a decrease and / or a modification of the constituent molecules such as collagen, hyaluronic acid, elastin or fibronectin.

The main factors responsible for this disorganization of the extracellular matrix are thus: - the decrease of the biosynthesis or the increase of the degradation of the constituent molecules; and / or increasing the modifications of these constituent molecules, in particular involving the glycation and hyperkeratinization phenomena.

Glycation, corresponding to a Maillard reaction, depends on the possibilities of encounter between the circulating glucose molecules and the free amino groups existing either at the N-terminus of the polypeptide chains or on the side chains of lysine. These reactions are more frequent in extracellular spaces and connective tissues where glucose circulates freely and where proteins such as collagen, elastin or fibronectin have relatively long lifetimes.

The glycation reaction proceeds in three steps: i) initiation with the formation of an Amadori product; ii) glycooxidation propagation; iii) termination with the formation of advanced glycation products (PGA) including pyrraline bound to an amino acid residue and pentosidine linked to two amino acids, thus creating cross-linking between two peptide chains. These crosslinks modify the biophysical properties of proteins. The glycated proteins are formed according to the chances of molecular encounters. The fixation of glucose at certain points of the molecules is such as to mask certain sequences important for the function of the proteins. Under certain conditions, the glycated groups can react with oxygen and become oxygenated radicals, thus constituting another progressive protein modifying factor explaining certain aging phenomena. Collagen is a preferred substrate for glycation. Glycation inhibits homotypic polymerization interactions between two collagen molecules but also heterotypic interactions between different macromolecules and can also alter interactions between extracellular matrix collagen and adjacent cells.

Keratin is a cytoskeleton protein belonging to the family of intermediate filament proteins. The supracellular filamentous protein networks composed of keratin partly ensure the resistance to repeated mechanical friction that the skin undergoes. Keratin also contributes to the differentiation of epithelia. Their expression, normally finely regulated, is greatly increased and more extensive as a result of skin changes (eg due to acne, wounds, aging, UV damage, drying, burning, erythema, or inflammations) or in stratified epithelia that undergo abnormal differentiation or hyperproliferation (following, for example, psoriasis, dermatofibroma or carcinoma). These hyperkeratinizations are responsible for modifications of the structure of the extracellular matrix correlated with an activation (proliferation and differentiation) of the keratinocytes thus inducing an alteration of the cutaneous structures and functions.

Identical phenomena occur at the level of the various cutaneous appendages such as the hair follicles, the apocrine sweat glands or the nails, and in particular at the level of the hair or the hairs. Thus, any phenomenon of glycation and / or hyperkeratinization is reflected in particular by a tissue relaxation and a loss of elasticity, firmness, tone, uniformity of the hue and / or skin surface texture and / or capillary.

As a consequence, in the context of the development of new cosmetic active agents that are useful for preventing or treating the signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, alteration of tissue regeneration, alteration of cutaneous and / or capillary microcirculation, alteration of skin detoxification, loss of uniformity and / or shine and / or the brilliance of the hue (cutaneous and / or capillary), the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), of the alteration of the cutaneous and / or capillary architecture (inducing in particular hair loss), it is desirable to identify compounds that make it possible to maintain the integrity of the constituent molecules of the extracellular matrix, in particular r by limiting the degradation of these constituent molecules, and / or compounds making it possible to reduce the glycation and / or hyperkeratinization processes.

Now, it has now been found, quite surprisingly, that the radical degradation of polysaccharides extracted from fenugreek made it possible to obtain oligosaccharides of low molecular weight, that is to say less than or equal to 100,000 Daltons, allowing no only a maintenance of the integrity of the constituent molecules of the extracellular matrix by limiting their degradation, but also a decrease in the processes of glycation and / or hyperkeratinization. It was so

totally unexpectedly found that the oligosaccharides thus prepared make it possible to delay intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, the alteration of tissue regeneration, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity

and / or brightness and / or shine of the hue (cutaneous and / or capillary), alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), the alteration of the cutaneous and / or capillary architecture (inducing in particular the fall of the hair).

The present invention therefore relates to an oligosaccharide of general formula (I) ~ R6 R7 R11 in which: - R 'to R'2 are chosen independently of each other and independently for each galactose unit and / or mannose as a group hydroxy, alkyloxy, carboxy, alkoxycarbonyl, acyloxy, sulfate or phosphate; or a group -OCH2Ra, wherein Ra is hydroxy, alkyloxy, acyloxy, sulfate or phosphate; n is an integer less than or equal to 100 and is chosen so that the molecular weight is less than or equal to 100,000 Daltons; as well as its pharmaceutically acceptable salt.

The oligosaccharides according to the present invention have never been described before. These compounds make it possible not only to maintain the integrity of the constituent molecules of the extracellular matrix by limiting their degradation, but also a reduction of the glycation and / or hyperkeratinization processes. These compounds can therefore be used in the context of the prevention and / or treatment of the intrinsic and / or extrinsic signs of skin and / or capillary aging, in particular the alteration of the cutaneous and / or capillary structures and functions, of the alteration of tissue regeneration, tissue loosening, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, shine and shine of the skin hue (cutaneous and / or capillary), alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), alteration of the architecture dermal and / or capillary (inducing in particular hair loss), or incorporated into cosmetic compositions useful for the treatment or prevention of such symptoms.

In the context of the present invention: the expression "molecular weight" refers indifferently to the molecule alone or to the mixture of molecules and then represents in this case a mean value; the term "pharmaceutically acceptable salt" means any addition salt with a mineral or organic acid by the action of such an acid in an organic or aqueous solvent such as an alcohol, a ketone, an ether or a solvent chlorine, which is acceptable from a pharmaceutical point of view. By way of example of such salts, mention may be made of the following salts: benzenesulphonate, hydrobromide, hydrochloride, citrate, ethanesulphonate, fumarate, gluconate, iodate, isethionate, maleate, methanesulphonate, methylene-bis-b-oxynaphthoate, nitrate, oxalate, palmoate, phosphate, salicylate, sulfate, tartrate, theophyllinacetate and p-toluenesulfonate; an alkyl group is understood to mean a saturated, monovalent, linear or branched hydrocarbon-based chain containing from 1 to 6 carbon atoms, such as the following groups: methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, pentyl, hexyl. the term "alkyl" as defined above retains the same definition when it includes the name of a group, for example in the alkyloxy group. Thus, among the alkyloxy groups, there may be mentioned methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentyloxy; - A "concentration under reduced pressure" step means a step for evaporating the water contained in an extract at a pressure of 50 to 100 mmHg and at a temperature ranging from 40 to 50 ° C.

Preferably, the subject of the present invention is an oligosaccharide of general formula (I) as defined above in which the substituents R 'to R' 2 are chosen independently of each other and independently for each galactose and / or mannose unit as being a hydroxy, alkyloxy, acyloxy, sulfate or phosphate group; or a group -OCH2Ra, Ra being as defined above.

Most preferably, the subject of the present invention is an oligosaccharide of general formula (I) as defined above in which the substituents R 'to R'2 are chosen for each galactose and / or mannose unit as being a hydroxy group.

The oligosaccharides of general formula (I) as defined above have an average molecular weight less than or equal to 100,000 Daltons. Preferably, the subject of the present invention is an oligosaccharide of general formula (I) as defined above in which n is chosen so that the molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 100,000 Daltons. More preferably, n is chosen so that the molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 50,000 Daltons. Most preferably, n is chosen so that the molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 10,000 Daltons.

The oligosaccharides according to the present invention are prepared by radical degradation of polysaccharides of galactomannan type extracted from fenugreek. The subject of the present invention is therefore a process for the preparation of oligosaccharides as defined above comprising the following steps: a) dispersion of the fenugreek powder in water at a temperature ranging from 20 ° C. to 100 ° C., the pH from the solution ranging from 4 to 8; b) gradual addition of hydrogen peroxide (H2O2), the solution temperature ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8; c) while stirring, the temperature of the solution ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8; d) filtration or centrifugation at room temperature; e) concentration under reduced pressure; f) purification of low molecular weight molecules obtained by: - tangential ultrafiltration on membranes of 5, 10 or 30 kDa and 5 atomization to fine powder; or - alcoholic precipitation, filtration, washing and drying of the precipitate obtained.

The degradation method according to the present invention makes it possible to obtain molecules of low molecular weight with a high production yield, of the order of 30% relative to the dry mass of fenugreek powder used.

Step a) of the degradation process according to the present invention consists of the dispersion of fenugreek powder in water. The fenugreek powder may be prepared from any part of the plant, including leaves, roots and / or seeds. Preferably, the powder is prepared from fenugreek seeds. Preferably the dispersion is carried out with stirring at a speed ranging from 500 to 2,500 revolutions / minute, preferably ranging from 1,000 to 2,000 revolutions / minute. The dispersion is carried out at a temperature of 20 ° C to 100 ° C, preferably at a temperature of 40 ° C to 90 ° C, more preferably at a temperature of 50 to 80 ° C. During the dispersion, the pH of the solution can vary from 4 to 8, preferably the pH of the solution varies from 5 to 7.5. To maintain the pH of the solution at these values, any means known to those skilled in the art can be used. Preferentially, 5N sodium hydroxide or 5N potassium hydroxide will be added. The concentration of polysaccharides in the water after dispersion may vary according to the needs of those skilled in the art and the equipment used. Preferably, the polysaccharide concentration may be from 1 to 1,000 g / l, more preferably from 1 to 100 g / l, most preferably from 10 to 50 g / l.

Step b) of the degradation process according to the present invention consists of the gradual addition of hydrogen peroxide. Preferably, the mass ratio between the fenugreek extract and the added hydrogen peroxide is 1/1. The added hydrogen peroxide will preferably be selected as 35% H2O2.

The addition of hydrogen peroxide is done gradually. Preferably, the addition of hydrogen peroxide will be continuous over a period of from 30 minutes to 5 hours. The addition of hydrogen peroxide is carried out at a temperature of from 20 ° C to 100 ° C, preferably at a temperature of from 40 ° C to 90 ° C, more preferably at a temperature of from 50 to 100 ° C. 80 ° C. During the addition of hydrogen peroxide, the pH of the solution can vary from 4 to 8, preferably the pH of the solution varies from 5 to 7.5. To maintain the pH of the solution at these values, any means known to those skilled in the art can be used. Preferentially, 5N sodium hydroxide or 5N potassium hydroxide will be added.

Step c) of the degradation process according to the present invention consists in stirring the mixture extracted from Fenugreek / hydrogen peroxide. Preferably the agitation is carried out at a rate of from 100 to 2,000 rpm, more preferably at a rate of from 300 to 1,000 rpm. Preferably, agitation will be maintained for a period of from 30 minutes to 5 hours, more preferably for a period of from 30 minutes to 3 hours, most preferably for a period of from 1 hour to 2 hours. The stirring is carried out at a temperature of from 20 ° C to 100 ° C, preferably at a temperature of from 40 ° C to 90 ° C, more preferably at a temperature of from 50 to 80 ° C. During stirring, the pH of the solution can vary from 4 to 8, preferably the pH of the solution varies from 5 to 7.5. To maintain the pH of the solution at these values, any means known to those skilled in the art can be used. Preferentially, 5N sodium hydroxide or 5N potassium hydroxide will be added.

Step d) of the degradation process according to the present invention consists of a filtration or centrifugation step to remove the insoluble particles from the solution. To carry out the filtration, any means known to those skilled in the art can be used. Preferably, the filtration of the medium will be carried out on diatom by frontal filtration. For centrifugation, any means known to those skilled in the art can be used. Preferably, the medium will be centrifuged for 10 minutes at 10,000 g and at room temperature.

Step e) of the degradation process according to the present invention consists of a step of concentration of the medium by evaporation of the water present in the extract. To carry out this concentration, any means known to those skilled in the art can be used. Preferably, this step will proceed under a pressure ranging from 50 and 100 mmHg, and at a temperature ranging from 40 to 50 ° C.

Step f) of the degradation method according to the present invention consists of purification of oligosaccharides of low molecular weight. In the case of a tangential ultrafiltration, this will preferably be carried out on membranes of 5, 10 or 30 kDaltons. In the case of alcoholic precipitation followed by filtration, washing and drying, any means known to those skilled in the art can be used. Preferably, the precipitation will be carried out using an alcohol which may for example be chosen as isopropanol or ethanol. Preferably, the precipitate obtained may be filtered on sintered or on canvas.

On the other hand, the degradation method according to the present invention can be conducted in the absence or in the presence of a catalyst. Thus, optionally, the degradation method according to the present invention may be conducted in the presence of a catalyst selected from divalent cations such as for example Cul + or Fe2 +. The catalyst may be added in step b), c) and / or d) of the process according to the invention.

The oligosaccharides according to the present invention can therefore be used in cosmetics to maintain the integrity of the constituent molecules of the extracellular matrix and / or to reduce the glycation and / or hyperkeratinization processes. Thus, another subject of the present invention relates to the cosmetic use of one or more oligosaccharides according to the present invention as an agent for the prevention and / or the treatment of intrinsic and / or extrinsic cutaneous or capillary aging, in particular the alteration of the cutaneous and / or capillary structures and functions, alteration of tissue regeneration, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity and / or brightness and / or shine of the hue (cutaneous and / or capillary), of the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), alteration of the cutaneous and / or capillary architecture (inducing in particular hair loss) are generally attributed to cellular aging.

Preferably, the subject of the present invention is the cosmetic use of one or more oligosaccharides according to the present invention as an agent for the prevention and / or treatment of the alteration of cutaneous and / or capillary structures and functions, of the alteration of tissue regeneration, loss of uniformity and / or brightness and / or shine of the dye (cutaneous and / or capillary), alteration of the cutaneous surface texture (appearance of wrinkles, pockets, dry skin, etc.) and / or capillary (brittle hair), alteration of the cutaneous and / or capillary architecture (inducing in particular hair loss).

The present invention also relates to a cosmetic composition comprising, as active principle, one or more oligosaccharides of low molecular weight as described above.

The compositions according to the present invention may be formulated in any dosage form suitable for their administration. The compositions according to the present invention can thus be formulated in the form of cream, gel, lotion, milk, oil-in-water emulsion or water-in-oil, solution, ointment, spray, body oil, aftershave, soap, lip protection stick. , stick and pencil for makeup. The compositions according to the present invention contain one or more oligosaccharides according to the present invention at contents ranging from 0.005% to 75% by total weight of the composition, preferably from 0.01% to 25%, preferentially still from 0.1% to 5%.

For the preparation of these compositions, one or more low molecular weight oligosaccharides according to the present invention or one or more of their pharmaceutically acceptable salts are mixed with the excipients conventionally used in the cosmetics field. The compositions according to the present invention may take the form of a cream in which one or more low molecular weight oligosaccharides according to the present invention or one or more of their pharmaceutically acceptable salts are combined with the excipients commonly used in cosmetology. The present invention may take the form of gels in suitable excipients such as cellulose esters or other gelling agents, such as carbopol, sepinov (polyacrylate), guar gum, and the like. The compositions according to the present invention may also take the form of a lotion or solution in which one or more low molecular weight oligosaccharides according to the present invention or one or more of their pharmaceutically acceptable salts are in encapsulated form. The microspheres according to the invention may for example consist of fatty substance, agar and water. One or more low molecular weight oligosaccharides according to the present invention or one or more of their pharmaceutically acceptable salts may be incorporated in liposomes, glycospheres, cyclodextrins, in chylomicrons, macro-, micro-nano-particles and as macro-, micro- and nanocapsules and also be absorbed on powdery organic polymers, talcs, bentonites and other mineral carriers.

These emulsions have good stability and can be stored for the time necessary for use at temperatures between 0 and 50 ° C without sedimentation of the constituents or phase separation. The compositions according to the present invention may also contain additives or adjuvants customary in cosmetologies, such as, for example, antimicrobial agents or perfumes, but also extraction or synthetic lipids, gelling and viscosifying polymers, surfactants and emulsifiers, hydro- or liposoluble active ingredients, plant extracts, tissue extracts, marine extracts, synthetic actives.

The compositions according to the present invention may also comprise other complementary active ingredients chosen for their action, for example for the slimming effect, the anti-cellulite effect, the firming effect, the moisturizing effect, the anti-cellulite effect, age, antimicrobial activity, antioxidant activity, anti-radical activity, healing effect, tensor effect, anti-wrinkle effect, chelating activity, complexing and sequestering activity, the soothing effect, the concealer effect, the anti-redness effect, the emollient activity, the hair conditioner effect, the anti-dandruff activity, the stimulating effect of the regrowth of the hair, the effect inhibiting hair loss, capillary gaining effect, depilatory activity, hair regrowth limiting activity, activity participating in cell renewal, activity modulating the inflammatory response, activity involved in maintaining the hair, oval of the face, but also the sun protection, has anti-irritant activity, cellular nutrition, cellular respiration, anti-seborrhoeic treatments, skin tone, hair protection. When the compositions according to the present invention contain complementary active ingredients, these are generally present in the composition at a sufficiently high concentration so that they can exert their activity.

The compositions according to the present invention are preferably used daily and applied one or more times per day.

The compositions according to the present invention are very well tolerated, they have no toxicity and their application to the skin for prolonged periods of time does not imply any systematic effect.

The compositions according to the present invention can therefore be used to maintain the integrity of the constituent molecules of the extracellular matrix and / or to reduce the glycation and / or hyperkeratinization processes and thus to prevent and / or treat the signs of cutaneous aging. and / or intrinsic and / or extrinsic capillaries, in particular the alteration of cutaneous and / or capillary structures and functions, the alteration of tissue regeneration, tissue relaxation, the alteration of cutaneous microcirculation and / or hair loss, skin detoxification, loss of uniformity, shine and shine of the dye (cutaneous and / or capillary), alteration of the cutaneous surface texture (appearance wrinkles, pockets, dry skin, etc.) and / or capillary (brittle hair), alteration of the cutaneous and / or capillary architecture (inducing in particular hair loss) ). The present invention is illustrated in a nonlimiting manner by the following examples.

EXAMPLE 1 Preparation of oli2osaccharides according to the invention Extraction of polysaccharides of galactomannan type

The extraction of the polysaccharides is carried out by dispersing 20 grams of fenugreek powder in 1 liter of water at 95 ° C. with vigorous stirring (1,000 rpm) for 2 hours.

The mixture is then filtered while hot (80 to 95 ° C.) on diatom (50 g) on a sintered glass 1.

The fenugreek extract is then precipitated in 3 volumes of ethanol 96 ° (at 4 ° C) with stirring (500 rpm) for 2 hours.

The precipitate is recovered on a filter cloth (porosity 500 microns) and then washed with 100 ml of ethanol 96 °, drained and dried (at 35 ° C for 12 hours).

Finally, the precipitate is crushed and then sieved on 500 ml in order to obtain a fine powder of polysaccharides of galactomannan type extracted from fenugreek. Production of oligosaccharides according to the invention

30 g of polysaccharides of galactomannan type thus obtained are dissolved in 1 liter of water (80 ° C., pH 7-7.5) with vigorous stirring (1,500 rpm). 133 ml of a 35% H 2 O 2 solution for 1 hour was added at a rate of 2.22 ml / min at 80 ° C and maintained at pH 7-7.5 by continuous addition of NaOH (5M).

After complete addition of the H2O2, the stirring is maintained for a further 3 hours (500 rpm) at 80 ° C while maintaining the pH between 7-7.5 by addition of NaOH (5M).

The medium is allowed to warm to 25 ° C and filtered through diatomaceous earth (or centrifuged at 10,000g, 10 minutes, 25 ° C) to remove the insolubles.

The filtrate is then concentrated under reduced pressure at 40 ° C. to a volume corresponding to 1/5 of the initial volume.

The concentrate is then precipitated in 5 volumes of ethanol 96 ° at 4 ° C with stirring (500 rpm) for 1 hour.

The precipitate is recovered by filtration on Fritted glass 2 (porosity less than 100 microns), triturated and then washed with 50 ml of ethanol for 30 minutes and then filtered on Sintered glass 2. Finally, the precipitate is dried in an oven (40 ° C.). C, 1 night) and then ground into fine powder. The production yield of oligosaccharides is 60-70%. Analysis Analysis of constituent sugars The oligosaccharides obtained with 1N TFA are hydrolysed and the constituent sugars are analyzed by ion chromatography (HPAEC) with reference to monosaccharide databases for identification.

Determination of Molecular Weight Analysis by Steric Exclusion Chromatography (SEC MALLS) with the Use of 2 OHPAK Chromatography Columns SB 804 and 806 HQ (Shodex). Galactomannans Example 2: Production of oli2osaccharides according to the invention continuously 15 Preparation 30g of fenugreek powder are dispersed in 1 liter of water at 90 ° C. with vigorous stirring (1,000 rpm) for 2 hours. The mixture is diatom-filtered (50 g) on sintered glass 1. The extracted galactomannan-type polysaccharide extract obtained is stirred vigorously (1,000 rpm) at 80 ° C. and pH 7-7.5. 40 ml of 35% H2O2 solution are added to the medium for 1 hour at a flow rate of 0.55 ml / min at 60 ° C. and maintaining at pH 7-7.5 by addition of NaOH (5M). After complete addition of 1 H 2 O 2 (in about 1 hour), stirring is maintained for 3 hours (500 rpm) at 80 ° C and pH 7-7.5. The filtrate is then concentrated under reduced pressure at 40 ° C. to a volume corresponding to 1 / 5th of the initial volume. The concentrate is then precipitated in 5 volumes of ethanol 96 ° at 4 ° C with stirring (500 rpm) for 1 hour. The precipitate obtained is recovered by filtration on glass Mass ratio Galactose Mannose Molecular weight (majority) X20 kDa Sintered 2 (porosity less than 100 microns), triturated and then washed with 50 ml of ethanol 96 ° for 30 minutes and then filtered on glass Sintered 2 Finally, the precipitate is dried in an oven (40 ° C, 1 night) and then ground to a fine powder. The production yield of oligosaccharides is 60-70%.

Analysis Analysis of the constituent sugars The oligosaccharides obtained with 1N TFA are hydrolysed and the constituent sugars are analyzed by ion chromatography (HPAEC) with reference to monosaccharide data bases for identification.

Determination of molecular weight Analysis by steric exclusion chromatography (SEC MALLS) with the use of 2 chromatography columns OHPAK SB 804 and 806 HQ (Shodex). Galactomannans Mass ratio Molecular weight (majority) Galactose Mannose k; 20 kDa Example 3: Production of oli2osaccharides according to the invention continuously and purification by UFT 30g of fenugreek powder are dispersed in 1 liter of water at 90 ° C. strong agitation (1,000 rpm) for 2 hours.

Then 40 ml of a 35% solution of H2O2 was added to the medium for 1 hour at a flow rate of 0.60 ml / min at 60 ° C and maintaining pH 5-8 by addition of NaOH (5M). After complete addition of H2O2 (in about 1 hour), stirring is maintained for 3 hours (500 rpm) at 80 ° C and pH 5-8.

The mixture is then hot-filtered on diatom (50 g) on sintered glass 1. The elimination of the insoluble particles can also be carried out by centrifugation (10,000 g, 15 minutes, 22 ° C.).

The filtrate is then concentrated by tangential ultrafiltration on a membrane of 5 kilo-5 daltons until a concentrated extract with a solids content of 10% is obtained.

The concentrate may finally be atomized in order to obtain a dry form (powder) of the oligosaccharides according to the invention.

Analysis Analysis of the constituent sugars The oligosaccharides obtained with 1N TFA are hydrolysed and the constituent sugars are analyzed by ion chromatography (HPAEC) with reference to monosaccharide databases for identification. Determination of molecular weight Analysis by steric exclusion chromatography (SEC MALLS) with the use of 2 chromatography columns OHPAK SB 804 and 806 HQ (Shodex). Galactomannans Example 4: Transcriptomic study carried out on human fibroblasts cultured in vitro

A transcriptomic study was carried out on human fibroblasts cultured in vitro in the absence or in the presence of oligosaccharides prepared according to Examples 1 to 3 at a concentration of 0.1% by weight (ie 0.1 g of oligosaccharides per 100 g). cell culture medium). Mass ratio Galactose Mannose Molecular weights (majority) k; 20 kDa When the two fibroblast cell cultures had reached confluence, their respective total RNAs were extracted and then reverse transcribed into respectively differentially labeled complementary mono-stranded (cDNA) DNAs (the CDNAs derived from the RNAs extracted from the fibroblasts cultured in the control condition are Cy3 cyanine labeled and the cDNAs derived from the RNAs extracted from the fibroblasts cultured in the test condition are labeled with cyanine Cy5). Both types of cDNA are pooled and hybridized on a DNA chip containing the 44,000 sequences of the human genome. The reading of the chip makes it possible to determine the overexpression (Cy5 labeling greater than the Cy3 labeling for the hybridized genetic sequence concerned) and the subexpression (Cy5 labeling less than the Cy3 labeling for the hybridized genetic sequence concerned) of the genes induced by the treatment of the genes. fibroblasts with oligosaccharides according to the present invention.

In this study, it was found that the oligosaccharides used lead in particular to the overexpression of the SAA1 gene and the sub-expression of the MAL genes coding for the proteins bearing the same names and involved in the metabolism of the constituent molecules of the extracellular matrix. . Indeed, the overexpression of the SAA1 gene allows an activation of the hyaluronic acid biosynthesis, one of the major constituents of the extracellular matrix and the under-expression of the MAL gene which thus allows an inactivation of matrix metalloproteinases involved in the degradation of collagens ( the second major constituent of the extracellular matrix). These two activities allow protection of the integrity of the extracellular matrix.

This study also revealed that the oligosaccharides used lead to the overexpression of the SOD2 gene and the under-expression of the HK3, ISM1 and KRT16 genes coding for proteins bearing the same names and involved in the modulation of the glycation and hyperkeratinization processes. . Indeed, overexpression SOD2 increases the protection of the body and therefore the skin against reactive oxygen molecules (free radicals and oxidizing molecules) responsible in particular for the initiation and amplification of the glycation phenomenon. Under-expression of the HK3 and ISM1 genes allows a decrease in glucose concentrations in the extracellular spaces and thus a reduction of the glycooxidation stage of the glycation phenomenon. This allows a modulation of the glycation phenomenon involved in the alteration of structures and cutaneous and / or capillary functions.

Under-expression of the KRT16 gene allows for a repression of the hyperkeratinization phenomenon occurring as a result of an alteration of the skin (acne, wounds, aging, UV lesions, drying out, burning, erythema, inflammation, external aggressions, etc.).

In conclusion, the transcriptomic study has made it possible to demonstrate that the compounds of the present invention make it possible to maintain the integrity of the constituent molecules of the extracellular matrix and a reduction in the processes of glycation and hyperkeratinization, thus allowing preservation of the structures and cutaneous and / or capillary functions.

EXAMPLE 5 In Vitro Study of the Vivoation on an Albumin Solution This study made it possible to study the effect of the oligosaccharides prepared according to one of Examples 1 to 3 on the formation of the advanced glycation products (PGA). presence of glucose and in comparison with aminoguanidine (reference molecule). In this study, five conditions were tested: - negative control condition: albumin solution (10 mg / ml in 20 mM phosphate buffer 200, pH 7.4) + glucose (200 mM), - positive control condition: solution albumin (10 mg / ml in 200 mM phosphate buffer, pH 7.4) + aminoguanidine (1 mM) + glucose (200 mM), - three test conditions with oligosaccharides: - albumin solution (10 mg / ml) in 200 mM phosphate buffer, pH 7.4) + 25 oligosaccharides according to the present invention at a concentration of 0.25% by weight + glucose (200 mM), - albumin solution (10 mg / ml in 200 mM phosphate buffer pH 7.4) + oligosaccharides according to the present invention at a concentration of 0.5% by weight + glucose (200 mM), albumin solution (10 mg / ml in 200 mM phosphate buffer, pH 7.4 ) + oligosaccharides according to the present invention at a concentration of 1% by weight + glucose (200mM).

These five conditions were incubated at 37 ° C for 14 days. Advanced glycation products (PGA) were determined using the Usen Life Science Inc. ELISA kit Wuhan. Briefly, the plate provided with Usen Life Science Inc. kit Wuhan has been pre - coated with a specific antibody for PGA. The standards or samples are then added to the appropriate wells of the plate with a biotin-conjugated polyclonal antibody specific for PGAs. Afterwards, the peroxidase conjugated avidin is added to each well of the microplate and incubated. A TMB substrate solution is then added to each well. Only wells that contain PGA, biotin-conjugated antibody and enzyme-conjugated avidin show a color change. The enzyme / substrate reaction is stopped by addition of a sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm. The concentration of PGA in the samples is then determined by comparing the optical density of the samples to the standard curve.

The results obtained show that aminoguanidine at 1 mM (positive control) leads to a significant decrease (53%) in the level of advanced glycation products (PGA) and that the compounds of the present invention at concentrations of 0.25 g, 0, 5 g and 1 g of compounds per 100 ml of cell culture medium (ie at 0.25%, 0.5% and 1% by weight) lead to a significant decrease in PGA of respectively 15%, 25% and 30%.

In conclusion, this study shows that the oligosaccharides of the present invention result in a decrease in the protein glycation process.

Example 6 In-vitro Study on Human Fibroblasts in Culture

An in vitro study was performed on human fibroblasts cultured in the absence of oligosaccharides prepared according to Examples 1 to 3 and in the presence of glucose at three concentrations (150 mM, 200 mM and 250 mM) (negative control condition). or in the presence of aminoguanidine (1 mM) and glucose at three concentrations (150 mM, 200 mM and 250 mM) (positive control condition), or in the presence of oligosaccharides prepared according to Examples 1 to 3 at the concentration 1% by weight (corresponding to 1 g of test molecules per 100 ml of cell culture medium) and in the presence of three glucose concentrations (150 mM, 200 mM and 250 mM).

Human fibroblasts were cultured according to the conditions previously described for 7 days. At the end of this period, the cells were removed and then sonicated three times for 5 seconds before being extracted with 1 ml of an ethanol: diethyl ether solution (3: 1 (v: v)). The supernatant was analyzed for PGA level by the Usen Life Science Inc. Wuhan ELISA kit, the use of which was described in the previous example.

This study evaluated the effect of tested compounds on the formation of advanced glycation products (PGA) in human fibroblasts in culture in the presence of different glucose concentrations.

The results obtained show that aminoguanidine at 1 mM (treatment for 7 days) results in a significant decrease (40%, 43% and 45%) in the level of the products of advanced glycation (PGA) respectively in the presence of the three glucose concentrations. (150 mM, 200 mM and 250 mM). Treatment of the cells, for 7 days, with the compounds of the present invention (at the concentration of 1% by weight) in the presence of 150, 200 or 250 mM glucose allows a significant decrease in PGA respectively by 22%, 24% and 22%.

In conclusion, this study shows that the oligosaccharides of the present invention cause a decrease in the glycation process in human cells. Example 7 In Vitro Study on Human Fibroblasts in Culture

An in-vitro study was carried out on human fibroblasts in culture in the absence of oligosaccharides prepared according to Examples 1 to 3 and in the presence of carbon-14 labeled glucose (negative control condition), or in the presence of aminoguanidine (1). mM) and carbon-14 radiolabeled glucose (positive control condition), or in the presence of different concentrations of oligosaccharides prepared according to Examples 1 to 3 and in the presence of carbon-14 radiolabeled glucose.

Human fibroblasts were cultured according to the conditions previously described for 7 days. At the end of this period, the cells were washed and then removed before being sonicated (3 times 5 seconds) and extracted with 1 ml of an ethanol: diethyl ether solution (3: 1 (v: v)). The supernatant was analyzed to determine the level of PGAs by detecting radioactivity. This study made it possible to evaluate the effect of the compounds tested on the incorporation of radiolabeled 14 carbon glucose in glycation products at the level of human fibroblasts in culture.

The results obtained show that aminoguanidine at 1 mM (treatment for 7 days) leads to a significant decrease (41%) in the incorporation of radiolabeled glucose at carbon 14, thus showing a significant decrease of 41% in the level of advanced glycation products ( PGA). The treatment of the cells, for 7 days, with the compounds of the present invention at concentrations of 0.25%, 0.5% and 1% by weight allows a significant decrease in the incorporation of radiolabelled glucose to carbon-14, hence the advanced glycation products, respectively 13%, 20% and 26%.

Example 8 In Vitro Study on Reconstituted Epidermis

An in-vitro study carried out on reconstituted epidermis cultured in the absence of oligosaccharides prepared according to Examples 1 to 3 (negative control condition), or in the presence of vitamin A brought to the concentration of 0.83 mM (positive control condition), or in the presence of test molecules at a concentration of 1% by weight (corresponding to 1 g of test molecules per 100 ml of cell culture medium).

This study made it possible to evaluate the effect of the tested compounds on epidermal differentiation (marking intensity of filaggrin).

The results obtained show that vitamin A at a concentration of 0.83 mM results in a significant decrease (75%) in the labeling intensity of filaggrin and that the compounds of the present invention at the concentration of 1% by weight (ie 1 g of compounds per 100 ml of cell culture medium) result in a significant decrease (50%) in the labeling intensity of filaggrin and thus in epidermal differentiation.

In conclusion, this study shows that the oligosaccharides of the present invention cause a decrease in epidermal differentiation. More generally, the results of the in vitro studies reported above demonstrate the important role of the compounds of the present invention in the protection against the process of glycation, and in the decrease of epidermal differentiation. This protection against the glycation process makes it possible to maintain the integrity of the extracellular matrix by preventing the modification of the constituent molecules. This correlated with a decrease in epidermal differentiation allows maintenance of epidermal and cutaneous integrity. The studies reported above also highlight the important role of the compounds of the present invention in limiting the degradation of the constituent molecules of the extracellular matrix thus allowing the maintenance of the integrity of these molecules and therefore the maintenance of the epidermal and cutaneous integrity.

Claims (10)

REVENDICATIONS1. in which: R 'to R'2 are independently selected. wherein: R 'to R'2 are independently selected from each other and independently for each galactose and / or mannose unit as hydroxy, alkyloxy, carboxy, alkoxycarbonyl, acyloxy, sulfate or phosphate; or a group -OCH2Ra, wherein Ra is hydroxy, alkyloxy, acyloxy, sulfate or phosphate; n is an integer less than or equal to 100 and is chosen so that the molecular weight is less than or equal to 100,000 Daltons; As well as its pharmaceutically acceptable salt. Oligosaccharide according to claim 1, characterized in that the substituents R 'to R' 2 are independently selected from each other and independently for each galactose and / or mannose unit as hydroxy, alkyloxy, acyloxy, sulfate or phosphate; or a group -OCH2Ra. 3. Oligosaccharide according to claim 2, characterized in that the substituents R 'to R'2 are chosen for each galactose unit and / or mannose as a hydroxy group. 1. Oligosaccharide of General Formula (I) ~ R6 R7 R11 24 25 4. Oligosaccharide according to one of claims 1 to 3, characterized in that n is chosen so that the average molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 100,000 Daltons. Oligosaccharide according to claim 4, characterized in that n is chosen so that the average molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 50,000 Daltons. An oligosaccharide according to claim 5, characterized in that n is selected so that the average molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 10,000 Daltons. 7. Process for the preparation of an oligosaccharide according to one of claims 1 to 6 comprising the following steps: a) dispersion of the fenugreek powder in water at a temperature ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8; b) gradual addition of hydrogen peroxide (H2O2), the solution temperature ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8; c) while stirring, the temperature of the solution ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8; d) filtration or centrifugation at room temperature; e) concentration under reduced pressure; f) purification of the molecules of low molecular weight obtained by: - tangential ultrafiltration on membranes of 5, 10 or 30 kDa and then atomization to fine powder; or - alcoholic precipitation, filtration, washing and drying of the precipitate obtained. 8. Cosmetic use of one or more oligosaccharides according to any one of claims 1 to 6 for maintaining the integrity of the constituent molecules of the extracellular matrix and / or reducing the glycation and / or hyperkeratinization processes. 9. Cosmetic use of one or more oligosaccharides according to any one of claims 1 to 6 for the prevention and / or treatment of the intrinsic and / or extrinsic signs of skin and / or capillary aging, in particular the alteration of the structures and cutaneous and / or capillary functions, alteration of tissue regeneration, tissue loosening, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity , the brightness and gloss of the hue, the alteration of the cutaneous and / or capillary surface texture, the alteration of the cutaneous and / or capillary architecture. 10. Cosmetic composition comprising, as active principle, one or more oligosaccharides according to one of claims 1 to 6.