WO2013150253A1

WO2013150253A1 - Novel oligosaccharide compounds and the cosmetic use thereof

Info

Publication number: WO2013150253A1
Application number: PCT/FR2013/050756
Authority: WO
Inventors: Laurent Rios; Cédric DELATTRE; Laurent CHAISEMARTIN; Jean-Yves Berthon
Original assignee: Greentech
Priority date: 2012-04-06
Filing date: 2013-04-05
Publication date: 2013-10-10
Also published as: JP6189928B2; KR20150002659A; JP2015515524A; KR102029078B1; FR2989087B1; EP2833863A1; FR2989087A1

Abstract

The subject of the present invention is an oligosaccharide of general formula (I) and also the pharmaceutically acceptable salt thereof, and the use of this compound for maintaining the integrity and the concentration of the constituent molecules of the extracellular matrix.

Description

NOVEL OLIGOSACCHARIDE COMPOUNDS AND THEIR COSMETIC USE

The present invention relates to novel oligosaccharide compounds extracted from the green alga Ulva Lactuca, 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 and concentration of the constituent molecules of the extracellular matrix. Ulva Lactuca (or sea lettuce) is a marine green, nitrophilous algae of the family Ulvaceae and the order of Ulvales.

Three polysaccharide families make up the wall of this green alga. The first family consists of a sulfated rhamnoglycuronan referred to as ulvan (Ray and Lahaye, 1995). It is soluble in water and differs from other polysaccharides contained in the wall of the seaweed by its content of rhamnose (30 to 50%), glucuronic acid (10 to 20%) and sulfate (16 to 19%) ). The second family consists of? - (1,4) -D-xyloglucans and? - (1,4) -D-glucuronans. Finally, the third family consists of amorphous cellulose.

Various extraction methods carried out on Ulva Lactuca have been described in the prior art. In general, the polysaccharides contained in this alga are obtained by aqueous extraction at temperatures ranging from 50 to 100 ° C., followed by an alcoholic precipitation. This is the case, for example, with the method described in Paradossi et al., "A physico-chemical study on the polysaccharide ulvan from hot water extraction of the macroalga Ulva", Int J Biol Macromol, 1999, 25 (4): 309- 15. However, this process does not make it possible to obtain oligosaccharides since no hydrolysis is carried out. Qi et al. for their part describe in "Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)", Journal of Applied Phycology (2005) 17: 527-534, an extraction method carried out on Ulva pertusa allowing to obtain sulfated heteropolysaccharides of molecular weights equal to 28.2 kDa, 58 kDa, 64.5 kDa and 151.7 kDa. However, unlike the compounds of the invention, the polysaccharides obtained consist not only of rhamnose units and glucuronic acid, but also xylose units, manoses, arabinoses and galactoses. On the other hand, the polysaccharides thus prepared are described as having an antioxidant activity, but nothing is said as to a possible activity for maintaining the integrity and concentration of the constituent molecules of the extracellular matrix by promoting their synthesis. and their organization, and by limiting their degradation

The patent application WO-A-2008/1461 16 describes the use of Ulva Lactuca or extracts of this alga for the preparation of cosmetic compositions having a triple action on the dermis: stimulation of the proliferation of fibroblasts, stimulation of elastin production and stimulation of the synthesis of type I collagen. Nevertheless, no indication is provided in this patent application concerning the extraction process used to prepare the extract of Ulva Lactuca, so that it is impossible to determine the exact composition. On the other hand, the Ulva lactuca extract only acts on the proliferation of fibroblasts. No stimulation of collagen and hyaluronic acid synthesis is associated with the use of Ulva lactuca extract alone.

However, the skin is constantly subject to many intrinsic and / or extrinsic stresses such as UV, pollution, heat, cold, various pathologies, injuries, inflammation, aging or hormonal activity. . 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 can be mainly attributed to disorganization of the extracellular matrix, itself induced by a decrease and / or a modification of the constituent molecules such as collagens, hyaluronic acid, elastin or fibronectin.

The main factors responsible for this disorganization of the extracellular matrix are thus the decrease of the biosynthesis and / or the increase of the degradation of these constituent molecules but also the bad disposition of these compounds within this matrix.

All this results in particular in a tissue relaxation and a loss of elasticity, firmness, tone, uniformity of the hue and / or cutaneous surface texture and / or capillary. Consequently, in the context of the development of new cosmetic active agents that are useful for inducing the biosynthesis and / or the organization of the constituent molecules of the extracellular matrix, and / or for reducing their degradation, thus enabling the establishment and maintenance of the architecture of the extracellular matrix and thus to prevent and / or treat the signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of the cutaneous and / or capillary structures and functions, the alteration tissue regeneration, tissue loosening, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine 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), and the alteration skin architecture and / or capillary (inducing in particular hair loss). Now, it has now been quite surprisingly found that the radical degradation of polysaccharides extracted from V Ulva Lactuca made it possible to obtain oligosaccharides of low molecular weight, that is to say less than or equal to 100,000 Daltons, allowing maintaining the integrity and concentration of the constituent molecules of the extracellular matrix by promoting their synthesis and organization, and limiting their degradation. It has thus been totally unexpectedly found that the oligosaccharides thus prepared make it possible to induce the biosynthesis and / or the organization of the constituent molecules of the extracellular matrix, and / or to reduce their degradation, thus allowing the introduction and maintaining the architecture of the extracellular matrix and thus preventing and / 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, tissue relaxation, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine of hue (dermal) and / or capillary), the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), and the alteration of the cutaneous and / or capillary architecture (inducing in particular the hair loss). The subject of the present invention is therefore an oligosaccharide of general formula (I)

(I) in which:

R ¹ to R ⁴ are independently selected from each other and independently for each rhamnose unit and / or glycuronic acid as hydroxy, alkyloxy, carboxy, alkoxycarbonyl, acyloxy, sulfate or phosphate; or an -OCH ₂ R ^{a group} , wherein R ^a is hydroxy, alkyloxy, acyloxy, sulfate or phosphate;

n is an integer less than or equal to 50 and 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;

as well as its pharmaceutically acceptable salt.

The oligosaccharides according to the present invention have never been described before. These compounds make it possible to maintain the integrity and concentration of the constituent molecules of the extracellular matrix by promoting their synthesis and their organization, and by limiting their degradation. These compounds can therefore be used to induce the biosynthesis and / or the organization of the constituent molecules of the extracellular matrix, and / or to reduce their degradation, thus allowing the establishment and maintenance of the architecture of the extracellular matrix and thus to prevent and / or treat the signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, the alteration of the tissue regeneration, the tissue relaxation, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine of the dye (cutaneous and / or capillary), alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), and the alteration of the cutaneous and / or capillary architecture (i including loss of hair). 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;

"Glycuronic acid" means glucuronic acid or iduronic acid (which is found in a small proportion);

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 ¹ to R ⁴ are chosen independently of each other and independently for each rhamnose unit and / or glycuronic acid as being a hydroxy, alkyloxy, acyloxy, sulfate or phosphate group; or a group -OCH ₂ R ^a , R ^a 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 ¹ to R ⁴ are chosen independently of each other and independently for each rhamnose unit and or glycuronic acid as a hydroxy group or a sulfate group. The oligosaccharides of general formula (I) as defined above have a molecular weight greater than or equal to 5,000 Daltons and 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 50,000 Daltons, preferably greater than or equal to 5,000 Daltons and less than or equal to 30,000 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 25,000 Daltons, preferably greater than or equal to 5,000 Daltons and less than or equal to 15,000 Daltons.

The oligosaccharides according to the present invention are prepared by radical degradation of polysaccharides extracted with Ulva Lactuca. The subject of the present invention is therefore a process for preparing oligosaccharides as defined above, comprising the following steps:

a) dispersing Ulva Lactuca seaweed 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 (H ₂ 0 ₂ ), the solution temperature ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 4 to 8;

c) keeping 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) the low molecular weight molecules can be purified by:

- tangential ultrafiltration then atomization into fine powder;

- Alcoholic precipitation, filtration, leaching and drying of oligosaccharides obtained.

The degradation method according to the present invention makes it possible to obtain oligosaccharides of low molecular weight with a high production yield, of the order of 25% relative to the dry weight of Ulva Lactuca seaweed powder used.

Step a) of the degradation process according to the present invention consists of the dispersion of the Ulva lactuca seaweed powder in water. Preferably the dissolution is carried out with stirring at a speed ranging from 500 to 2500 rpm, preferably from 1000 to 2000 rpm. The dissolution 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 the dissolution, 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 dissolution 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 1000 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 weight ratio between the Ulva lactuca extract and the added hydrogen peroxide is 1/1. The added hydrogen peroxide will preferably be selected as 35% H ₂ 0 ₂ .

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 ranging from 20 ° C. to 100 ° C., preferably at a temperature ranging from 40 ° C. to 90 ° C., more preferably at a temperature ranging from 50 ° to 80 ° C. ° 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 of stirring the mixture extracted from Ulva / actwca / hydrogen peroxide.

Preferably the agitation is carried out at a speed ranging from 100 to 2000 rpm, more preferably at a speed ranging from 300 to 1000 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 may 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 process according to the present invention consists of the purification of oligosaccharides of low molecular weight by:

In the case of tangential ultrafiltration, this will preferably be carried out on membranes of 5, 10 or 30 kDa.

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 method of degradation according to the present invention may be conducted in the presence of a catalyst selected from divalent cations such as for example Cu ²⁺ or Fe ²⁺ . The catalyst may be added in step b), c) or d) of the process according to the invention. The degradation method according to the present invention makes it possible to obtain low molecular weight molecules with a high production yield, of the order of 20 to 25% relative to the dry weight of Ulva lactuca powder used.

The oligosaccharides according to the present invention can therefore be used in cosmetics to induce the biosynthesis and / or the organization of the constituent molecules of the extracellular matrix, and / or to reduce their degradation, thus enabling the establishment and maintenance of the architecture of the extracellular matrix and thus to prevent and / or treat the signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of the structures and cutaneous and / or capillary functions, the alteration of the regeneration tissue, tissue relaxation, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine of dye (cutaneous and / or capillary). ), the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), and the alteration n cutaneous and / or capillary architecture (inducing in particular hair loss).

The subject of the present invention is therefore also the cosmetic use of one or more oligosaccharides according to the present invention as an agent for the prevention and / or treatment of the intrinsic and / or extrinsic signs of skin and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, alteration of tissue regeneration, tissue loosening, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity , brightness and shine 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 ), and the alteration of the cutaneous and / or capillary architecture (inducing in particular the 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 associated with excipients commonly used in cosmetology.

The compositions according to 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-seborrheic 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 exercise 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 exhibit 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 induce the biosynthesis and / or the organization of the constituent molecules of the matrix. extracellular, and / or to reduce their degradation, thus enabling the establishment and maintenance of the architecture of the extracellular matrix and thus prevent and / or treat intrinsic and / or extrinsic signs of skin and / or capillary aging. , including alteration of cutaneous and / or capillary structures and functions, alteration of tissue regeneration, tissue relaxation, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine of the dye (cutaneous and / or capillary), the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), and the alteration of the cutaneous and / or capillary architecture (inducing in particular the hair loss).

The present invention is illustrated in a nonlimiting manner by the following examples. Example 1 Preparation of oligosaccharides according to the invention

Extraction

The extraction of the polysaccharides is carried out by dispersing 50 grams of Ulva lactuca seaweed powder in 1 liter of water at 95 ° C. with vigorous stirring (1000 rpm) for 2 hours. The mixture is then diatom-filtered (50 g) on a sintered glass 1. (Removal of the algae can also be carried out by centrifugation (10000 g, 30 minutes, 22 ° C.)). The Ulva lactuca extract is then precipitated in 3 volumes of 96 ° ethanol (at 4 ° C.) with stirring (500 rpm) for 2 hours. The precipitate is recovered on a filter cloth (porosity 500 μιη) then washed with 100 ml of ethanol 96 °, drained and dried (at 40 ° C for 15 hours). Finally, the precipitate is ground and sieved over 500 μιη in order to obtain a fine powder of polysaccharides extracted from Ulva Lactuca.

Production of oligosaccharides of low molecular weight

50 g of polysaccharides (extracts with 'Ulva lactuca) thus obtained are dissolved in 1 liter of water (80 ° C., pH 7-7.5) with vigorous stirring (1500 rpm). 150 ml of a 35% H 2 O 2 solution for 1 hour at a flow rate of 2.5 ml / min at 80 ° C and maintaining at pH 7-7.5 are added by continuous addition of NaOH (5M). After complete addition of Γ Η2Ο2, the stirring is maintained for a further 2 hours (500 rpm) at 80 ° C. while maintaining the pH between 7-7.5 by addition of NaOH (5M).

The temperature of the medium is allowed to return to 25 ° C and then filtered through diatomaceous earth (or centrifuged at 10,000g, 30 minutes, 25 ° C) to remove the insolubles.

The filtrate is then concentrated under reduced pressure at 40 ° C. to a volume corresponding to 1/4 of the initial volume.

The concentrate is then precipitated in 5 volumes of ethanol 96 ° at 4 ° C with stirring (500 rpm) for 2 hours.

The precipitate is recovered by filtration on sintered glass 3 (porosity <100 microns), triturated and then washed with 50 ml of ethanol for 30 minutes and then filtered on Sintered glass 3. Finally, the precipitate is dried in an oven (40.degree. 15 hours) and then ground into fine powder.

The production yield of oligosaccharides of low molecular weight is 80%.

Analysis

Analysis of constituent sugars

The hydrolysis of the oligosaccharides is carried out with TF A 2N. The constituent sugars are analyzed by ion chromatography (HPAEC) with reference to monosaccharide databases (glucuronic acid, Rhamnose) for identification. Determination of molecular weight

Analysis by size exclusion chromatography (SEC MALLS) with the use of 2 OHPAK chromatography columns SB 804 and 806 HQ (Shodex).

Determination of sulfate groups

The determination of the sulfate level was carried out according to the turbidimetric barium / gelatin method of Dodgson and Price (1962). % relative Molecular weight (majority)

Rhamnose Sulfate Acid

glucuronic

¾10 kDa

Oligosaccharides 60 25 15

Example 2 Production of oligosaccharides according to the invention continuously

Preparation

50 g of Ulva lactuca powder are dispersed in 1 liter of water at 90 ° C. with vigorous stirring (1000 rpm) for 2 hours. The mixture is hot-filtered on diatom (50 g) on sintered glass 1. The elimination of insoluble particles can also be carried out by centrifugation (10000 g, 15 minutes, 22 ° C.).

The polysaccharide extract of Ulva lactuca is stirred vigorously (1000 rpm) at 80 ° C. and pH 7-7.5. To the medium is added 60 ml of a 35% H 2 O 2 solution for 1 hour at a flow rate of 1 ml / min at 60 ° C and maintaining at pH 7-7.5 by addition of NaOH (5M).

After complete addition of H2O2 (in about 1 hour), stirring is maintained for 2 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 1/5 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 obtained is recovered by filtration on sintered glass 3 (porosity <100 microns), triturated and then washed with 50 ml of ethanol 96 ° for 30 minutes and then filtered on sintered glass 3.

Finally, the precipitate is dried in an oven (40 ° C., 15 hours) and then ground to a fine powder.

The production yield of low molecular weight oligosaccharides is 80%.

Analysis Analysis of constituent sugars

The hydrolysis of the oligosaccharides is carried out with 2N TFA. The constituent sugars are analyzed by ion chromatography (HPAEC) with reference to monosaccharide databases (glucuronic acid, Rhamnose) for identification.

Determination of molecular weight

Analysis by size exclusion chromatography (SEC MALLS) with the use of 2 OHPAK chromatography columns SB 804 and 806 HQ (Shodex). Determination of sulfate groups

The determination of the sulfate level is carried out according to the turbidimetric barium / gelatin method of Dodgson and Price (1962).

EXAMPLE 3 Production of oligosaccharides according to the invention continuously and purification by Ultra Tangential Filtration 50 g of powder of algae of Ulva lactuca are dispersed in 1 liter of water at 90 ° C. with vigorous stirring (1000 rpm) during 2 hours. Then 60 ml of 35% H 2 O 2 solution was added to the medium for 1 hour at a flow rate of 1 ml / min at 80 ° C and maintained at pH 5-8 by addition of NaOH (5M). After complete addition of H2O2 (in 1 hour), stirring is maintained for 3 hours (500 rpm) at 80 ° C and pH 5-8. The mixture is then heat-filtered on diatom (50 g) on sintered glass 1. The elimination of the insoluble particles can also be carried out by centrifugation (10000 g, 15 minutes, 22 ° C.).

The filtrate is then concentrated by tangential ultrafiltration on a membrane of 5 kilodaltons until a concentrated extract with 10% dry matter is obtained. The concentrate is finally atomized into a fine powder of oligosaccharides of low molecular weight.

Example 4 Transcriptomic Study Performed 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 0.05% by weight (ie 0.05 g of oligosaccharides per 100 g of cell culture medium).

When both fibroblast cell cultures have reached confluence, their respective total ANs are extracted and then reverse transcribed into respective differentially labeled mono-stranded complementary (cDNA) DNAs (the cDNAs derived from the RNAs extracted from the fibroblasts grown in the control condition are labeled Cy3 cyanine 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 has been demonstrated that the oligosaccharides used in particular cause the overexpression of the FGF1, CCNA2 and RHAMM genes and the under-expression of the ADAMTS, TMEM27 and ITH5 genes. All these genes encode the proteins bearing the same names and involved in the metabolism and the establishment of the constituent molecules of the extracellular matrix. Indeed, the FGF1 and CCNA2 proteins allow the mobilization of fibroblasts and induce the stimulation of the biosynthesis, by these cells, of the two main types of constituent molecules of the extracellular matrix (collagens and hyaluronic acid). As for the RHAMM protein, the latter coupled to the hyaluronic acid receptor participates in the fixation and organization of the latter thus allowing the establishment and maintenance of the architecture of the extracellular matrix.

In addition, the under-expression of the ADAMTS and TMEM27 genes induces the decrease in catabolism (degradation) of collagen molecules by inhibiting the activity of Matrix Metalloproteinases (MMPs). Also, the subexpression of the ITH5 gene induces the inhibition of catabolism of hyaluronic acid.

In conclusion, the transcriptomic study has made it possible to demonstrate that the compounds of the present invention allow the establishment and maintenance of the integrity of the extracellular matrix i) by inducing the biosynthesis of the collagen molecules and the hyaluronic acid (thanks to the overexpression of the FGF1 and CCNA2 genes), ii) by inhibiting the degradation of these constituent molecules (thanks to the under-expression of the ADAMTS, TMEM27 and ITH5 genes), iii) by promoting the fixation and the organization of the hyaluronic acid molecules (thanks to the overexpression of the RHAMM gene).

Example 5 In-vitro Study on Human Fibroblasts in Culture

A second in-vitro study performed on human fibroblasts cultured in the absence of oligosaccharides according to the present invention (control condition) or in the presence of three concentrations of these molecules 0.01% - 0.02% and 0.05% by weight. weight (respectively 0.01g - 0.02g and 0.05g - of oligosaccharides per 100g of culture medium) (conditions treated).

The fibroblast cultures are established from skin of human foreskins harvested during circumcisions and are amplified in RPMI 1640 culture medium supplemented with fetal calf serum, L-glutamine and gentamicin. The tests are performed on fibroblasts, between the 2 ^nd and 4 ^th passage, in order to ensure reproducibility between different experiments. The fibroblasts are seeded in 25 cm ² dishes at a rate of 10 ⁶ cells per milliliter. They were then incubated for 24 hours in the absence (control condition) or in the presence of three concentrations of oligosaccharides of the present invention (conditions treated). ^• Bachelor Apr 24 hour incubation, fibroblasts were recovered by centrifugation and t are measured:

the Matrix Metalloproteinases MMP1 and M M P 3 by immunohistochemical analysis using the ELISA technique,

hyaluronic acid by immunohistochemical analysis using the ELISA technique,

total collagens by chromatographic assay. Indeed, once centrifuged, the pellets containing fibroblasts are digested with collagenase (1 mg / cell pellet) in acetic acid 0.5 ml / 1 for 24 hours at 4 ° C. After centrifugation at 10000 g, the collagens are precipitated with 1M sodium chloride, the precipitate is resuspended and then dialyzed. The primary amino acids are derived by ophthaldehyde acid (OPA) thus eliminating their interference. Hydroxyproline and proline are then derived by NBD-Cl by coupling of the amino groups to NBD-Cl. The NBD-Hyp is separated and identified by reverse phase HPLC. For the development of the separation of amino acid derivatives, a coupling to NBD-C1 of a standard containing hydroxyproline is first performed. Hydroxyproline is assayed by fluorescence measurement after separation by reverse phase HPLC.

All tests are performed in triplicate and the results are presented in the table below. The results presented in this table are calculated in comparison with the control condition (untreated).

The results obtained show that the oligosaccharides of the present invention induce an increase in the biosynthesis of the main constituent molecules of the extracellular matrix, namely collagens and hyaluronic acid. Also, these results demonstrate that these oligosaccharides of the present invention induce a decrease in the production of Matrix Metalloproteinases (MMP1 and MMP3) responsible for the degradation of collagen molecules.

These oligosaccharides thus cause biosynthesis and prevent the degradation of the main constituent molecules of the extracellular matrix.

The compounds of the present invention can therefore be used to induce the biosynthesis and / or organization of the constituent molecules of the extracellular matrix, and / or to reduce their degradation, thus enabling the establishment and maintenance of the the extracellular matrix and therefore to prevent and / or treat the signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, of the alteration of the tissue regeneration, tissue laxity, alteration of cutaneous and / or capillary microcirculation, alteration of cutaneous detoxification, loss of uniformity, brightness and shine of dye (cutaneous and / or capillary), the alteration of the cutaneous surface texture (appearance of wrinkles, puffiness, dry skin, etc.) and / or capillary (brittle hair), the alteration of the arch cutaneous and / or capillary itecture (inducing in particular hair loss).

Example 6: Clinical Study on Human Volunteers A double-blind clinical study was carried out on 18 human volunteers (18 Caucasian women aged 56 ± 6 years) in order to estimate, at the level of the face, the effects remodeling and re-sculting oligosaccharides according to the present invention. During 56 days, each volunteer applied, on one of its hemispheres, a cosmetic formulation containing 2% by weight of oligosaccharides according to the present invention (ie 2 g of oligosaccharides according to the present invention per 100 g of formulation) ; on its other hemi-face a placebo cosmetic formulation (formulation of identical composition but not containing oligosaccharides according to the present invention). The distribution of the hemispaces was performed in a randomized manner. The remodeling and re-sculting effects in the face were evaluated by two complementary techniques:

the projection of fringes to study the reduction of sagging skin in the jowls

and ballistometry to analyze skin firmness and elasticity.

The projection of fringes allows a direct and immediate acquisition of the morphology of the face in three dimensions and thus to evaluate its volume. This technique allows an evaluation of the reduction of the relaxation of the oval of the face (remodeling effect, re-scultant). The three-dimensional information is calculated by the deformation of the fringes projected on the surface of the face and recorded by a digital camera. The analysis consists of a superposition of images taken at different measurement times. Thus, a region of interest is defined at the level of sagging skin. This zone is located in the same place for all measurement times in the same subject. It makes it possible to calculate a volume between this zone of interest and its projection on a reference plane. The parameter analyzed is the relative volume of the jowls, expressed in mm ³ . A decrease in this parameter is synonymous with a remodeling effect of the tested product.

The second technique used, ballistometry, is based on the use of a mass impacting the surface of the skin. The biomechanical properties of the skin are then measured through skin-mass interactions. In other words, a vibrational motion is imposed on the skin and is recorded for 3 seconds then translated into electrical signals, then quantified and finally evaluated in terms of amplitude. The measurements therefore make it possible to evaluate skin firmness and elasticity.

These measurements are made using a template allowing accurate repositioning of the measuring device for all measurement times. The measured parameters are:

the indentation (in mm) corresponding to the peak recording height of penetration of the tip of the probe into the skin during the first impact. The indentation shows the resistance of the skin during the penetration of the probe, and thus allows an estimate of the firmness of the skin. The lower the indentation, the firmer the skin. Thus, a decrease in indentation reveals a firming effect;

alpha (without unit) measures the damping energy. The alpha describes the rebound energy of the probe and therefore the density of the skin. More energy is strong, the more the skin is dense. Thus, an increase in alpha shows a denser skin;

the area (in mm ² ) corresponding to the area under the curve, ie the area between the rebound profile and the x-axis. The area represents the bounces of the probe and therefore the softness (the flaccid aspect) of the skin: its relaxation. The smaller the area, the firmer the skin. Thus, a decrease in the area reveals a reduction in relaxation (increased firmness).

All results obtained are shown in the table below. The results presented in this table are calculated in comparison with the measurements recorded at the start of the study (tO), before any application of cosmetic formulations.

The results obtained show that the oligosaccharides of the present invention induce:

a remodeling effect (decrease of the volume of relaxation of the lower cheeks, measured by the technique of projection of fringes),

a resuscitating effect, inducing a firming effect (decrease of the indentation parameter, measured by ballistometry), by improving the cutaneous density (increase of the alpha parameter, measured by ballistometry) and by decreasing the cutaneous relaxation (decrease of the parameter area, measured by ballistometry).

Claims

1. Oligosaccharide of general formula (I)

in which :

as well as its pharmaceutically acceptable salt.

An oligosaccharide according to claim 1, characterized in that the substituents R ¹ to R ⁴ are independently selected from each other and independently for each rhamnose unit and / or glycuronic acid as hydroxy, alkyloxy, acyloxy, sulfate or phosphate; or a group -OCH ₂ R ^a .

3. Oligosaccharide according to claim 2, characterized in that the substituents R ¹ to R ⁴ are independently selected from each other and independently for each rhamnose unit and / or glycuronic acid as a hydroxy group or a sulphate group.

4. Oligosaccharide according to one of claims 1 to 3, characterized in that 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.

An oligosaccharide according to claim 4, characterized in that n is chosen such that the molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 30,000 Daltons.

6. An oligosaccharide according to claim 4, characterized in that n is chosen so that the molecular weight is greater than or equal to 5,000 Daltons and less than or equal to 25,000 Daltons.

7. Process for preparing an oligosaccharide according to one of claims 1 to 6 comprising the following steps:

d) filtration or centrifugation at room temperature;

e) concentration under reduced pressure;

f) the molecules of low molecular weight can be purified by:

tangential ultrafiltration then atomization into fine powder;

- Alcohol precipitation, filtration, washing and drying of the oligosaccharides obtained.

8. Cosmetic use of one or more oligosaccharides according to any one of claims 1 to 6 to induce the biosynthesis and / or the organization of the constituent molecules of the extracellular matrix, and / or to reduce their degradation.

9. Cosmetic use according to claim 8 for the prevention and / or treatment of the intrinsic and / or extrinsic signs of skin and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, the alteration of tissue regeneration, tissue relaxation, alteration of cutaneous and / or capillary micro-circulation, alteration of cutaneous detoxification, loss of uniformity, radiance and shine of cutaneous and / or capillary tinge, alteration of cutaneous surface texture, in particular the appearance of wrinkles, puffiness and cutaneous dryness, the alteration of the hair texture, in particular the brittle hair, and the alteration of the cutaneous and / or capillary architecture, inducing in particular the fall hair.

10. Cosmetic composition comprising, as active principle, one or more oligosaccharides according to one of claims 1 to 6.