EP1631594A1

EP1631594A1 - Novel agent for stimulating the release of beta-endorphins, cosmetic and/or dermatological compositions containing same and uses thereof

Info

Publication number: EP1631594A1
Application number: EP03718827A
Authority: EP
Inventors: Sophie Leclere; Jean-François MOLINA; Laurent Lassalle; Jean-Luc Philbe
Original assignee: Societe La Biochimie Appliquee SOLABIA SAS
Current assignee: Societe La Biochimie Appliquee SOLABIA SAS
Priority date: 2003-02-04
Filing date: 2003-02-04
Publication date: 2006-03-08
Also published as: WO2004078789A1

Abstract

The invention relates to the field of essential goods, particularly the fields of cosmetics and pharmaceutics and, more specifically, dermatology. In particular, the invention relates to novel active compounds which stimulate the release of β-endorphins by keratinocytes, consisting of a polysaccharide and/or a saccharide derivative comprising repeating saccharide units containing at least one rhamnose molecule which is fixed in a branched manner, said sequence preferably comprising mainly rhamnose. The invention also relates to the use of such polysaccharide compounds in order to stimulate the release of β-endorphins by keratinocytes. The invention further relates to cosmetic and/or pharmaceutical compositions, particularly dermatological compositions, containing the inventive active compound which may be used together with or in a mixture with one or more suitable pharmaceutical and/or cosmetic vehicles or excipients. The aforementioned compositions can be used, for example, to care for and/or treat the skin and/or skin appendages.

Description

New agent stimulating the release of β-endorphins, cosmetic and / or dermatological compositions containing them and their applications.

The present invention relates to the field of basic necessities of life and more particularly to the fields of cosmetology, pharmacy and more specifically dermatology.

The present invention relates to active compounds stimulating the release of β-endorphins by keratinocytes, comprising repetitive saccharide sequences and containing at least one rhamnose molecule attached in a branched manner.

More specifically, the subject of the invention is a polysaccharide and / or a derivative thereof comprising repetitive saccharide sequences, containing at least one rhamnose molecule attached in a branched manner, said sequence consisting, as a whole, mainly of rhamnose . Such a polysaccharide and / or derivative is characterized in that it is formed of repeating elements comprising at least three molecules of rhamnose, one of which is branched, and two other sugars of which at least one of the two is a uronic acid . The new active principles according to the invention are characterized in that the repeating elements, mainly containing rhamnose, comprise at least the following constituents, and correspond to the general formula I:

in which Rli is a rhamnose molecule, Rh * is a rhamnose molecule linked in a branched manner to the neighboring element, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and n is between 1 and 100, preferably from 5 to 65.

The present invention also relates to the use of compounds comprising repetitive saccharide sequences containing at least one molecule of rhamnose attached in a branched manner and preferably containing predominantly rhamnose, as cosmetic and / or pharmaceutical agent, in particular dermatological, stimulating the release of β - endorphins.

The present invention also relates to cosmetic and / or pharmaceutical compositions which contain at least one of these new active compounds, of formula I, optionally in combination or in mixture with one or more excipients or vehicles cosmetics and / or pharmaceuticals suitable for application to the skin, mucous membranes or integuments.

Such compositions relate in particular to the care and / or treatment of the skin, including the lips and integuments (nails, hair, hair, etc.).

Β-endorphins are neuropeptides derived from the pro-opiomelanocortin (POMC) precursor and have already been the subject of numerous studies published in the literature. In particular, the presence of β-endorphins and their μ receptor has been demonstrated within the epidermis, and more particularly within keratinocytes (Bigliardi P.L, et al., 1998). Thus, it has been shown that keratinocytes are both sensitive to many neuromediators, in particular to β-endorphins, synthesized by nerve cells or by other epidermal cells, but are also capable of synthesizing them. This particular link between nervous and epidermal tissues could be explained by their common ectoblastic embryonic origin.

The β-endorphins synthesized by the skin represent more than 50% of the rate of circulating endorphins and are therefore particularly advantageous for attenuating or eliminating painful phenomena.

The release of β-endorphins can be triggered in response to numerous signals, in particular in response to cellular stress, to the secretion of other mediators themselves induced by stress (IL-1, IL-6, TNF, etc. ..) or following significant physical effort. In addition, other mechanisms of cellular release of β-endorphins exist in the absence of cellular stress.

From a physiological point of view, β-endorphins are chemical analgesics naturally produced by the body which, by interacting with opiate receptors, bring the body a feeling of well-being. This sensation is accompanied by an immunomodulatory effect. Recent work has highlighted the numerous interconnections of the epidemic and of the dermis with the immune and nervous systems which are associated with it, respectively through, inter alia, Langerhans cells and nerve endings. Β-Endorphins are also responsible for the initiation of a number of biological reactions that manifest themselves at all levels of the body, for example, by stimulation of the vascular and cardiac system, or by muscle relaxation.

These neuromediators thus constitute real "pleasure" molecules because their release is accompanied by a soothing or relieving effect, locally and generally.

Thus, the development of active principles capable of stimulating, without requiring cellular stress, the release of β-endorphins, is of great interest.

We have already identified, without further details, several extracts of algae or plants capable of inducing the release of β-endorphins. Thus, a cocoa extract (Theobroma cacao), especially in the form of 30/70 titled glycolic extract caffeine and theobromine and marketed under the brand Caobromine ^® (CEP) is likely to trigger a release of β-endorphins. Likewise, in patent FR-A1-2,774,292, an extract of a Cyclotella alga capable of triggering a release of β-endorphins, 16 times greater than that of a control, has been described.

According to the present invention, the Applicant has unexpectedly discovered that polysaccharides and their derivatives, comprising saccharide repeating sequences containing at least one molecule of rhamnose attached in a branched manner, have the property of stimulating the cutaneous release of β-endorphins.

Polysaccharides as well as their derivatives of low molecular weight, of vegetable or bacterial origin, algae extracts or products by synthesis, have many biological activities (immunomodulation, anti-inflammation, ...) and have been used for many years for their potential properties in numerous fields of application, for example as a gelling agent in the food industry, moisturizer, makeup remover, texturing agent, etc. in cosmetics. However, the actual influence of the composition and nature of these polysaccharide active principles and / or their derivatives, on said biological properties, still remained to be determined.

We have already described, in document WO-A 1-0203945, the use of oligosaccharides of 2 to 6 sugars containing at least two galactose units, in particular those extracted from the Tephrosia plant in a cosmetic and / or dermatological composition to stimulate the release of β-endorphins from the skin. According to the present invention, new polysaccharides have been identified, quite unexpectedly, capable of stimulating, without cellular stress, the secretion of β-endorphins by keratinocytes. These new cosmetic and / or dermatological agents are, unlike the prior art, made up of repeating elements containing at least one molecule of rhamnose attached in a branched manner, and preferably containing predominantly rhamnose which, in addition, their confer a high affinity for keratinocytes.

Studies have shown that human keratinocytes have a specific affinity for rhamnose, greater than that which they can have for other sugars such as fucose, galactose, N-aeetylglucosamine, due to the presence at the surface of the membranes of keratinocytes, of lectin receptors specific for rhamnose (Cerdan et al., Bio. cell. (1991) 73, 35-42). In patent document WO-A1 -90/11069, the use of rhamnose in a cosmetic and / or dermatological composition has also been described as a specific binding vector for an active compound on the membrane of a keratinocyte by means of 'a ligand-receptor bond. Rhamnose was also known for its ability to limit the biological phenomena leading to allergy of the immune type (patent FR-A1 -2756735) or used, in free form, obtained after hydrolysis of exopolysaccharides, as a flavoring agent or as flavorings (patent FR-A 1-2624522). However, none of these studies previously carried out on rhamnose made it possible to predict such a stimulating effect of rhamnose in the form attached in a branched manner to a saccharide chain, on the cutaneous secretion of endorphins.

According to the invention, the new active ingredients are characterized in that the repeating elements, mainly containing rhamnose, comprise at least the components of general formula I:

in which Rh is a molecule of rhamnose, Rh * is a molecule of rhamnose attached in a branched manner, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and n is between 1 and 100, preferably between 5 and 65.

By repetitive elements containing mainly rhamnose, one understands a branched chain containing, approximately, at least 50% of rhamnose of series D and / or L, as well as its isomers α and / or β. According to the invention, the term “derivatives” groups together the hydrolysis products of the polysaccharides and also the sugar derivatives obtained by chemical modification of the structure of the saccharides according to the invention. For the derivative molecule to be active on the secretion of β-endorphins, it is however necessary that the rhamnose branching and / or the rhamnose molecule attached in a ramified manner to the saccharide chain are not globally altered by the chemical reactions in order that the branched rhamnose remains accessible. For the derivative molecule to be active, the rhamnose molecule initially in the branched position, can quite, following a chemical modification, be located in the terminal position of the derivative molecule.

Thus, according to the invention, the new derivatives are defined as any compound of saccharide nature as long as it contains at least one accessible rhamnose molecule located at the end of the chain or branched and consists mainly of rhamnose. The new derivatives according to the invention then have the same structure and / or are derived from the new polysaccharide according to the invention, which leads to compounds for which n has a lower value. It can therefore particularly be oligosaccharide hydrolysis products. These hydrolysates or fractions can in particular be obtained from a polysaccharide of higher molecular weight, by known degradation techniques, in particular by enzymatic hydrolysis or chemical hydrolysis.

The sugar derivatives obtained by chemical modification can be amino, acid derivatives and saccharide salts and / or predefined hydrolysis products.

Sugar O can in particular be chosen from fucose, galactose, ribose, arabinose, xylose and mannose.

By uronic acid U, is meant any hexose oxidized on its primary alcohol function to carboxylic acid, in particular glucuronic acid, galacturonic acid, mannuronic acid or iduronic acid

According to a particular embodiment of the invention, the branched rhamnose molecule can be fixed by an osidic bond from its carbon 1 to a free carbon of a sugar O or uronic acid U or Rh rhamnose molecules. the saccharide chain, in particular on carbons 2 or 3. According to another particular embodiment, the repeating elements can in particular consist of the sequence of general formula II:

in which Rh is a molecule of rhamnose, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and the ramification of rhamnose on the osm O is done according to an osidic bond (1 → 2) or (l- → 3).

According to a particularly advantageous embodiment, the sugar O is galactose and the uronic acid U is glucuronic acid. Preferably, the sequence has a sequence containing 3 molecules of rhamnose, one of which is branched, 2 molecules of galactose and one molecule of glucuronic acid. A particularly advantageous polysaccharide according to the invention has been identified and is presented in Example 1. According to formula II, n represents a value such that this polysaccharide has a molecular weight of the order of 50,000 daltons. It can be obtained from cultures of Klebsiella type bacteria, in particular Klebsiella pneumoniae and in particular strain 1-714 (N ° CNCM I-2743-National Collection of Culture of Microorganisms) according to a process described below. Advantageously, this polysaccharide has the rhamnose branching on the galactose in position V. It appears from the analyzes presented in Example 1 that this polysaccharide is more particularly made up of the following repeating unit: → 4) - α-L-Rha /? (l → 3) - ^ -D-Gal ^ (l- → 2) - α-L-Rha /? (l → 4) - β-D- GlcpA (l → 3) - [α-L-Rha / > (l → 2)] - α-D-Galp (l— ”. The hydrolysis of this polysaccharide also makes it possible to obtain a mixture of fractions of lower molecular weight, in particular the majority fractions of 5,000 daltons and of 13,000 daltons, possibly purifiable and particularly interesting according to the invention.

According to another particular embodiment, the repeating elements can in particular consist of the sequence of general formula III:

Rh in which Rh is a molecule of rhamnose, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and the branching of rhamnose on rhamnose is done according to an osidic bond (1 → 3). According to a particularly advantageous embodiment, the sugar O is glucose and the uronic acid U is glucuronic acid, preferably according to a sequence containing 3 molecules of rhamnose including one branched, one molecule of glucose and one molecule of acid glucuronic. Such a polysaccharide can in particular be obtained according to the method described below from a culture of bacteria of the Klebsiella planticola type, in particular the strain 1-2743 (N ° CNCM I-2743 - National Collection of Culture of Microorganisms). Advantageously, this polysaccharide, as described in Example 2 and called BEC291, has the rhamnose branching on the rhamnose in position III. It appears from the analyzes presented in Example 2 that this polysaccharide more particularly consists of the following repeating unit: → 3) - β-L- Rha /? (L → 4) - /? - D-Glcp (l → 2) - [α-L-Rha /? (L → 3)] - -L-Rha (1 → 4) - α-D-Glc /? A (ι → -

The hydrolysis of this polysaccharide also makes it possible to obtain a mixture of fractions of lower molecular weight, in particular the majority fraction of 5,000 daltons, optionally purifiable and particularly advantageous according to the invention.

In general, the polysaccharides or saccharide derivatives can be of bacterial or vegetable origin, ... They can be obtained by conventional techniques for producing polysaccharides (chemical synthesis, enzymatic extraction of exopolysaccharides, etc.). According to an advantageous embodiment, the polysaccharides are exopolysaccharides obtained by fermentation of a bacterial strain by producing, of the encapsulated bacteria type, according to a production process such as that detailed in patent FR-B 1-264522. This process is defined in that a strain of Klebsiella type bacteria is cultured in a nutritive medium comprising a carbon source, a preferential nitrogen source and appropriate mineral salts, at a pH of approximately 6 to 8 , at a temperature of about 30 to 35 ° C, with stirring and aeration, for 4 to 12 days. The carbon / nitrogen ratio is advantageously greater than 5 in order to promote the secretion of the polysaccharide. The polysaccharide can then be isolated by subjecting the fermentation at a heat treatment at approximately 70-120 ° C, for 10 minutes to approximately 1 hour, then separating it, for example by centrifuging in the cold. Exopolysaccharides and cellular polysaccharides are found entirely in the clear supernatant phase. If necessary, the polysaccharides can be purified by precipitation by adding a non-solvent organic liquid such as acetone or a lower alcohol such as ethanol or propanol, and separated by filtration or centrifugation before being dried.

The isolated polysaccharides can thus easily be incorporated into a composition, as such or in hydrolyzed form. In this case, the hydrolysis can be carried out before drying by common methods such as acid hydrolysis. It can be carried out using a proton donor usually used, such as hydrochloric acid, at a temperature ranging from 50 to 100 ° C. and for 30 minutes to 4 hours, depending on the desired size of the fractions. The oligosaccharide fractions thus obtained can be recovered and purified if necessary according to conventional methods.

This protocol can be carried out using bacterial strains producing exopolysaccharides rich in rhamnose, in particular encapsulated bacteria. According to a preferred embodiment of the invention, a strain of Klebsiella bacteria, preferably Klebsiella pneumoniae or Klebsiella planticola, is used.

According to the invention, it is possible to use the polysaccharide alone or a mixture of heterogeneous polysaccharides and / or a mixture of their derivatives.

The polysaccharides alone and / or the mixture of polysaccharides and / or their derivatives can also, in the context of the invention, be used as active ingredient in grafted form, in particular by chemical substitution means on the free carboxylic function uronic acid U, on a chemical, biochemical or biological residue which does not modify the stimulating effect of the secretion of β-endorphins. For example, they may be fatty chains containing from 6 to 30 carbon atoms or chains of amino acids. It is also possible to incorporate polysaccharides and / or their derivatives according to the invention in a cyclodextrin.

In addition, it has been discovered that the polysaccharides and / or their derivatives according to the invention have other remarkable properties, in particular of inhibition of the cellular adhesion of neutrophils to keratinocytes (40% inhibition at 2% of the compound described in Example 1) and have a very good affinity for keratinocytes, at least equal to that of rhamnose (60% inhibition of neoglycoprotein binding obtained with 8% of the compound described in Example 1). These two properties have been demonstrated in particular in Examples 4 and 5.

The polysaccharides and / or their derivatives previously defined are therefore particularly suitable for use as a cosmetic and / or pharmaceutical and more particularly dermatological agent, in particular for stimulating the cutaneous release of β-endorphins.

A subject of the invention is therefore also cosmetic and / or pharmaceutical and in particular dermatological compositions comprising, as active principle, at least one polysaccharide compound and / or a derivative as defined above, optionally in combination or in mixture with a or more suitable cosmetic and / or pharmaceutical excipients or vehicles. Said polysaccharides and / or derivatives can be incorporated into these compositions at a concentration varying between 0.001% and 20% by weight, and preferably between 0.01% and 10% by total weight of the composition.

According to a particularly advantageous embodiment, the composition contains, as active principle, Rhamnosoft ^® polysaccharide of Example 1 and / or BEC291 polysaccharide of Example 2.

The compositions according to the invention may be in any form suitable for skin application, in particular in the form of a gel, of an emulsion, for example, an oil-in-water or water-in-oil emulsion, or in the form of a multiple emulsion, with liquid crystals or not. They can also be in the form of aqueous solution, lotion, milk, cream, mask, foam. It is also possible to use them in the form of patches.

According to the invention, the composition can also comprise the usual acceptable pharmaceutical and / or cosmetic excipients. Among these excipients, mention may in particular be made of sun protection agents, in particular for carrying out sun care, hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, gums, resins, surfactants, solvents, fillers such as as rice starch, pigments, preservatives, essential oils, antioxidants, dyes, pigments, pearlescent agents, glitter, perfumes, odor absorbers, pH regulating agents or neutralizing agents, penetrating agents, thickening agents, such as those usually used.

The choice and / or the quantity of the ingredients complementary to the composition will naturally be determined by the choice of physical properties and of the desired consistency for the composition according to the invention. The various adjuvants are used in proportions conventionally used in the field of cosmetics, namely for example from 0.01 to 20% of the total weight of the composition, the complement being constituted by a vehicle such as water or a water-alcohol mixture .

Any other positively influence the release of β-endorphins may also be incorporated in the composition according to the invention, including cocoa extract commercialized under the name ^® Caobromine (CEP).

In addition, the composition may also contain a second compatible active principle, in particular for dermatological use, in order to confer a double property on the composition or in order to limit the uncomfortable sensations linked to the application of this second active principle. As examples of active principles, mention may in particular be made of local antiseptics such as benzoyl peroxide, local anti-acne drugs such as tretinoin, a local antibiotic such as sulfacetamide to reduce the side effects of local irritation, a local anti-inflammatory such as ibuprofen. The second active ingredient is added to the composition in a usually effective therapeutic amount.

The composition according to the invention can be produced in a conventional manner, according to the known methods usually used in the field of cosmetology and / or dermatology.

In the cosmetic field, the compositions can be used for daily care and / or treatment and / or protection of the skin of the body and / or of the face and / or the integuments (nails, hair, hair, etc.). By stimulating the production of β-endorphins, these compositions have soothing and calming properties, in particular skin discomfort which may result, for example, from irritations, aggressions of the skin, in particular by external agents such as certain therapeutic treatments. , climatic agents (wind, cold, sunburn, etc.), waxing, shaving, insect bites ... and thus provide a feeling of well-being. They can therefore be particularly used for the care of sensitive or sensitized skin. Such compositions also provide a local analgesic effect and can also be used in the pharmaceutical and more particularly dermatological field to relieve skin and / or subcutaneous pain, to suppress the unpleasant perception of skin reactions, the feelings of discomfort. the skin which manifests itself in particular in the case of allergies, pruritus, itching, insect bites.

A subject of the invention is therefore also the use of the polysaccharide agents according to the invention for the production of a medicament intended to relieve skin and / or subcutaneous pain, and / or to suppress the sensations of discomfort of the skin (allergies, pruritus, irritations, skin pain linked to burns, stings, chapping, cuts ...) by local application.

According to another aspect, the invention also relates to a cosmetic care process comprising the application to an affected area of the skin and / or integuments of at least one active compound previously defined in a cosmetically acceptable excipient or optionally in the form of one of the cosmetic compositions previously defined.

The compositions according to the invention have no toxicity and do not cause any local intolerance. They are also not allergenic.

The following examples and preparations illustrate the invention without limiting it in any way. In the examples and the preparations, the proportions indicated are percentages in olume / volume.

Example 1: Example of the structure of the repeating unit of the exopolysaccharide produced by Klebsiella pneumoniae 1-714 and called Rhamnosoft ^® :

The composition of Rhamnosoft ^® was studied by the analysis procedure by GC gas chromatography and by gas chromatography coupled with mass spectrometry (GC / MS) of the degradation products after permethylation of the native molecule. The method of degradation of uronic acids by β-elimination after permethylation (described by Aspinall GO et al. Carbohydr Res. 1977 May; 55: ll-9) was used in particular to confirm the position and the nature of the substituents of the galactose residue. I The Rhamnosoft sequence was determined by nuclear magnetic resonance (NMR). With a branched structure, this polymer with a molecular weight of the order of 50,000 daltons therefore has a saccharide sequence comprising three molecules of rhamnose (I, III, VI), two molecules of galactose (II, V), one molecule of acid glucuronic (IV). Rhamnose therefore constitutes 50% of the polysaccharide. The polysaccharide has a rhamnose VI branch on the galactose in position V.

The structure of the repeating unit is in this case:

→ 4) - -L-Rha (l → 3) - β-O-Ga \ p (l → 2) - -L-Rha;? (L → 4) - /? - D-GlcpA (ι ^→ 3) - [α-L-

Rrιa _/ l → 2)] - aO-Galp (l →.

It responds to the formula detailed below:

VI

) In the following tests (Examples 3 to 7), the product according to the invention is used in an aqueous solution of 2.5% (w / w) in dry matter. Example 2: Example of the structure of the repeating unit of the exopolysaccharide produced by Klebsiella planticola 1-2743 and called BEC 291:

The composition of BEC 291 was studied by the analysis procedure by GC gas chromatography and by gas chromatography coupled with mass spectrometry (GC / MS) of the degradation products after permethylation of the native molecule. The method of degradation of uronic acids by β-elimination after permethylation (described by Aspinall GO et al. Carbohydr Res. 1977 May; 55: 11-19) was used. The BEC 291 sequence was determined by nuclear magnetic resonance (NMR).

Of branched structure, this polymer therefore has a saccharide sequence comprising three molecules of rhamnose (I, III, V), a molecule of glucose (II), a molecule of glucuronic acid (IV). This polysaccharide therefore has a rhamnose V branch on the rhamnose in position III.

The structure of the repeating unit in this case is:

→ 3) - yS-L-Rha;? (L → 4) - -D-Glçp (l → 2 [α-L-Rha ^ (l → 3)] - ct-L-Rhap (l → 4) - α-D-

GlcpA (l →.

It responds to the formula detailed below:

Example 3 Study of the effect of the polysaccharides according to the invention containing 50% of rhamnose on the release of β-endorphins by the keratinocytes.

^π Procedure: Normal human keratinocytes are cultured for 48 hours. They are treated for 36 hours with the product to be tested. The supernatants, containing β-endorphin, are collected and stored at -20 ° C until assay. The control of the test is carried out by treating the keratinocytes with the excipient (butylene glycol 70%) at 2%. The assay is carried out with an ELISA kit (spectrophotometric assay at 450 nm). The results are expressed in programs per milliliter of synthesized β-endorphin. Prior cytotoxicity studies were systematically carried out in these examples in order to show that the effects of the product are not linked to a cytotoxic effect. A positive control for the release of β-endorphins was carried out using PMA (Phorbol 12-Myristate 13-Acetate) which, as an agent which attacks keratinocytes, induces the release of β-endorphins by a cellular stress mechanism.

a) Rhamnosoft effect

Rhamnosoft ^® is tested at concentrations of 1% and 5%.

Effect of Rhamnosoft on the production of β-endorphins ((*: validation: mean significantly different compared to the control (p <0.05))

It is apparent from these results that Rhamnosoft ^®, at concentrations of 1 and 5% causes CGCE of β-endorphin secretion by keratinocytes. Preliminary studies have demonstrated the absence of cytotoxicity of this type of product with respect to human keratinocytes, regardless of the concentrations of Rhamnosoft ^® tested.

We can consider that the effect of Rhamnosoft ^® on keratinocytes therefore occurs by a mechanism different from that previously described and independent of cellular stress.

b) Effect of hydrolyzed fractions of Rhamnosoft ^® of 13 kdaltons and 5 kdaltons. The hydrolyzed fractions of Rhamnosoft ^® of 13kdaltons and 5 kdaltons are mainly obtained during the acid hydrolysis of Rhamnosoft "carried out in the presence of hydrochloric acid at a concentration of the order of 0.1 to 2.5 mol / liter, at a temperature from 50 to 100 ° C for 30 minutes and 4 hours respectively. They can be purified if necessary by ethanolic precipitation or by physical methods.

Effect of hydrolyzed fractions of Rhamnosoft, respectively 13 kd and 5 kd, on the production of β-endorphins ((* mean validation significantly different compared to the control (p <0.05))

It is clear from the results that the 13 kd and 5 kd fractions, hydrolysis products of Rhamnosoft ^® , at concentrations of 0.01 and 0.05% cause a significant increase in the release of β-endorphins by keratinocytes. Preliminary studies have shown the absence of cytotoxicity and therefore confirm that the effect of the hydrolyzed fractions of Rhamnosoft ^® is not due to cell stress but rather to an interaction of the saccharide fractions with the keratinocyte receptors

c) Effect of a BEC 291 type polysaccharide.

BEC 291 has been tested at concentrations of 0.001% and 0.02%

Tab 3 Effect of the polysaccharide BEC 291 on the production of β-endorphins ((* mean validation significantly different compared to the control (p <0.05))

It appears that the polysaccharide BEC291 also has the capacity to stimulate the release of β-endorphins by human keratinocytes. Preliminary studies have demonstrated the absence of cytotoxicity with respect to keratinocytes, regardless of the concentrations of BEC 291. Here again, the effect of the polysaccharide BEC 291 on the secretion of β-endorphins is not due to cellular stress but indeed an interaction between the saccharide fractions and the keratinocyte receptors.

d) Effect of FrBEC 291, hydrolyzed fraction of BEC 291 of 5 kd. This FrBEC291 fraction is mainly obtained by acid hydrolysis of BEC 291, carried out in the presence of hydrochloric acid at a concentration of the order of 0.1 to 2.5 mol / liter, at a temperature of 50 ° C to 100 ° C for 4 hours.

Tab 3 Effect of pofysacchm ide BEC 291 on the production of β-endorphins {(* mean validation significantly different from the control (p <0.05))

It appears that the oligosaccharide fraction of BEC291, at all the concentrations tested, induces a strong stimulation of the release of β-endorphins by human keratinocytes. Preliminary studies have also shown that this fraction has no cytotoxic effect. Its effect can therefore only be explained by a mechanism independent of cellular stress, truly linked to an interaction between the saccharide fractions and the keratinocyte receptors.

Furthermore, these tests show that the polysaccharides and / or their derivatives according to the invention, containing a branched rhamnose and sufficiently "rich" in rhamnose, and more particularly containing predominantly rhamnose, stimulate the release of β-endorphins by keratinocytes. All these products devoid of cytotoxicity with respect to keratinocytes act by a mechanism of action which is not linked to cellular stress.

Example 4 Study of the affinity of keratinocytes with respect to polysaccharides according to the invention containing 50% of rhamnose.

Procedure: Experiment 1: The specificity of osidic recognition of keratinocyte membrane receptors was determined using fluorescent neoglycoproteins (NGP). To do this, human keratinocytes were incubated with different types of neoglycoproteins. Bovine Serum Albumin (BSA) was used as the carrier protein. NGP uptake was determined by measuring the fluorescence index.

Experiment 2: Experiment 1 was repeated in the presence of Rhamnosoft ^{® in} order to determine its inhibitory effect on the binding of fluorescent NGPs on human keratinocytes. To do this, human keratinocytes were pre-incubated for 15 min with different concentrations of Rhamnosoft ^® . At the end of this period, the fluorescent NGPs were added to the concentration of 100 μg / ml and experiment 1 was "renewed. Experiment 3: The specific binding of Rhamnosoft ^® to human keratinocytes was studied by the technique of labeling Rhamnosoft ^® with a fluorescent derivative and by the technique of fixing fluorescent latex beads impregnated with Rhamnosoft ". Rhamnosoft ^® has everything First marked with fluorine escheine isothiocyanate (FTC). Human keratinocytes were incubated in the presence of Rhamnosoft "thus polymerized, at different concentrations ranging in particular from 0.025% to 0.4%. Binding was determined by measuring the index fluorescence and binding obtained for a concentration of 0.4% of Rhamnosoft ^® -FTC was visualized by fluorescence microscopy BioRad MRC1025 at 488nm and 520nm and phase contrast. The technique using latex beads was carried out using 0.2 μm diameter Polysciences latex beads in activated -COOH form. Keratinocytes were incubated for 30 min at 37 ° C in the presence of beads on which Rhamnosoft ^® was immobilized. The cells are then washed and fixed with paraformaldehyde (1% w / v in PBS) for 30 minutes at 20 ° C. The results were visualized using a Biorad MRC1025 fluorescence microscope at 488nm and 520nm and in phase contrast.

Note: In all of these experiments, all incubations with human keratinocytes were carried out for 20 minutes at 37 ° C, unless otherwise stated. All the tests were carried out in triplicate for several concentrations of the product tested. The fluorescence index indicating the fixation has always been evaluated by flow cytometry (FACS 440 Becton Dickinson).

Results:

-Experiment 1 confirmed that human keratinocytes have a preferential affinity for rhamnose. In fact, at concentrations between 10 μg / ml and 200 μg / ml, the fluorescence index of the neoglycoprotein containing rhamnose is approximately twice as high as that of the other neoglycoproteins containing in particular fucose, glucose, or Nacetylglucosamine. A phenomenon of saturation of the receptors has also appeared from 200 μg / ml for rhamnose.

-Experiment 2 made it possible to demonstrate the very high affinity of Rhamnosoft ^® for keratinocytes. Indeed, the binding of all NGPs was inhibited by approximately 10%) from a 3% v / v oncentration of Rhamnosoft ^® . In addition, this action was particularly important on NGP with rhamnose since approximately 10% to 60% of inhibition was observed DOUΓ of the respective concentrations of 0.5% to 8% v / v of Rhamnosoft ". -Experiment 3 made it possible to verify that the results obtained during previous experiments were not due to a direct interaction between Rhamnosoft ^® and NGP. The results are as follows:

Likewise, Rhamnosoft ^® has been clearly demonstrated in the membranes of keratinocytes, by fluorescence and by phase contrast.

Assessment: It thus appears clearly that the polysaccharide according to the invention binds specifically on the membrane receptors of human keratinocytes with a particularly high affinity.

EXAMPLE 5 Study of the Effect of the Polysaccharides According to the Invention Containing 50% of Rhamnose on the Adhesion of Neutrophils to Keratinocytes

Procedure: The keratinocytes were pre-incubated for 24 hours in the presence of Rhamnosoft ^® at different concentrations and in the presence of 3 μg / ml of lipopolysaccharides (LPS). Human neutrophils previously made fluorescent by treatment with calcein are then added. After 2 hours of contact, the keratinocytes are washed and lysed. The inhibition of neutrophil adhesion is determined by measuring the fluorescence index.

Results:

LPS is an agent that induces inflammation.

It therefore appears that the polysaccharide according to the invention induces an inhibition of the cellular adhesion of neutrophils to keratinocytes. Example 6: Examples of cosmetic formulations according to the invention.

These preparations were obtained by simple mixing of the ingredients.

Moisturizing gel for sensitive skin

Phase A:

Demineralized water qs 100%

Dihydrogenoethylenediamine disodium tetraacetate 0.2%

Urea 3.00%

Glycerol 1.50%

Carbomer 1.00%

Rhamnosoft 3.00% preservatives qs triethanolamine eqs

Phase B:

Mixture of PCA * of Sodium, Magnesium, Zinc and Manganese 1.00%

Polysorbate 20 3.00%

Perfume 0.15%

Sun milk

Phase A:

PEG-30 Dipolyhydroxystearate 2.00%

Isohexadecane 8.00%

Octylmethoxycinnamate 5.00%

Monoi oil 4.00%

Butyl Methoxydibenzoylmethane 1.50% preservatives qs

Phase B:

Demineralized water qs 100%

Disodium Dihydrogenoethylenediamine tetraacetate 0.2%

Glycerol 1.50%

Magnesium sulfate heptahydrate 0.70%

Rhamnosoft ^® 3.00% preservatives qs

Phase C:

Demineralized water 15.00%

Phenylbenzimidazole Sulfonic Acid 1.50%

Soda qs pH

Phase D:

Fucogel 1000 / Polysaccharide rich in fucose 4.00%

Phase E:

Silica Dimethyl Silylate 0.30%

: PCA = pyrrolidone-carboxylic acid

Example 7: Examples of dermatological formulations according to the invention.

These preparations were obtained by simple mixing of the ingredients.

Anti-mosquito wellness ointment:

Rhamnosoft 4% repellent 3535 (ethylbutylacetylaminopropionate) 15% UV B sunscreen 2% lavender essence 0.4% enoxolone 0.5% excipient qs 100

Soothing antibiotic skin cream for injured, infected skin, paris-p-aminophenyl sulfonamide 2%

Rhamnosoft 3%

Butylhydroxyanisole 0.05%

Cetyl alcohol 1%

Glycerol 0.75%

Liquid paraffin 2%

0.02% potassium sorbate

Polysorbate 60 0.5%

Vaseline 0.4%

Purified water qs 100

Claims

1. New polysaccharides stimulating the release of β-endorphins, characterized in that they consist of repeating elements which mainly contain rhamnose and contain at least the following components of general formula I:

in which Rh is a molecule of rhamnose, Rh * is a molecule of rhamnose attached in a branched manner, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and n is between 1 and 100, and preferably between 5 and 65.

2. Polysaccharides according to claim 1, characterized in that the ramification of the Rh * rhamnose takes place according to an osidic bond (1 → 2) or (1 → 3) on the saccharide chain.

3. Polysaccharides according to claim 1 or 2, characterized in that the sugar O is chosen from fucose, galactose, glucose, ribose, arabinose, xylose and mannose.

4. Polysaccharides according to one of claims 1 to 3, characterized in that the repeating elements consist of the following sequence of formula II:

Rh

in which Rh is a molecule of rhamnose, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and the ramification of rhamnose on the osm O is done according to an osidic bond (1—> 2 ) or (1 → 3).

5. Polysaccharides according to one of claims 1 to 4, characterized in that the repeating element is composed of a sequence consisting of 3 molecules of rhamnose, one of which is branched, of 2 molecules of galactose and of one acid molecule glucuronic.

6. Polysaccharide according to one of claims 1 to 5, characterized in that the repeating elements have the following sequence: → 4) - α-L-Rhaj7 (l—> 3) - β-D-Galp (l → 2 ) - -L- Rha? (L → 4) -? -D-GlqcA (l → 3) - [-L-Rhap (l- → 2) j- α-D-Galp (l— *., N representative a value such that the molecular weight is approximately 50,000 daltons.

7. Polysaccharides according to claim 6, characterized in that said polysaccharides are in the form of hydrolyzate of fractions of 5,000 and / or 13,000 daltons, purified or in mixture.

8. Polysaccharides according to one of claims 1 to 3, characterized in that the repeating elements consist of the following sequence of formula III:

Rh

in which Rh is a molecule of rhamnose, O is a molecule of a hexosidic or pentosidic sugar, U is a molecule of uronic acid and the ramification of rhamnose on rhamnose is done according to an osidic bond (1 → 3).

9. Polysaccharides according to claim 8 or according to one of claims 1 to 3, characterized in that the repeating element is composed of a chain consisting of 3 molecules of rhamnose including one branched, of one molecule of glucose and d 'a molecule of glucuronic acid.

10. Polysaccharide according to claim 9, characterized in that the repeating elements have the following sequence: → 3) - /? - L-Rhaj? (L → 4) -? -D-Glcp (l → 2) - [α -L-Rha (l— * 3) j- α-L-Rhaj? (L → 4) - α-D-GlcpA (l →.

11. Polysaccharides according to claim 8, characterized in that said polysaccharides are in the form of hydrolyzate of fractions of 5000 daltons, purified or in mixture.

12. Polysaccharides according to one of the preceding claims, characterized in that they are obtained by fermentation of a bacterium of the genus Klebsiella in a nutritive medium comprising a carbon source, a preferential nitrogen source and mineral salts, followed heat treatment and separation.

13. Oligosaccharides, derivatives of polysaccharides according to one of the preceding claims, characterized in that they comprise at least one molecule of rhamnose accessible at the end of the chain or attached in a branched manner and in that they consist mainly of rhamnose.

14. Oligosaccharides according to claim 13, characterized in that they are obtained by hydrolysis of the polysaccharides according to one of claims 1 to 11.

15. Oligosaccharides according to claim 14, characterized in that they are obtained by hydrolysis of the polysaccharide according to claim 6 and have a molecular weight of 5kDa or 13kDa.

16. Oligosaccharides according to claim 14, characterized in that they are obtained by hydrolysis of the polysaccharide according to claim 10 and have a molecular weight of 5kDa.

17. Polysaccharides and / or derivatives of polysaccharides according to one of the preceding claims, characterized in that they are grafted on fatty chains of 6 to 30 carbon atoms.

18. Cosmetic and / or pharmaceutical composition, in particular dermatological composition, stimulating the release of β-endorphins, characterized in that it contains at least one active compound of polysaccharide type and / or one of its derivatives according to one of claims 1 to 17, alone or in mixture with one or more excipient (s) or suitable cosmetic and / or pharmaceutical vehicle (s).

19. Composition according to Claim 18, characterized in that it contains between 0.001% o and 20%> of said polysaccharide and / or derivative, by weight.

20. Composition according to claim 19, characterized in that it contains between 0.01% and 10% of said polysaccharide and / or derivative, by weight.

21. Use of at least one polysaccharide and / or derivative thereof comprising saccharide repeating elements containing at least one molecule of rhamnose attached in a branched manner and preferably containing predominantly rhamnose, as cosmetic and / or pharmaceutical agent, in particular dermatological, stimulating the release of β-endorphins.

22. Use of a composition as defined according to one of claims 18 to 20 for the care of skin appendages and / or of the skin, in particular of sensitive skin.

23. Use of a composition according to one of claims 18 to 20 for the cosmetic treatment of uncomfortable reactions of the skin and / or integuments.

24. Use of at least one polysaccharide and / or a derivative according to one of claims 1 to 17 for the manufacture of a medicament intended to treat uncomfortable reactions and / or relieve pain in the skin and / or appendages by topical application.

25. Cosmetic care method, characterized in that it comprises the application to a zone of the skin and / or integuments concerned, of a polysaccharide and / or of a derivative defined according to one of claims 1 to 17 optionally mixed with one or more suitable cosmetic excipients or vehicles.