WO2011117547A1

WO2011117547A1 - Oligosaccharide extracted from halymenia durvillaei, method for preparing such an oligosaccharide, and cosmetic use of said oligosaccharide

Info

Publication number: WO2011117547A1
Application number: PCT/FR2011/050631
Authority: WO
Inventors: Laurent Rios; Cédric DELATTRE; Jean-Yves Berthon
Original assignee: Greentech
Priority date: 2010-03-25
Filing date: 2011-03-24
Publication date: 2011-09-29
Also published as: FR2957923A1; FR2957923B1

Abstract

The invention relates to a novel oligosaccharide extracted from Halymenia durvillaei, to a method for preparing such an oligosaccharide, and to the cosmetic use of said oligosaccharide, particularly in the field of preventing and/or treating intrinsic and/or extrinsic skin and/or hair aging.

Description

OLIGOSACCHARIDE EXTRACT OF HALYMENIA DURVILLAEI, PROCESS FOR PREPARING SUCH OLIGOSACCHARIDE AND COSMETIC USE THEREOF

The present invention relates to a novel oligosaccharide extracted from Halymenia durvillaei, a process for preparing this oligosaccharide, and its cosmetic use, in particular in the context of the prevention and / or treatment of intrinsic cutaneous and / or capillary aging and / or extrinsic.

Halymenia durvillaei is a red alga belonging to the family Cryptonemiaceae0 (Halymeniaceae Order Cryptonemiales). It is abundantly distributed on the coasts of several islands near the Malagasy coasts but also in a large part of the Indian Ocean.

Various extraction methods carried out on Halymenia durvillaei have been described in the prior art. These processes allow the extraction of certain polysaccharides contained in Halymenia durvillaei but do not allow their degradation in oligosaccharides of low molecular weight.

Thus, Briones A.V. et al. describe in The Philippine Journal of Science, 2000, Vol.129 (1) 15-17 a process for the preparation of sulfated polysaccharides derived from carrageenan. These polysaccharides are obtained from Halymenia durvillaei by heating at temperatures ranging from 60 to 100 ° C followed by extraction with isopropyl alcohol. No specific use of the extract thus obtained is described in this document.

Similarly, Japanese Patent Applications JP 2001/354518, JP 2001-172130 and JP5 2003-160486 each describe an extraction process carried out on Halymenia durvillaei and consisting of mixing the algae with water and stirring at room temperature. ambient, and then to concentrate and lyophilize the aqueous extract obtained. The extracts thus obtained consist of polysaccharides but no oligosaccharides, the operating conditions used being too "mild" to degrade the polysaccharides extracted into oligosaccharides.

Intrinsic and / or extrinsic cutaneous and capillary aging, including alteration of cutaneous and / or capillary structures and functions, tissue relaxation, loss of uniformity of hue, alteration of cutaneous surface texture ( appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair), the alteration of the cutaneous and / or capillary architecture (inducing in particular the hair loss) are generally attributed to the process of cell division involving mainly cellular proliferation and differentiation processes. Thus, in the context of the development of new cosmetic active agents useful for combating intrinsic and / or extrinsic cutaneous and capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, tissue relaxation, the loss of uniformity of the hue, the alteration of the cutaneous surface texture (appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair), the alteration of the cutaneous and / or capillary architecture (inducing in particular the hair loss), it is desirable to identify compounds that slow down cell division.

Now, it has now been found, quite surprisingly, that the radical degradation of the polysaccharides extracted from Halymenia durvillaei made it possible to obtain oligosaccharides of low molecular weight, that is to say predominantly less than or equal to 100,000 Daltons, having an activity on cell division. It has indeed been found totally unexpectedly that the oligosaccharides thus prepared induce a slowing down of cell division, thus delaying intrinsic and / or extrinsic cutaneous and capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions. , tissue relaxation, loss of uniformity of the hue, alteration of the cutaneous surface texture (appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair), the alteration of the cutaneous and / or capillary architecture (inducing in particular hair loss).

This is all the more surprising since the polysaccharide extracts obtained by the conventional extraction methods on Halymenia durvillaei have never been described as having any activity on cell division. Thus, Japanese Patent Application JP 2001-354518 describes the use of polysaccharide extracts of Halymenia durvillaei as an antioxidant active principle, which makes it possible to promote epidermal keratinization or to promote the synthesis of dermal collagen.

Japanese patent applications JP 2001-172130 and JP 2003-160486 describe the use of polysaccharide extracts of Halymenia durvillaei as an active ingredient for the protection or improvement of the quality of the hair or as a promoter of the grows hair. Such activity does not presage any activity on cell division, quite the contrary. Indeed, the hair is constantly renewed cyclically, each cycle comprising an anagen phase, a catagen phase and a telogen phase. In the course of life, the number of Pilaris cycles is limited on average to 20 to 30 cycles. Thus, a hair growth promoting agent will accelerate the anagen phase. In contrast, a compound slowing down cell division, i.e., reducing cellular proliferation and differentiation, will extend the duration of the anagen phase and, thereby, the total duration of the hair cycle. This lengthening of the hair cycle thus decreases the frequency of the cycles and preserves in fact the "pilaire youth capital", that is to say the number of cycles remaining. An asset allowing a slowing down of cell division will therefore have a more effective and lasting action than an active agent simply acting on the growth of the hair.

The subject of the present invention is therefore an oligosaccharide of general formula (I):

in which :

R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ , which are identical or different, are chosen independently for each galactose unit as being a hydrogen atom or an SO ₃ ^"group , it being understood that 10 to 50 % of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are chosen as SO ₃ ^" ; and

n is chosen so that the molecular weight is less than or equal to 100,000 Daltons;

as well as its pharmaceutically acceptable salt.

The low molecular weight oligosaccharides according to the present invention are novel and their chemical structure could be determined by proton NMR analyzes; HPLC analyzes to define the constituent sugars; GPC analyzes to define the type of glycosidic linkage and colorimetric assays to quantify the percentages of grafted sulfate. The compounds according to the present invention make it possible to act favorably on cell division by slowing down it and can therefore be used in cosmetics in the context of the prevention and / or treatment of intrinsic signs of skin and / or capillary aging and / or extrinsic. It has indeed been discovered quite surprisingly in a transcriptomic study performed on human fibroblasts cultured in vitro in the absence or in the presence of oligosaccharides according to the present invention that they in particular, lead to the overexpression of the LM02, TCF7 genes and the under-expression of the EDN1, MAPK3, KIF20A genes coding for the proteins bearing the same names and involved in the processes of cell division and proliferation. Indeed, the LM02 and TCF genes are described as being negative modulators of cell division and proliferation. Their overexpression amplifies the decrease or even the inhibition of cell division and proliferation. Conversely, the MAPK3, EDN1 and KIF20A genes are described as stimulators of cell division and proliferation. Their under-expression therefore leads to an absence of stimulation of cell division and proliferation. The resultant of these gene over- and subexpressions is thus a negative modulation of cell division and proliferation.

The transcriptomic study carried out also unexpectedly revealed that, in parallel with this modulatory effect on cell division and proliferation, the compounds according to the present invention did not induce any action, i.e., none. stimulation and nor any inhibition, on the apoptotic function at the origin of cell death. Consequently, the compounds according to the present invention will not risk blocking the apoptotic process if it proves necessary to eliminate damaged or drifting cells to a tumor stage. In addition, the compounds according to the present invention do not induce the apoptotic process in an anarchic manner. It has therefore been demonstrated that the compounds according to the present invention act on the reduction of cell division and proliferation and thus on the preservation of cellular youth capital, without causing negative effects via stimulation or inhibition of apoptotic function. In the context of the present invention:

the expression "molecular weight" used hereinafter 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; - A "concentration under reduced pressure" step means a step for evaporating the water contained in the extract.

Preferably, the subject of the present invention is a compound of formula (I) as defined above, and in which the following characteristics are chosen alone, or in combination:

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 or equal to 10,000 Daltons; and or

- 20% to 50%, preferably 30%> to 50%>, more preferably 40 to 50%> R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are selected as SO ₃ ^"

On the other hand, the present invention also relates to a process for the preparation of oligosaccharide as defined above by radical degradation of polysaccharides extracted from Halymenia durvillaei, comprising the following steps:

a) dissolving the polysaccharides extracted from Halymenia durvillaei in water at a temperature ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 6 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 6 to 8;

c) while stirring, the temperature of the solution ranging from 20 ° C to 100 ° C, the pH of the solution ranging from 6 to 8;

d) filtration or centrifugation at room temperature;

e) concentration under reduced pressure;

f) precipitation of low molecular weight oligosaccharides; and

g) filtration, washing and drying of the precipitate 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 60%> to 70%>. The oligosaccharides thus obtained allow slowing down of cell division and can therefore be used in cosmetics in the context of the prevention and / or treatment of signs of intrinsic and / or extrinsic cutaneous and / or capillary aging.

Step a) of the degradation process according to the present invention consists in the dissolution of the extracts of Halymenia durvillaei in water. Preferably the dissolution 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 dissolution is carried out at a temperature of 20 ° C to 100 ° C, preferably at a temperature of 40 ° C to 80 ° C, more preferably at a temperature of 50 to 70 ° C.

During the dissolution, the pH of the solution can vary from 6 to 8, preferably the pH of the solution varies from 7 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 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 Halymenia durvillaei extract and the added hydrogen peroxide is 1/1. The added hydrogen peroxide will preferably be selected as 30% H ₂ 0 ₂ .

The addition of hydrogen peroxide is done gradually. Preferably, the addition of hydrogen peroxide will be continuous over a period ranging from 30 minutes to 3 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 80 ° C., more preferably at a temperature ranging from 50 to 70 ° C. ° C.

During the addition of hydrogen peroxide, the pH of the solution can vary from 6 to 8, preferably the pH of the solution varies from 7 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 Halymenia durvillaei / hydrogen peroxide.

Preferably the agitation is carried out at a speed ranging from 100 to 2,000 rpm, more preferably at a speed of from 300 to 1,000 rpm.

Preferably, agitation will be maintained for a period of from 30 minutes to 4 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 80 ° C, more preferably at a temperature of from 50 to 70 ° C.

During stirring, the pH of the solution can vary from 6 to 8, preferably the pH of the solution varies from 7 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 precipitation of low molecular weight oligosaccharides. To do this, any means known to those skilled in the art can be used. Preferably, this precipitation will be carried out using an alcohol which may for example be chosen as isopropanol or ethanol.

Stage g) of the degradation process according to the present invention consists in filtering, washing and drying the precipitate obtained. To do this, any means known to those skilled in the art can be used.

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 can 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 present invention also relates to a process for the preparation of oligosaccharide as defined above from Halymenia durvillaei comprising the following steps:

1) dispersion of the alga Halymenia durvillaei in water at a temperature ranging from 20 ° C to 100 ° C;

2) stirring, the temperature of the solution ranging from 20 ° C to 100 ° C;

3) filtration or centrifugation of the mixture at a temperature ranging from 20 ° C to 100 ° C;

4) precipitation of polysaccharides extracted from Halymenia durvillaei;

5) radical degradation of the polysaccharides thus obtained according to the process described above.

Step 1) of the extraction process according to the present invention consists of the dispersion of the alga Halymenia durvillaei in water at a temperature ranging from 20 ° C to 100 ° C. Preferably, the dispersion is carried out at a temperature of from 60 ° C to 100 ° C, more preferably from 80 ° C to 100 ° C.

Step 2) of the extraction process according to the present invention consists of stirring the solution. Preferably, stirring is maintained for a period of from 30 minutes to 4 hours. More preferably, the prepared solution is stirred for 1 to 2 hours.

Preferably stirring is carried out at a speed ranging from 300 to 2,000 rpm, preferably from 800 to 1,500 rpm.

The stirring is carried out at a temperature ranging from 20 ° C. to 100 ° C. Preferably, the agitation is carried out at a temperature of from 60 ° C to 100 ° C, more preferably from 80 ° C to 100 ° C.

Step 3) of the extraction process according to the present invention consists of a filtration or centrifugation step intended to eliminate the algae present in solution. To proceed to filtration, any means known to those skilled in the art can be used. Preferably, filtration of the medium will be carried out on diatom by frontal filtration.

Step 4) of the extraction process according to the present invention consists in the precipitation of the polysaccharides thus extracted from Halymenia durvillaei. To do this, any means known to those skilled in the art can be used. Preferably, this precipitation will be carried out using an alcohol which may for example be chosen as isopropanol or ethanol.

The extraction method carried out on Halymenia durvillaei according to the present invention can be carried out continuously, that is to say that the degradation step (step 5) is carried out immediately following the filtration or centrifugation of the extract (step 4). In this case, the degradation (step 5) is performed on the filtrate (or supernatant). An alternative to this continuous process consists of filtering, washing, drying and optionally grinding the precipitate obtained following the filtration or centrifugation step of the extract (step 4), and then to carry out the degradation (step 5 ) on the dry extract. To do this, any means known to those skilled in the art can be used.

The oligosaccharides of low molecular weight according to the present invention can therefore be used to slow down the frequency of cell division, and thus promote the maintenance of the integrity of cellular and / or molecular structures, in particular telomeres, which degrade as and when cell division cycles thus participating in the process of intrinsic and / or extrinsic cutaneous and capillary aging. Thus, another subject of the present invention relates to the use of one or more low molecular weight oligosaccharides according to the present invention as an agent for preventing and / or treating 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 relaxation of the tissue, the loss of the uniformity of the hue, the alteration of the cutaneous surface texture (appearance of wrinkles, pockets, etc.). ) and / or capillary (brittle hair), 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 according to the present invention. The compositions according to the present invention may be formulated in any dosage form suitable for their administration such as cream, gel, lotion, milk, oil-in-water emulsion or water-in-oil, solution, ointment, spray, body oil, aftershave, soap, lip stick, stick and pencil for makeup.

The compositions according to the present invention contain one or more low molecular weight 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%. 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 generally employed in the cosmetic art. 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 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 than macro-, micro- and nanocapsules and also be absorbed on powdery organic polymers, talcs, bentonites and other mineral supports.

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 antimicrobial activity, the antioxidant activity, the antiradical activity, the healing effect, the tensor effect, the anti-wrinkle effect, the chelating activity, the complexing and sequestering activity, the soothing effect, the anti-radical effect, -cernes, 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 the fall of the hair, the effect capillary gain, the depilatory activity, the activity limiting the regrowth of hair, the activity participating in cell renewal, the activity modulating the inflammatory response, the activity involved in maintaining the oval of the face, but also the protection solar, anti-ir activity laughing, cellular nutrition, cellular respiration, anti-seborrhoeic treatments, cutaneous tonicity, protection of the hair.

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 to be used daily by applying them 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 or the hair for prolonged periods of time does not imply any systematic effect. The compositions according to the present invention can therefore be used to slow down the cell division, causing the degradation of cellular structures and or molecules, including telomeres, and thus participate in the process of intrinsic and / or extrinsic cutaneous and capillary aging. Thus, another subject of the present invention relates to the cosmetic use of a composition according to the present invention for preventing and / or treating signs of intrinsic and / or extrinsic cutaneous and / or capillary aging, in particular the alteration of the structures and cutaneous and / or capillary functions, tissue relaxation, loss of uniformity of hue, alteration of cutaneous surface texture (appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair) , the alteration of the cutaneous and / or capillary architecture (inducing in particular the fall of the hair).

In view of the effect on cell division during the use of the compositions according to the present invention, they may especially be used in the context of the prevention and / or treatment of the alteration of structures and functions. cutaneous and / or capillary, tissue relaxation, loss of uniformity of hue, alteration of cutaneous surface texture (appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair), l alteration of the cutaneous and / or capillary architecture (inducing in particular the fall of the hair).

The present invention is illustrated in a nonlimiting manner by the following examples.

Example 1 Extraction

The extraction of the polysaccharides is carried out by dispersing 30 grams of algae (Halymenia Durvillaei) in 1 liter of water at 90 ° C. with vigorous stirring (1,000 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 (10,000 g, 15 minutes, 22 ° C.)). The Halymenia Durvillaei extract is then precipitated in 3 liters of isopropanol (at 4 ° C.) with stirring (500 rpm) for 2 hours. The precipitate is recovered on a filter cloth (porosity 500 μιη) and then washed with 100 ml of isopropanol, drained and dried (35 ° C, 12H). Finally, the precipitate is crushed and sieved over 500 μm to obtain a fine powder of polysaccharides extracted from Halymenia Durvillaei. Production of oligosaccharides of low molecular weight

40 g of polysaccharides (Halymenia Durvillaei extracts) thus obtained are dissolved in 1 liter of water (60 ° C., pH 7-7.5) with vigorous stirring (1,500 rpm). 133 ml of a 30% H 2 O 2 solution for 1 hour was added at a rate of 2.22 ml / min at 60 ° C and maintained at pH 7-7.5 by continuous addition of NaOH (5M).

After complete addition of ΓΗ2Ο2, the stirring is maintained for an additional hour (500 rpm) at 60 ° 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 7 volumes of 96% ethanol at 4 ° C. with stirring (500 rpm) for 1 hour.

The precipitate is recovered by filtration on Fritted glass 2 (porosity <100 microns), 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., 1 ° C.). night) and then ground into fine powder.

The production yield of low molecular weight oligosaccharides is 60-70%.

Analysis of oligosaccharides

Analysis of constituent sugars

Oligosaccharides are hydrolysed to TFA IN and constituent sugar analyzes are carried out by ion chromatography (HPAEC) with reference to monosaccharide databases for identification.

Determination of sulfate%: by colorimetric BaC12 / gelatin assay using dextran sulfate as a reference. 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).

oligosaccharides

EXAMPLE 2 Production of oligosaccharides continuously 30 g of algae (Halymenia Durvillaei) are dispersed in 1 liter of water at 90 ° C. with vigorous stirring (1,000 rpm) for 2 hours. The mixture is hot-filtered on diatom (50 g) on sintered glass 1. The elimination of the algae can also be carried out by centrifugation (10,000 g, 15 minutes, 22 ° C.).

The Halymenia Durvillaei polysaccharide extract is stirred vigorously (1,000 rpm) at 60 ° C. and pH 7-7.5. 33 ml of 30% H 2 O 2 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 H2O2 (in about 1 hour), stirring is maintained for 1 hour (500 rpm) at 60 ° C and pH 7-7.5.

The concentrate is then precipitated in 7 volumes of 96% ethanol at 4 ° C. with stirring (500 rpm) for 1 hour. The precipitate obtained is recovered by filtration on Sintered glass 2 (porosity <100 microns), 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, 1 night) and then ground into a fine powder. The production yield of low molecular weight oligosaccharides is 60-70%. Analysis of oligosaccharides

Analysis of constituent sugars

Determination of sulfate%: by colorimetric BaC12 / gelatin assay using dextran sulfate as a reference.

Determination of molecular weight

Composition Weight

(% mass) molecular

(Majority)

Gay S03-

Oligosaccharides 50 44 <10kDa

Example 3 Activity on division and cell proliferation

In vitro studies were performed on human keratinocytes in culture in absence (control condition) or in the presence of different concentrations of test molecules. The evaluation of the effect of the low molecular weight oligosaccharides according to the present invention on the appearance of apoptotic cells and the lengthening of the cell cycle is carried out by flow cytometry after immunofluorescence labeling of Bromodeoxyuridine (BrdU).

Immunofluorescence staining of the incorporated BrdU and flow cytometric analysis provide a high resolution technique for determining the frequency and nature of individual cells that synthesized DNA.

In this method, BrdU (a DNA precursor thymidine analogue) is incorporated into the newly synthesized DNA by cells entering and progressing through the S phase (DNA synthesis) of the cell cycle. Embedded BrdU is labeled with a fluorescent anti-BrdU specific antibody. The rate of BrdU-associated cells is then measured by flow cytometry. A second staining with a probe that binds the total DNA, such as 7-amino-actinomycin D (7-AAD), is coupled with immunofluorescent BrdU labeling. With this combination, two-color flow cytometry analysis allows the enumeration and characterization of cells that actively synthesized DNA (BrdU incorporation) as a function of their position in the cell cycle (GO / 1 phase). , S or G2 + M defined by the 7-AAD labeling intensity). Extended exposures of BrdU cells make it possible to identify and analyze the different cell fractions actively in the cycle or, conversely, not in the cell division phase.

The study made it possible to demonstrate that even for low concentrations (0.02 g of low molecular weight oligosaccharides according to the present invention per 100 ml of cell culture medium (ie at 0.02% by weight), the division and cell proliferation is strongly slowed compared to the control condition (in the absence of treatment), because there is a brake on the progression of cells in the cell cycle The rate of cells in the G0 / G1 phase (quiescent cells therefore not in the course of cell division) is more important in the treated condition than in the control condition:

76.9% of the cells treated for 1 hour are in the G0 / G1 phase, compared to 58.5% of the untreated cells, and

57.8% o cells treated for 24 hours are in phase G0 / G 1, against 37.3%> untreated cells.

This is to the detriment of the cells in phase S (cells synthesizing DNA therefore during cell division) which are much less numerous than in the control condition:

7.8% o cells treated for 1 hour are in phase S, against 17.7% of the untreated cells, and

16% of the cells treated for 24 hours are in phase S, compared to 51.7% of the untreated cells.

The study also showed that the effect of the oligosaccharides tested on division and cell proliferation is reversible. Indeed, less than 24 hours after stopping treatment, the cells found a progression in the cell cycle identical to that of the control cells. This result therefore makes it possible to envisage a use in the cosmetics sector. Moreover, the same study was carried out in the absence and in the presence of an inducer of apoptosis (hydrogen peroxide), this in order to verify that the treatment of the cells with the oligosaccharides did not prevent the setting up of the apoptotic process when it was necessary.

This study showed that even for high concentrations of oligosaccharides (1 g of low molecular weight oligosaccharides according to the present invention per 100 ml of cell culture medium (ie 1% by weight), there was no inhibition or stimulation of the establishment of the apoptotic function.

In addition, a study carried out on reconstituted epidermis made it possible to show that for concentrations of 0.1 g and 0.5 g of low molecular weight oligosaccharides according to the present invention per 100 ml of culture medium (ie 0, 1% and 0.5% by weight), there is a decrease in the proliferation and differentiation of keratinocytes, which results in:

by an accumulation, at the level of the basal lamina, of keratinocytes in the quiescent phase (phase G0 / G1) respectively of 15% and 17% compared to the control condition (without treatment), and

by an increase in the epidermis thickness of 9% and 12%, respectively, relative to the control condition correlated with an increase in cell cohesion.

As a result, there is an improvement in the cutaneous barrier function and preservation of the cutaneous architecture. All these results show that the oligosaccharides according to the present invention induce a slowing of the progression within the cell cycle without inhibiting the apoptotic process if it is to be put in place. This slowing down of cell division and proliferation makes it possible to preserve cellular youth capital by preserving in particular the integrity of telomeres (ends of chromosomes degrading at each cell division).

These properties are particularly useful in cosmetics in the prevention and / or treatment of intrinsic and / or extrinsic signs of skin and / or capillary aging, in particular the alteration of cutaneous and / or capillary structures and functions, tissue relaxation, loss of uniformity of the hue, the alteration of the cutaneous surface texture (appearance of wrinkles, pockets, etc.) and / or capillary (brittle hair), the alteration of the cutaneous and / or capillary architecture .

Claims

1. Oligosaccharide of general formula (I):

in which :

as well as its pharmaceutically acceptable salt.

2. Oligosaccharide according to claim 1, 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 100,000 Daltons.

3. Oligosaccharide according to claim 2, 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 10,000 Daltons.

4. Oligosaccharide according to one of claims 1 to 3, characterized in that 20%> to 50%) of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are chosen as SO ₃ ^" .

5. Oligosaccharide according to claim 4, characterized in that 40%> to 50%> of the groups R ¹ , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are chosen as SO ₃ ^" .

6. Process for preparing an oligosaccharide according to one of claims 1 to 5 by radical degradation of polysaccharides extracted from Halymenia durvillaei comprising the following steps:

d) filtration or centrifugation at room temperature;

e) concentration under reduced pressure;

f) precipitation of low molecular weight oligosaccharides; and

g) filtration, washing and drying of the precipitate obtained.

7. Process according to claim 6, characterized in that step a), b) and / or c) is carried out at a temperature ranging from 40 ° C to 80 ° C.

8. Method according to claim 6 or 7, characterized in that for step a), b) and / or c), the pH of the solution varies from 7 to 7.5.

9. Method according to one of claims 6 to 8, characterized in that the stirring is maintained for a period of from 30 minutes to 3 hours.

10. Method according to one of claims 6 to 9, characterized in that the precipitation of low molecular weight oligosaccharides is carried out using an alcohol

11. The process for preparing an oligosaccharide according to one of claims 1 to 5 by extraction carried out on Halymenia durvillaei comprising the following steps:

2) stirring, the temperature of the solution being from 20 ° C to 100 ° C;

3) filtration or centrifugation of the mixture at a temperature ranging from 20 ° C. to 100 ° C.

4) precipitation polysaccharides extracted from Halymenia durvillaei; 5) radical degradation of the polysaccharides thus obtained according to the process according to one of claims 6 to 10.

Process according to claim 11, characterized in that step 1), 2) and / or 3) is conducted at a temperature of from 60 ° C to 100 ° C.

13. The method of claim 1 1 or 12, characterized in that the precipitation of polysaccharides is carried out using an alcohol.

14. Cosmetic use of one or more oligosaccharides according to one of claims 1 to 5 as an agent for preventing and / or treating signs of intrinsic and / or extrinsic cutaneous and / or capillary aging.

15. Cosmetic composition comprising, as active ingredient, one or more oligosaccharides as defined in one of claims 1 to 5.