Exopolysaccharides and oligosaccharides, in particular for cosmetics

Abstract

The present invention relates to exopolysaccharides, oligosaccharides obtainable by cleavage of said exopolysaccharides, microorganisms for their preparation, methods of obtaining them, compositions containing them, and uses, in particular cosmetic uses. The oligosaccharides are particularly suitable for improving skin gloss, bulge, compactness and hydration and protecting the skin from aging and / or contamination effects.

Description

[0001] The present invention relates to exopolysaccharides, oligosaccharides that can be obtained by cleavage of said exopolysaccharides, microorganisms for their preparation, processes for obtaining them, compositions containing them, and uses, in particular cosmetic uses.

[0002] The field of application of the present invention is mainly the chemical industry, for example the food, cosmetic, pharmaceutical, veterinary, agricultural, environmental or paints and coatings industry.

[0003] The present invention can be applied more particularly in the field of the cosmetic and dermatopharmaceutical industry, which prepares and / or uses products intended for treating the skin (including the scalp), the mucous membranes and the appendages (such as body hair, eyelashes, eyebrows, nails, hair) of mammals, animals or humans, for improving their appearance and / or general condition.

[0004] Polysaccharides consist of longer or shorter chains of monosaccharides and / or monosaccharide acids linked by glycosidic bonds. They can be linear or branched. They exist in nature in plants, mammals or microorganisms, their role being in particular energy storage, structure or support of tissues, regulation of tissue viscoelasticity, water retention, protection against bacterial or protease attacks, and / or coagulation.

[0005] The family of polysaccharides contains a particular class consisting of exopolysaccharides, which are characterized by their production by microorganisms living in ecosystems in extreme conditions and by their extracellular secretion.

[0006] One of the advantages of exopolysaccharides for industry is that the microorganisms that produce them can be cultivated in high quantities under controlled conditions. This facilitates the production of exopolysaccharides on a large scale, relative to polysaccharides that usually require complex extraction from natural sources such as plants or algae.

[0007] Various exopolysaccharides have been described for use in the cosmetic and / or dermatological field.

[0008] Patent application US 2009 / 069213 describes various exopolysaccharides obtained from a strain of microalgae from the Porphyridium genus ( Porphyridium ). An exopolysaccharide obtained from a strain of Porphyridium ( Porphyridium cruentum ) is presented, which consists of three monosaccharides (xylose, glucose and galactose) and a glucuronic acid, and is recommended for its anti-wrinkle and moisturizing properties.

[0009] Another example is given in patent application WO 2012 / 072245, which describes an exopolysaccharide obtained from a Pseudoalteromonas sp. strain deposited under the number I-4150 ( Pseudoalteromonas spExopolysaccharide isolated from a strain of Pseudomonas sp. (Pseudomonas sp. CECT 8437) which is a specific non-thermophilic strain. This exopolysaccharide is composed of five monosaccharides, mannose, glucose, galactose, rhamnose and N-acetylglucosamine, and two uronic acids, glucuronic acid and galacturonic acid. It is recommended as a substitute of hyaluronic acid, again for its anti-wrinkle and moisturizing effects.

[0010] A further example is given in patent application WO2015 / 117985 which describes an exopolysaccharide obtained from a culture of Pseudomonas sp. (Pseudomonas sp. CECT 8437) deposited under the number CECT 8437. This exopolysaccharide is composed of three monosaccharides, glucose, galactose and fucose, and a glycoside uronic acid. It is recommended in the cosmetic and food field for its emulsifying activity. Pseudomonas sp

[0011] Furthermore, with the help of appropriate cleavage, the exopolysaccharides can advantageously be used as a source of sugar monomers or oligosaccharides. For the skilled person, oligosaccharides are small polysaccharides, which are generally characterized in that they comprise less than 25 monosaccharides and / or monosaccharide acids. The advantage of oligosaccharides is that they more easily penetrate the skin barrier and, due to their reduced size, exhibit a better stability in formulations.

[0012] Thus, for example, the above patent application WO2015 / 117985 describes the hydrolysis of the exopolysaccharide into oligosaccharides and monosaccharides which can be used in cosmetic or pharmaceutical products. No particular activity is presented.

[0013] Another patent application WO98 / 50013 describes the use of oligosaccharides without specifying their source. The described oligosaccharides have from two to six glycosidic residues, including a galactose residue at the terminal position. They are recommended in cosmetic compositions for increasing the synthesis and / or reducing the degradation of proteoglycans in the connective tissue, allowing to moisturize the skin, and for combating the effects of free radicals.

[0014] However, there is still a high commercial demand for new active ingredients to address the current sustainability issues, in particular to provide products obtained from biotechnological processes that protect natural resources.

[0015] It is a particular object of the present invention to meet this demand.

[0016] For this purpose, according to First aspect The present invention provides an exopolysaccharide characterized in that it comprises at least one monosaccharide unit which is an N-acetyl quino sugar amine (NAcQ).

[0017] Thanks to the presence of the N-acetyl quino sugar amine, this new exopolysaccharide advantageously constitutes a source of new oligosaccharides which exhibit unique cosmetic properties, as shown by the test results set out in the following description.

[0018] ​Moreover, the new exopolysaccharide can advantageously be used in the above-mentioned technical fields, in a native or partially lysed state. For example, it can be used as a rheological or film-forming agent in formulations. In the cosmetics field, it can be used to improve the overall condition of the skin and appendages, to prevent water loss by forming a protective barrier and to produce a soft and comfortable sensation. It can also have anti-aging properties via a firming effect.

[0019] According to other characteristics, the exopolysaccharide has a molecular weight greater than or equal to 260 000 Da, more particularly greater than or equal to 500 000 Da.

[0020] According to yet other particular characteristics of the exopolysaccharide according to the application:

[0021] - it also comprises at least one monosaccharide acid unit chosen from glucuronic acid (Glu) and galacturonic acid (Gal), and preferably comprises both of these monosaccharide acid units; and / or

[0022] - it also comprises at least one monosaccharide unit that is rhamnose (Rham); and / or

[0023] - it comprises 15% to 55%, preferably substantially 25%, of N-acetylquinovosamine; and / or

[0024] - it comprises 15% to 55%, preferably substantially 25%, of glucuronic acid; and / or

[0025] - it comprises 15% to 55%, preferably substantially 25%, of galacturonic acid; and / or

[0026] - it comprises 15% to 55%, preferably substantially 25%, of rhamnose; and / or

[0027] - it comprises a concatenation of the three following saccharide units: glucuronic acid, galacturonic acid and N-acetylquinovosamine (Glu-Gal-NAcQ); and / or

[0028] - it also comprises rhamnose, with a concatenation of: glucuronic acid, galacturonic acid, N-acetylquinovosamine and rhamnose (Glu-Gal-NAcQ-Rham); and / or

[0029] - it consists of a repetition of glucuronic acid, galacturonic acid, N-acetylquinovosamine, rhamnose (Glu-Gal-NAcQ-Rham) concatenated, each saccharide unit being present in the exopolysaccharide substantially in equimolar amounts.

[0030] All the percentages expressed in the above paragraphs are by weight relative to the total weight of the exopolysaccharide.

[0031] Furthermore, when a carboxylic acid functional group is present, the extracellular polysaccharide can be derivatized on the carboxylic acid functional group; in particular, the extracellular polysaccharide can be derivatized such that at least one carboxylic acid terminus of glucuronic acid or galacturonic acid is, for example, esterified or acylated.

[0032] Based on other characteristics, the extracellular polysaccharide is derived from a bacterial strain of the genus *Pseudomonas* deposited by the applicant under the Budapest Treaty at the National Center for Microbial Collection (CNCM) in Paris, under the number I-5893. Pseudoalteromonas sp The bacterial strain was obtained from warm waters collected in the Bimini Islands of the Bahamas. Pleuxaura The octocortic corals of this genus are branched, candelabra-shaped corals that separate on the surface. Due to their marine origin, the unspored bacteria are rod-shaped and grow as colonies in saline environments.

[0033] As mentioned above, the extracellular polysaccharides according to the present invention can be used as a source of oligosaccharides.

[0034] Therefore, according to Second aspect The present invention also provides oligosaccharides that can be obtained by cleavage of extracellular polysaccharides according to the invention and consist of at least one monosaccharide unit as N-acetylquinolamine.

[0035] The oligosaccharides according to the present invention can also be synthesized from monosaccharides by chemical or enzymatic means.

[0036] Advantageously, the oligosaccharides according to the invention are presented in the detailed description as having particularly attractive and unique cosmetic activities as well as conventional moisturizing and anti-wrinkle properties. In particular, test results indicate that the oligosaccharides help combat cellular hypoxia for a very rapid cosmetic benefit of radiance to the skin.

[0037] According to specific characteristics, the oligosaccharides of the present invention are:

[0038] - Having a degree of polymerization of 2 to 12, preferably 3, 4, 7 or 11; and / or

[0039] - In addition to N-acetylquinolamine, it also contains at least one monosaccharide acid unit selected from glucuronic acid and galacturonic acid, and preferably contains both of these monosaccharide acid units; and / or

[0040] - Contains linked glucuronic acid, galacturonic acid, and N-acetylquinolamine (Glu-Gal-NAcQ); and / or

[0041] - It also contains rhamnose, which has the following links: glucuronic acid, galacturonic acid, N-acetylquinoline, and rhamnose (Glu-Gal-NAcQ-Rham).

[0042] According to other features, since the average molecular weight of the sugar units of the exopolysaccharide according to the application is of about 200 Da (corresponding to the average of the mass of one unit of N-acetyl-quinovosamine, glucuronic acid, galacturonic acid and rhamnose), the oligosaccharides according to the application preferably have a molecular weight less than or equal to 5000 Da (corresponding to a degree of polymerization of 25), more preferably less than or equal to 3000 Da (corresponding to a degree of polymerization of 15), more preferably less than or equal to 2200 Da (corresponding to a degree of polymerization of 11), more preferably less than or equal to 1400 Da (corresponding to a degree of polymerization of 7), more preferably less than or equal to 800 Da for a degree of polymerization less than or equal to 4, more preferably less than or equal to 600 Da for a degree of polymerization less than or equal to 3 and more preferably less than or equal to 400 Da for a degree of polymerization less than or equal to 2.

[0043] The preferred oligosaccharides according to the application are:

[0044] - a trisaccharide having a degree of polymerization of 3 comprising one N-acetyl-quinovosamine unit, one glucuronic acid unit and one galacturonic acid unit and having a molecular weight of 557 Da, more particularly consisting of the sequence Glu-Gal-NAcQ, and / or

[0045] - a heptasaccharide having a degree of polymerization of 7 comprising two N-acetyl-quinovosamine units, two glucuronic acid units, two galacturonic acid units and one rhamnose unit and having a molecular weight of 1242 Da, more particularly consisting of the sequence Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ, and / or

[0046] - an undecasaccharide having a degree of polymerization of 11 comprising three N-acetyl-quinovosamine units, three glucuronic acid units, three galacturonic acid units and two rhamnose units and having a molecular weight of 1927 Da, more particularly consisting of the sequence Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ.

[0047] Moreover, when carboxylic acid functions are present, the oligosaccharides according to the application can be derivatized on the carboxylic acid functions; in particular the oligosaccharides can be derivatized such that at least one carboxylic acid end of the glucuronic acid or galacturonic acid is esterified or acylated, for example.

[0048] As presented above, the exopolysaccharide according to the application and thus the resulting oligosaccharides can be obtained by a fermentation process, in particular starting from the strain I-5893 deposited at the CNCM.

[0049] According to other features, Third aspectThe present application thus provides a bacterial strain Pseudoalteromonas sp. which has been deposited by the Applicant at the Collection Nationale de Cultures de Microorganismes (CNCM) in Paris under the number I-5893.

[0050] According to other features, the bacterial strain Pseudoalteromonas sp. I-5893 is cultivated in a first step in a culture medium suitable for producing bacteria secreting exopolysaccharides in the culture medium. Fourth aspect The present application provides a method for cultivating the strain for producing exopolysaccharides according to the first aspect and oligosaccharides according to the second aspect, said method comprising the following steps:

[0051] - a first step of cultivating the bacterial strain Pseudoalteromonas sp. I-5893 deposited at the CNCM in a culture medium suitable for producing bacteria secreting exopolysaccharides in the culture medium; and

[0052] - a second step of recovering the exopolysaccharides isolated from the culture medium.

[0053] Advantageously, in the first step, the amount of bacterial strain increases while the bacterial strain secretes exopolysaccharides in the culture medium.

[0054] The culture medium is liquid and can contain macroelements, microelements, yeast extract, vitamins and sugars. They are chosen to promote growth and the production of exopolysaccharides according to the application.

[0055] Mineral salts can be used as microelements for the culture. For example, but not limited to, salts providing the ions Na + , K + , NH4 + , Ca 2 + , Mg 2+ , PO4 3- , SO4 2- , Cl - and / or CO3 2- , or trace elements such as Cu, Mn, Fe and / or Zn, or mixtures thereof. These salts are chosen to promote growth and the production of exopolysaccharides.

[0056] Similarly, sugars can be used as carbon source to feed the bacterial strain, such as, but not limited to, galactose, glucose, mannose, amygdaline, cellobiose, maltose, starch, glycogen, lactose or mixtures thereof. The addition of sugars also increases the production of exopolysaccharides.

[0057] According to other features, in the second step, the isolation of the exopolysaccharides from the culture medium is carried out in two stages.

[0058] In a first stage, the exopolysaccharide present in the liquid medium is separated from the biomass which is a solid, by a method which makes it possible to separate the liquid phase from the solid. The separation can be carried out, for example but not exclusively, by centrifugation, ultrafiltration, tangential filtration or pressure filtration. The biomass present in the solid phase can be removed or conserved in order to be added to the exopolysaccharide obtained at the end of the process after extraction, with the aim of enriching it with proteins and with sugars, which are compounds present in the biomass.

[0059] In a second stage, the exopolysaccharide is purified by separating it from the liquid medium containing other molecules, which can be salts, proteins, etc. For example, these molecules can be precipitated by virtue of a decrease in temperature, known as cold precipitation, or by virtue of a water-miscible solvent in which the exopolysaccharide is insoluble, for example methanol, isopropanol, ethanol or a mixture thereof, or tangential filtration can be carried out.

[0060] Preferably, the exopolysaccharide is separated from the medium by virtue of tangential filtration. More particularly, a filtration membrane having a cut-off value lower than that of the exopolysaccharide is used. More particularly, the filtration membrane has a cut-off value lower than 150 000 Da.

[0061] In a further step, the exopolysaccharide obtained by the above process can be cleaved in order to obtain at least one oligosaccharide according to the application. This cleavage can be carried out by chemical or enzymatic hydrolysis.

[0062] Chemical hydrolysis in an acid medium can be carried out by a strong acid, such as hydrochloric acid, nitric acid, sulphuric acid or trifluoroacetic acid.

[0063] Enzymatic hydrolysis can be carried out by specific hydrolytic enzymes.

[0064] For these different types of hydrolysis, the concentration of solvent or enzyme, the reaction time and the temperature are defined as depending on the size of the oligosaccharide which is desired to be obtained. For example, the higher the acid concentration or the longer the reaction time, the greater the extent of small oligosaccharides which will be obtained.

[0065] Acid hydrolysis is the preferred process, and the solvent which is preferably used is hydrochloric acid at a concentration of 0.2 M.

[0066] During the hydrolysis, precipitates can appear in the liquid medium containing the oligosaccharides. These precipitates can consist of salts, proteins, cell fragments, etc., and an attempt is made to remove these precipitates in order to purify the liquid phase. To achieve this, any method which makes it possible to separate the liquid phase from the solid is employed. For example, the separation can be carried out by centrifugation, ultrafiltration, tangential filtration or pressure filtration.

[0067] In the exopolysaccharide according to the application, the chemical bonds around the rhamnose are cleaved as the most fragile bonds. Since the exopolysaccharide has repeating linkages of glucuronic acid, galacturonic acid, N-acetyl quinozamine and rhamnose, the following oligosaccharides can in particular be obtained:

[0068] - a trisaccharide with a linkage of glucuronic acid, galacturonic acid and N-acetyl quinozamine (Glu-Gal-NAcQ);

[0069] - other oligosaccharides with a linkage of trisaccharide and rhamnose between each trisaccharide, such as in particular:

[0070] - a heptasaccharide corresponding to two trisaccharides Glu-Gal-NAcQ linked by a rhamnose (Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ);

[0071] - an undecasaccharide corresponding to three trisaccharides Glu-Gal-NAcQ each linked by a rhamnose (Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ).

[0072] According to Fifth aspect , the application provides a mixture of oligosaccharides, which can be obtained by the method of the fourth aspect of the application, preferably consisting mainly of the trisaccharide and / or the heptasaccharide according to the application, i.e. more than 50%, more particularly more than 80%. The percentages are expressed by weight relative to the total weight of the mixture of oligosaccharides.

[0073] Each oligosaccharide according to the application can be isolated from the mixture of oligosaccharides obtained by the method according to the application by appropriate techniques, for example by flash chromatography on silica.

[0074] The exopolysaccharide and the oligosaccharides according to the application, in particular obtained by the method described above, can be used as active ingredients in cosmetic or dermatological compositions.

[0075] Thus, according to Sixth aspect , the application provides a cosmetic or dermatological composition comprising, as active agent, the exopolysaccharide according to the application and a physiologically acceptable medium.

[0076] Thus, likewise, according to Seventh aspect , the application provides a cosmetic or dermatological composition comprising, as active ingredient, at least one oligosaccharide according to the application according to the application and a physiologically acceptable medium.

[0077] The term "physiologically acceptable medium" according to the application means, but is not limited to, an aqueous or hydroalcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a microemulsion, an aqueous gel, an anhydrous gel, a serum, a dispersion of vesicles, or a powder.

[0078] The term "physiologically acceptable" means a composition suitable for topical or transdermal use, in contact with the mucosae, the nails (ungual), the scalp, the hair, the body hair and the skin of a mammal and more particularly of a human being, which can be ingested or injected into the skin without risk of toxicity, incompatibility, instability, allergy, etc.

[0079] Preferably, the physiologically acceptable medium can be an aqueous solution, a hydroglycolic solution or a hydroalcoholic solution, or a water-in-oil emulsion, an oil-in-water emulsion, or a microemulsion, or a mixture thereof. Preferably, it is a mixture of water, glycol and diol.

[0080] According to other advantageous features, the exopolysaccharide and / or at least one oligosaccharide can be combined with one or more other active ingredients in effective concentrations, which can act synergistically or additively for enhancing and achieving the desired effects described in the present application, such as the following ingredients: filtered radiation, in particular UVA, UVB, IR or radiation generated by blue light, hydration, moisturizing, humectant, calming, muscle relaxant, slimming, restructuring, firming, re-plumping, lifting, smoothing, acting on blood microcirculation, inflammation, free radicals, anti-aging, anti-fine lines and wrinkles, lightening, acting on skin tone, anti-glycation, anti-carbonylation, pro-pigmentation, acting on the stratum corneum, acting on the dermal-epidermal junction, acting on HSP protein production, acting on the firmness, elasticity and tone of the skin, acting on hair growth or anti-regeneration (including eyelashes and eyebrows), acting on the eye contour (dark circles and bags), promoting blood circulation, regeneration, favorably or unfavorably acting on the skin microbiota and its appendages, promoting hair growth, promoting the elimination of toxins, etc. These active ingredients can be obtained from plant materials, such as plant extracts or plant cultures or products of fermentation production processes.

[0081] More specifically, the extracellular polysaccharide and / or at least one oligosaccharide according to the invention can be combined with at least one compound selected from: vitamin B3 compounds, such as nicotinamide or tocopherol compounds, retinoids such as retinol (especially in encapsulated form), hexamidine, α-lipoic acid, resveratrol or DHEA, hyaluronic acid, ceramide or ceramide analogs (synthetically or biotechnically obtained), peptides, especially N-acetyl-Tyr-Arg-O-hexadecyl, VW, Pal-PP, Biot-GHK, Ela-KFK, Pal-KPK, Pal-KHG, Myr-SPA, Pal-VGVAPG (SEQ ID NO: 1), Pal-KTTKS (SEQ ID NO: 2), Pal-GHK, Pal KMO2K, Pal-GQPR (SEQ ID NO: 3), Pal K(Ac)HG, Pal K(P)HG and Pal KTSKS (SEQ ID NO: 3). 4) Pal KTFK (SEQ ID NO: 5), PalYGGFL (SEQ ID NO: 6), cyclic linolenic acid, plant extracts, especially senna (Salvia splendens). Marrubium vulgare Extracts, and Edelweiss obtained through in vitro cell culture ( Leontopodium Alpinum ) extract, four o'clock flower ( Mirabilis jalapa ) extracts, or mixtures thereof, such as rosemary ( Rosmarinus officinalis ), and Pal-GQPR (SEQ ID NO: 3), which are known active ingredients that can be used in topical cosmetic or dermatological pharmaceutical compositions.

[0082] according to Eighth aspect The present invention provides the use of one of the extracellular polysaccharides and / or at least one oligosaccharide and / or compositions containing the present invention in the industrial fields mentioned above (i.e., primarily the chemical industry, for example, the food, cosmetic, pharmaceutical, veterinary, agricultural, environmental, or paint and coating industries).

[0083] More particularly, the present invention provides the use of at least one oligosaccharide and / or compositions containing it according to various aspects of the invention for non-therapeutic cosmetic treatments of the skin and its appendages. Preferably, according to the invention, the treatment is localized.

[0084] Preferably, the treatment is carried out using a mixture of oligosaccharides according to the fifth aspect of the invention.

[0085] According to the present invention, “topical treatment” or “topical application” means an application intended to have an effect at the site of application: skin, mucous membranes and / or appendages.

[0086] More particularly, the cosmetic treatment provided by the application aims to combat the signs of unattractive skin resulting from the loss of cell vitality and the loss or reduction of certain fundamental skin functions.

[0087] The loss of radiance, firmness, moisturization and / or the appearance of roughness of the skin tone form part of these aesthetic signs. They are due to the normal chronological aging of the skin and / or to premature aging involving extrinsic factors such as, for example, aggressive external exposure, in particular in the form of atmospheric pollutants or radiation. Over time and / or in the presence of these extrinsic factors, the important functions of the skin cells and certain functions at the level of the skin layers will also deteriorate.

[0088] According to a particular feature, the cosmetic treatment implementing at least one oligosaccharide according to the application is suitable for:

[0089] - improving the radiance of the skin tone, also known as luminosity;

[0090] - improving the relief of the skin;

[0091] - improving the firmness of the skin; and / or

[0092] - improving the moisturization of the skin.

[0093] The tests carried out in vitro and presented below in the detailed description show that the oligosaccharide-based treatment according to the application makes it possible to advantageously:

[0094] - act at the level of the skin cells: by combating oxidative and cellular hypoxia, by stimulating the production of energy, the consumption of oxygen and cellular respiration; and

[0095] - act at the level of the skin: by enhancing the barrier function, the moisturization function, the function of the dermal-epidermal junction (DEJ) and the microbiota function.

[0096] More specifically at the level of the skin cells :

[0097] The tests show that the treatment according to the application confers Beneficial antioxidant action .

[0098] Under oxidative stress, the protein encoded by the gene Nrf2 enters the nucleus of the cell and induces the activity of certain genes such as NQO-1 and HMOX-1.

[0099] NQO-1 is a gene encoding an antioxidant protein that serves to maintain the balance of the oxidative state within the cell and also to detoxify aromatic-type pollutants. This protein is synthesized when there is an increase in ROS in the cell.

[0100] HMOX-1 is the gene encoding the protein heme oxygenase (HO-1), which is rapidly induced after exposure to free radicals. HMOX-1 exhibits a protective effect against oxidative stress, essentially due to its ability to degrade heme into carbon monoxide and biliverdin (a precursor of bilirubin, which is a powerful antioxidant).

[0101] Thanks to this antioxidant effect, skin aging is limited, in particular via preservation of the dermal extracellular matrix (ECM). Oxidation has a negative effect on the synthesis of molecules such as fibroblasts responsible for the synthesis of collagen, elastin and other components of the ECM, which allow the structure to be maintained in the skin, wrinkles and fine lines to be avoided, allowing firmness and elasticity to be maintained.

[0102] Tests show that the treatment according to the application has an activity on Cellular hypoxia has

[0103] - the mitochondrial potential is increased, which generates energy in cells that need it in order to make proteins, operate enzymes, detoxify cells from accumulated pollutants, for skin moisturizing, etc.

[0104] - the consumption of oxygen also generates energy. Indeed, oxygen is consumed to enable the removal of CO2 and the generation of energy;

[0105] - the production of adenosine triphosphate (ATP) is increased, which activates cellular respiration by stimulating detoxification mechanisms and by increasing the production of cells.

[0106] More energy and oxygenation of the cells leads to detoxification and regeneration of the cells, improving the quality of the epidermis and enhancing the skin barrier for a better radiance of the complexion.

[0107] More specifically at the level of the skin :

[0108] In vitro tests show that the treatment according to the application has an activity on Enhancement of the skin barrier Stratum corneum the horny layer.

[0109] The horny layer ( Moisturizing function of the skin has ), or horny layer, is the outermost layer of the epidermis and constitutes a physical and functional barrier, which acts to prevent the penetration of harmful external agents, such as allergens, pathogens and pollutants, in one direction, and to prevent the dehydration of the skin, in the other direction.

[0110] The horny layer is composed of keratinocytes, lipids, lamellar bodies and natural moisturizing factors (NMF - substances capable of fixing moisture in the horny layer), which maintain the degree of hydration of the skin at optimal levels. ​

[0111] Transdermal water loss (TEWL) characterizes the evaporation of water contained in the skin: the greater the damage to the skin barrier, the easier it is for water to evaporate.

[0112] Improved differentiation of keratinocytes leads to a stronger skin barrier, and thus helps to better protect the epidermis and better moisturize it.

[0113] Keratinocytes are living cells with a lipid profile in the epidermis that migrate from the basal layer to the outermost layer, differentiating themselves into small, flat, brick-like keratinocytes without a nucleus. Proteins such as inner laminarin, fimbriaein, and sealing protein-1 are involved in the formation and migration of keratinocytes into keratinocytes. Furthermore, keratinocytes are strongly connected to each other by corneodesmosomes, where the main protein is corneodesmosin, and are “cemented” by a complex layer of organized extracellular lipids: cholesterol, ceramides, and neutral lipids.

[0114] Inner lamina protein is a protein synthesized in the cytoplasm of keratinocytes in the granular layer. By binding to the protein lamina protein, inner lamina protein helps protect the formation of the cell capsule of keratinocytes in the skin.

[0115] Protein sealant protein-1 is a component of the tight junctions of the granular layer, which forms a selective barrier controlling the paracellular transport of molecules and inflammatory cells. These tight junctions are also crucial for limiting water loss.

[0116] Tests revealed the effects of these essential proteins: inner lining protein, keratin desmosome, and sealing protein-1.

[0117] Tests also showed that the treatment according to the invention, by stimulating the synthesis of aquaporin-3 (AQP-3) (a known marker for moisturizing and present in the epidermis) and also of filinin (which plays a key role in maintaining barrier function and has degradation products essential for pH regulation and skin moisturizing), has an effect on... Function of the DEJ It is active. Therefore, advantageously, due to the treatment according to the invention, not only is skin dehydration prevented with an enhanced skin barrier, but the moisturizing function itself is stimulated by increasing the synthesis of one of the macromolecules responsible for moisturizing in the epidermis. Better moisturized skin is plumper, softer, and smoother (less rough).

[0118] Due to the treatment according to the present invention, the skin is better protected and better moisturized, and therefore more radiant.

[0119] The treatment according to the invention also works by ensuring that keratinocytes are effectively anchored to the basal layer and by improving communication between cells.Skin microbiota function allowing keratinocytes to polarize. The synthesis of the main components of the DEJ (collagen IV, collagen VII and laminin) decreases with age and therefore has a significant impact on the firmness of the skin.

[0120] Tests have shown that the treatment according to the application is active on the markers of the DEJ, i.e. collagen IV and collagen VII, for improving the firmness of the skin. These molecules are also involved in the reinforcement of the skin barrier.

[0121] In addition, the treatment according to the application acts on Staphylococcus epidermidis .

[0122] This function is mainly ensured by the presence of strains of Staphylococcus epidermidis at the surface of the skin Figure 1 ). This is a bacterium that colonizes the skin and is known to have a beneficial effect on the skin immunity. In addition, it has been demonstrated that Staphylococcus epidermidis is favorable to the homeostasis of the barrier function due to its ability to cut sphingomyelin 1) into phosphocholine, which acts as a nutritional substrate for the bacterium and allows it to colonize the skin space, and 2) into ceramide type 2, which promotes the quality of the skin barrier.

[0123] The colonization of the skin surface by Staphylococcus epidermidis enhances the effect of the production of ceramides, which are known to improve the skin barrier, prevent dehydration and make the skin smooth, the skin appearing more luminous.

[0124] Tests have shown that the treatment according to the application is active on the growth of Staphylococcus epidermidis and on the production of ceramides by the skin and by Staphylococcus epidermidis.

[0125] Advantageously and synergistically, all these effects can fight against the dull complexion of the skin (loss of luminosity), the loss of skin firmness and the lack of hydration, fighting against the aesthetic signs on the skin.

[0126] Advantageously, the treatment according to the application is particularly suitable for fighting against pollution, in particular atmospheric pollution.

[0127] The results of specific tests on cell hypoxia and cell detoxification carried out in the presence of a solution of urban atmospheric microparticles (UMPs) to simulate atmospheric pollutants are given below in the detailed description.

[0128] In vivo tests have been carried out, demonstrating the cosmetic benefits according to the application, in particular tests on dull complexion (or luminosity), tests on microrelief (demonstrating the smoothing effect), hydration tests and firmness tests.

[0129] Thus, the present application provides a non-therapeutic cosmetic treatment method for enhancing or improving the appearance and general condition of the skin and / or its appendages and for treating imperfections in a subject in need thereof, which comprises the application of an effective amount of an oligosaccharide or a mixture of oligosaccharides according to the application or of a composition comprising it in a physiologically acceptable excipient.

[0130] The "effective" amount according to the application, i.e. its dose in the composition, depends on the intended use of the composition. It depends on various factors such as the age and condition of the patient and the severity of the disorder. By effective amount is meant a non-toxic amount that is sufficient to obtain the desired effect.

[0131] In the cosmetic composition according to the application containing at least one oligosaccharide in an effective amount, it is generally present in an amount ranging from 0.000001 % to 15%, preferably ranging from 0.00001 % to 10%, based on the total weight of the composition, depending on the use of the composition and the more or less pronounced desired effect. More preferentially, the effective amount is from 0.0001 % to 5% based on the total weight of the composition.

[0132] According to other characteristics, the cosmetic treatment method according to the application can be combined with one or more other treatment methods for the skin such as phototherapy, thermal or aromatic therapy treatments.

[0133] According to the application, a device with multiple compartments or kits can be proposed to apply the above-described methods, which can comprise, for example and not limitatively, a first compartment containing a composition comprising the oligosaccharides of the application and, in a second compartment, it contains additional active ingredients, in which case the compositions contained in the first and second compartments are considered to be a combined composition for simultaneous, separate or stepwise use in time, in particular in one of the treatment methods described above.

[0134] For reference, the European standard dose of a cream for a cosmetic facial treatment is 2.72 mg / cm2 / day / person and the European standard dose of a lotion for a cosmetic body treatment is 0.5 mg / cm2 / day / person.

[0135] The present application also relates to the use of an exopolysaccharide and / or oligosaccharide and / or mixture of oligosaccharides according to the application for the preparation of a composition for a cosmetic treatment as described above.

[0136] The composition for use according to the application can be provided in any galenic form as defined according to the use of the composition and the site of application (examples are given below).

[0137] The compositions according to the invention can be applied to the face, body, neckline, scalp, hair, eyelashes, and body hair in any form or medium known to those skilled in the art, particularly as a solution, dispersion, emulsion, paste, or powder alone or as a premix, or as a medium alone or as a premix in combination, incorporated into or adsorbed in a carrier such as macrocapsules, microcapsules, or nanocapsules, macrospheres, microspheres, or nanospheres, liposomes, oleosomes, or chylomicrons, macroparticles, microparticles, or nanoparticles, or macrosponges, microsponges, or nanosponges, microemulsions, or nanoemulsions, or adsorbed on organic polymer powders, talc, bentonite, spores, or exoskeletons and other inorganic or organic carriers.

[0138] In cosmetics, it can be specifically applied in the areas of skin care for the face, body, hair and body hair, as well as in the areas of makeup treatments (especially eyelashes and eyebrows).

[0139] For example, the composition may be in the form of an emulsion, cream, lotion, milk, solid form, foam, gel, deodorant, antiperspirant, shampoo, conditioner, hair mask, face mask, shower gel, etc.

[0140] Galen formulations can be used in various product ranges for personal care and / or beautification products, including skin care, cleansing, makeup, cleansing, sun protection, artificial tanning, pre-shave, during-shave or post-shave, moisturizers, humectants, emollients, conditioning agents, exfoliants, astringents, depilatory or antiperspirant, deodorants, etc.

[0141] The composition can be incorporated into nonwoven or woven materials having natural or synthetic fibers, wool, or any material intended to come into contact with the skin and which can be used in clothing, including tights and socks, shorts, day or night underwear, tissues, handkerchiefs, or fabrics, to exert its cosmetic effects and achieve continuous local delivery (cosmetic textiles) through contact with the skin / textiles.

[0142] Based on the features of particular interest, cosmetic treatments using oligosaccharides according to the present invention are suitable for:

[0143] - Improves skin radiance;

[0144] - Improves skin texture;

[0145] - Improves skin firmness; and / or

[0146] - Improves skin hydration.

[0147] More specifically, cosmetic treatments can be procedures that protect the skin from the following:

[0148] - Aging; and / or

[0149] - Pollution, especially air pollution.

[0150] More specifically, the treatment is as follows:

[0151] - Improve skin quality by strengthening the skin barrier, resulting in firmer, softer, smoother, and more moisturized skin, and thus a more radiant complexion; and / or

[0152] - Combats cellular hypoxia for a very quick beauty benefit of radiant skin tone.

[0153] According to the present invention, these cosmetic effects can be conceived individually or in combination, advantageously providing uses that combine beautifying and sensory effects.

[0154] Detailed Explanation

[0155] A better understanding of the invention will be obtained from the following detailed description of the working embodiments, studies, and accompanying drawings.

[0156] [ Acmella oleracea This graph shows the changes in skin deformation over time under the influence of mechanical deformation.

[0157] 1. An example of preparing extracellular polysaccharides from strain I-5893

[0158] First step: Production of extracellular polysaccharides.

[0159] The strain *Pseudomonas* species number I-5893 was inoculated in a bioreactor containing culture medium for a period of time sufficient to allow the strain to multiply.

[0160] The culture medium is then transferred to a larger bioreactor, and fresh medium may be added to the reactor to increase the production of the strain. Incubation should continue for a period sufficient to obtain even larger quantities of *Pseudorhynchospora* species. This step may be repeated optionally.

[0161] The culture medium used contains:

[0162] - 0.1% to 0.5% yeast extract;

[0163] - 0.1% to 0.5% KH2PO4;

[0164] - 0.1% to 0.5% Na2HPO4;

[0165] - 0.1% to 0.5% NH4Cl;

[0166] - 0.001% to 0.01% KCl;

[0167] - 0.001% to 0.01% ferric citrate;

[0168] - At least 0.05% defoamer; and

[0169] - 1% to 5% sea salt.

[0170] 1% to 5% glucose (carbon source) was also added to the culture medium to increase the production of extracellular polysaccharides.

[0171] Optionally, fresh culture medium may be added to the bioreactor during incubation and / or minerals and / or sugars may be added during incubation.

[0172] Under these conditions, the culture is placed for a sufficient time to allow the strain to secrete the extracellular polysaccharide according to the invention into the culture medium and obtain the desired amount.

[0173] Preferably, incubation is carried out at 25°C to 35°C (e.g., 30°C), pH 6.5 to 8, and dissolved oxygen pressure (pO2) of 10% to 50% by increased agitation. When the bacteria are in the exponential phase, pO2 is adjusted to stimulate the secretion of extracellular polysaccharides into the culture medium.

[0174] During incubation, the bacterial strain secretes extracellular polysaccharides into the culture medium.

[0175] The second step: recovery of extracellular polysaccharides.

[0176] At the end of the incubation period, the culture medium (liquid) containing extracellular polysaccharides was separated from the biomass (solid). The liquid and solid phases were separated by continuous centrifugation at a speed of 9000 g.

[0177] The biomass contained in the solid phase can be removed or retained to be added to the extracellular polysaccharide obtained at the end of the method according to the invention or to a mixture of oligosaccharides after extraction.

[0178] When the liquid phase containing the extracellular polysaccharide has been recovered, the extracellular polysaccharide according to the invention is separated from other molecules contained in the liquid phase. In the case of a 100,000 Da membrane, tangential filtration is used.

[0179] Tangential filtration allows liquid molecules to be separated according to their size: molecules with a size less than 100,000 Da pass through the membrane and are removed. Extracellular polysaccharides have a size greater than 260,000 Da; they do not pass through the membrane and can therefore be recovered.

[0180] Thus, extracellular polysaccharides were obtained through unexpected molecular washing and concentration.

[0181] Optionally, the obtained extracellular polysaccharides may then undergo various types of processing: dilution, preservation, purification, separation by precipitation or by chromatographic fractionation, freeze drying, zeodration, or spray drying.

[0182] Characterization of extracellular polysaccharides

[0183] During fermentation, the secretion of extracellular polysaccharides can be monitored by size exclusion chromatography (SEC) in the aqueous phase with detection by an evaporative light scattering detector (ELSD).

[0184] The obtained extracellular polysaccharides were characterized by nuclear magnetic resonance (NMR) spectroscopy and direct injection high-resolution (HR) Orbitrap mass spectrometry.

[0185] Cross-analysis of the spectra identified the repetitive sequence that makes up the extracellular polysaccharide: Glu-Gal-NAcQ-Rham.

[0186] 2. Examples of preparing mixtures of oligosaccharides from extracellular polysaccharides

[0187] The extracellular polysaccharide obtained according to the invention by the method described in Part 1 above can be cleaved to obtain an oligosaccharide.

[0188] Cleavage was carried out via an acid hydrolysis step. The extracellular polysaccharide was mixed with a 0.2 M concentrated hydrochloric acid solution with a pH <1. The mixture was heated at 85°C to 95°C (e.g., 90°C) for 2 hours to 4 hours and 30 minutes.

[0189] Hydrolysis is terminated by using NaOH solution to raise the pH of the mixture to, for example, pH 3.

[0190] During the hydrolysis step, precipitates form in the liquid medium containing oligosaccharides. Therefore, a clarification step is performed. These precipitates can be removed by centrifugation, ultrafiltration, tangential filtration, or pressure filtration.

[0191] A mixture of oligosaccharides is obtained, which mainly contains the trisaccharides and / or heptasaccharides according to the invention.

[0192] Optionally, each oligosaccharide according to the invention can be separated from the remainder of the mixture of oligosaccharides by rapid chromatography on silica.

[0193] Characterization of the obtained oligosaccharide mixture

[0194] The oligosaccharides present in the mixture have a degree of polymerization greater than 2 and less than 12 and correspond to the assembly of sugar units of extracellular polysaccharides, such as N-acetylquinolamine, galacturonic acid and glucuronic acid, and rhamnose in the appropriate case.

[0195] The mixture of oligosaccharides contains approximately 85% oligosaccharides with the sequence Glu-Gal-NAcQ, primarily a primary sequence corresponding to a trisaccharide, and / or a secondary sequence corresponding to a heptasaccharide.

[0196] The major sequence Glu-Gal-NAcQ present in the oligosaccharide mixture was characterized by NMR spectroscopy and ORBITRAP ultra-high performance liquid chromatography-high resolution mass spectrometry.

[0197] Cross-analysis of the spectra determined:

[0198] - The structure of oligosaccharides is a linking of extracellular polysaccharide units: glucuronic acid, galacturonic acid, N-acetylquinolamine, and rhamnose in appropriate cases.

[0199] - Most of the oligosaccharides in the mixture of oligosaccharides are:

[0200] - Trisaccharides with the following linkages: Glu-Gal-NAcQ; and / or

[0201] - A hepta-saccharide with the following linkage: Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ;

[0202] - The mixture of oligosaccharides contains minor amounts of other oligosaccharides, such as:

[0203] - A tetrasaccharide with the following linkage: Glu-Gal-NAcQ-Rham;

[0204] - Undecanoic sugars with the following linkage: Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ.

[0205] 3. Examples of the preparation of active ingredients containing oligosaccharide mixtures, particularly for use in cosmetics.

[0206] The mixture of oligosaccharides according to the invention obtained in Part 2 above is dissolved in a physiologically acceptable medium containing water, a humectant, and preservation promoters such as pentylene glycol and propylene glycol to obtain an oligosaccharide concentration of 4,000 ppm to 7,000 ppm—for example, 5,000 ppm.

[0207] 4. Examples of compositions containing active ingredients

[0208] The following describes various cosmetic preparations containing the active ingredients according to the invention as described above in Part 3.

[0209] Where appropriate, additional active ingredients may be added to the suitable phase to support and / or supplement activity, depending on their hydrophobic, hydrophilic, or amphiphilic properties. These ingredients can belong to any category, such as terrestrial, benthic, marine, aerial, plant, or microbial sources such as bacteria, archaea, or fungi.

[0210] Where appropriate, these formulations may contain additional active ingredients to support and / or supplement the activity of the active ingredients according to the invention. These ingredients may belong to any category, depending on one or more of their functions, application site (body, face, neck, chest, hands, scalp, hair, body hair, etc.), desired end effect, and target consumer, such as anti-wrinkle, moisturizing, concealing, firming, anti-glycation, plumping, soothing, muscle relaxation, anti-redness, detoxification, etc.

[0211] In the context of this invention, the following additional active ingredients sold by Sederma are particularly advantageous:

[0212] - To enhance the activity of the active ingredients according to the present invention, such as BB-BIONT TM Or skin shine and smoothness; due to its anti-pollution activity, CITYSTEM™, PACIFEEL™ and / or SYNCHROLIFE TM AQUALANCE™ for its moisturizing properties; one or more products from the CERAMIDE 2™, CERAMIDE A2™, CERAMIDE HO3™ and / or DS-CERAMIDE™ series for their skin barrier-enhancing activity;

[0213] - As a supplement to the activity of the active ingredients according to the invention, such as MEL[O]STEM™ for its white and brown anti-spot activity; CALMOSENSINE for its skin-soothing activity. TM And / or PACIFEEL™; due to its anti-aging activity, MAJESTEM™ DRYPURE TM ; and especially due to its activity on dermal EMC, one or more products in the MATRIXYL™ series.

[0214] The International Cosmetic Ingredient Dictionary & Handbook, published by the Cosmetic, Toiletry, and Fragrance Association, Inc. (CTFA) of Washington, DC, describes a variety of non-limiting cosmetic and pharmaceutical ingredients commonly used in the skincare industry, suitable for use as additional ingredients in compositions according to the invention, provided they are physically and chemically compatible with the other ingredients of the composition, and especially with the active agents of the invention. Furthermore, the properties of these additional ingredients must not impair the benefits of the active agents of the invention. These additional ingredients may be synthetic or natural, such as plant extracts, or derived from bio-fermentation methods.

[0215] Other skin care active agents that can be used in combination with the compositions according to the invention can be found in the sales literature of Sederma, Crodarom and Alban Muller and on the website www.croda.fr.

[0216] As examples, the following commercial active agents could also be mentioned: betaine, glycerin, Actimoist Bio 2™ (Active Organics), AquaCacteen™ (Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia), Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (Arch Chemicals, Inc), Hydra'Flow™ (Sochibo), Hydromoist L™ (Symrise), RenovHyal™ (Soliance), Seamoss™ (Biotech Marine), Argireline™ (trade name for acetyl hexapeptide-3 from Lipotec), genistein, or longan chrysanthemum ( Boswellia serrata Extracts of ) (known as Gatuline Expression™ (Gattefossé)), frankincense ( HederaExtracts of Boswellin™, Deepaline PVB™ (SEPPIC), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), PhytoCellTec™ Argan (Mibelle), Papilactyl D™ (Silab), Preventhelia™ (Lipotec), or one or more of the following active ingredients sold by Sederma: Subliskin™, Venuceane™, Moist 24™, Vegesome Moist 24™, Essenskin™, Juvinity™, Revitat™, Resistem™, Chronodyn™, Kombuchka™, Chromocare™, Calmosensine™, Glycokin factor S™, Biobustyl™, Idealift™, Ceramide 2™, Ceramide A2™, Ceramide HO3™, Legance™, Intenslim™, Prodizia™, Beautifeye™, Pacifeel TM , Zingerslim™, Meiritage™, Sebuless™, Apiscalp™, Rubistem™, Citystem TM Neonyca TM ,NG Shea Butter Unsaponifiables TM Majestem TM Hydrogenesis TM Poretect TM Crystallide TM Amberstem TM , Synchrolife™, Sylverfree™, Feminage™, Ameyezing™, BB-Biont™, Revitalide™, Mel[o]stem TM Or a mixture thereof.

[0217] Among plant extracts (in the form of conventional extracts or prepared by in vitro methods) that can be used as additional active agents, the following extracts may be specifically mentioned: ivy (e.g., climbing ivy (English ivy)). helix Bupleurum chinensis ))), Bupleurum chinense ( Bupleurum falcatum ), Altai Bupleurum ( Arnica montanaArnica (Yangju) Rosmarinus officinalis L.)), rosemary (rosemary ( Calendula officinalis N.)), Calendula (Calendula ( Salvia officinalis )), Sage (European Sage ( Panax ginseng L.)), ginseng (ginseng ( Ginkgo biloba )),ginkgo( Hyperycum St. John's wort (St ... perforatum Ruscus aculeatus )) 、 Butcher's broom (butcher's broom) Filipendula ulmaria L.), Spiraea (Zootopia 'Zanthoxylum') Orthosiphon stamincus L.)), cat's whiskers (cat's whiskers ( Cynara scolymus Benth.), artichoke (Artichoke ( Fucus vesiculosus ), seaweed (fucus vesiculosus) Betula ), birch (white birch ( alba Cola nitida ), green tea, cola nuts (white cola ( Centella asiatica Horse chestnut, bamboo, centella asiatica ()), horse chestnut, bamboo, centella asiatica () Chrysanthellum indicum ), photinia, algae (wrack), willow, pilosella, horse chestnut extract, atractylodes extract, golden chamomile ( Armeniacea ) extract, apricot ( Atractylodis ) plants, platicodon Sinnomenum , Flemingia pharbitidis, genus *Pharbitis* C. forskohlii ), Coleus (such as Coleus pubescens) C. blumei ), Coleus ( C. esquirolii ), hairy calyx sheath stamen ( C. scutellaroides ), Five-colored Su ( C. xanthantus ), Yellow-sheathed stamen flower ( C. barbatus ) and hairy throat coleopterus ( Guioa Such as extracts from the roots of Coleus forsythia, extracts from Prunus armeniaca, and extracts from the genus Tripterygium (…). Davallia ), Drynaria fortunei ( Terminalia ), Terminalia genus ( Barringtonia ), genus *Gnaphalium* ( Trema ), genus *Hemp* ( Antirobia ), Cecropia genus *Horntree* ( Argania ), genus Caprifolia ( Dioscoreae ), Dioscorea ( Dioscorea opposita Such as yam ( Dioscorea mexicana ) or Mexican tortoise-shell dragon ( Ammi visnaga ), toothpick celery ( Siegesbeckia Extracts of ) and Siegesbeckia ( Siegesbeckia orientalis Especially Siegesbeckia orientalis (Ericaceae ) 、 From Ericaceae ( Vaccinium angustifolium Plant extracts (especially European blueberry (dwarf blueberry)) Arctostaphylos uva ursi )), Bearberry ( ​ Extracts of aloe vera ( ) Aloe vera Plants containing sterols (especially phytosterols) Manjistha (Madder genus) Rubia Plants, especially madder ( Rubia cordifolia (extracts) Guggal (Myrrh) Commiphora ) plants, especially myrrh (mukur myrrh) Commiphora mukul Extracts of ), Kola extract, chamomile, red clover, kava pepper ( Piper methysticum (From Sederma's Kava Kava), False Purslane ( Bacopa monieri (Bacocalmine™, Sederma) and sea whip, licorice ( Glycyrrhiza glabra ), mulberry, Melaleuca genus ( Melaleuca (Tea tree), Extremely forked Laria ( Larrea divaricata ), broken rice strips ( Rabdosia rubescens ), slender Euglena ( Euglena gracilis ) 、 Heavenly Vine ( Fibraurea recisa hirudinea ), Jungle sorghum ( Chaparral sorghum Sunflower, Green Flower Ennan Tomato ( Enantia chlorantha ), Buttongrass genus ( Spermacocea )of Mitracarpe , Buchu barosma , frothy flowers ( Lawsonia inermis L.), maidenhair fern ( Adiantium capillus-veneris L.), Celandine ( Chelidonium majus ), loofah ( Luffa cylindrica ), Japanese mandarin (Wenzhou mandarin ( Citrus reticulata blanco var. unshiu)), tea Camelia sinensis ), White Grass ( Imperata cylindrica ), Yellow Flower Sea Poppy ( Glaucium flavum Mediterranean cypress ( Cupressus sempervirens ), Kowloon Ring Polygonatum multiflorum ), Lovely Hemsleya, Elderberry ( Sambucus nigra ), cotton beans ( Phaseolus lunatus ), genus *Lysimachia* ( Centaurium Giant kelp Macrocystis pyrifera ), creeping clock flower (Turnera diffusa ), Anemarrhena asphodeloides ( Anemarrhena asphodeloides ), hairy purslane ( Portulaca pilosa ), Hops ( Humulus lupulus Small-bean coffee Coffea arabica Paraguayan holly ( Ilex paraguariensis ), Heart-leaved Ground Flower ( Globularia cordifolia ), acid wood ( Oxydendron arboreum ), Albizia ( Albizzia julibrissin ), red ginger ( Zingiber zerumbet Smith), Astragalus ( Astragalus membranaceus Atractylodes macrocephala ( ) Atractylodes macrocephalae ), longleaf plantain ( Plantago lanceolata ), Alpine Edelweiss ( Leontopodium alpinum (or eldelweiss), four o'clock flower ( Mirabilis jalapa ), dry celery ( Apium graveolens ), European summer solstice ( Marrubium vulgare ), large-leaved Buddleja ( Buddleja davidii Franch), American mint ( Monarda didyma ),Lavender( Lavandula angustifolia Orchid.

[0218] The compositions of the present invention may comprise peptides, including but not limited to dipeptides, tripeptides, tetrapeptides, pentapeptides, and hexapeptides and their derivatives. According to a particular embodiment, the concentration of one or more additional peptides in the composition ranges from 1 x 10⁻⁶. -7 weight% to 20% by weight, preferably 1x10 -6 From % to 10% by weight, preferably 1x10 -5 From 5% to 5% by weight.

[0219] In the context of this invention, the term "peptide" refers to peptides containing 10 or fewer amino acids, their derivatives, isomers, and complexes with other substances such as metal ions (e.g., copper, zinc, manganese, magnesium, etc.). The term "peptide" refers to both natural peptides and (bio)synthetic peptides. It also refers to compositions containing peptides that are found in nature and / or are commercially available.

[0220] Non-limiting examples of dipeptides used in the context of this invention include carnosine (β-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK, TT, PA, PM, or PP.

[0221] Non-limiting examples of tripeptides are RKR, HGG, GHK, GGH, GHG, GKH, KPK, KFK, KavaK, KβAK, KabuK, KacaK, KPK, KMOK, KMO2K (MO2 is sulfoxide methionine), PPL, PPR, SPR, QPA, LPA, or SPA.

[0222] Non-limiting examples of tetrapeptides are KTFK (SEQ ID NO: 7), GQPR (SEQ ID NO: 8), RSRK (SEQ ID NO: 9), KTAK (SEQ ID NO: 10), KAYK (SEQ ID NO: 11), KFYK (SEQ ID NO: 12), TKPR (SEQ ID NO: 13), AVPG (SEQ ID NO: 14), VPGA (SEQ ID NO: 15), LKLE (SEQ ID NO: 16), ELED (SEQ ID NO: 17), or LLAN (SEQ ID NO: 18).

[0223] Non-limiting examples of pentapeptides are KTTKS (SEQ ID NO: 19) and KTSKS (SEQ ID NO: 20).

[0224] Non-limiting examples of hexapeptides are GKTTKS (SEQ ID NO: 21) or VGVAPG (SEQ ID NO: 22).

[0225] Other peptides used in the context of this invention may be selected from (this list is not limiting): lipophilic derivatives of peptides, preferably palmitoyl (Pal) derivatives or myristoyl (Myr) derivatives, and metal complexes as described above (e.g., copper complexes of tripeptides HGG or GHK).

[0226] Preferred dipeptides include, for example, N-palmitoyl-β-Ala-His, Pal-KT, and Pal-RT (Sederma). Preferred tripeptide derivatives include, for example, copper derivatives of HGG (Lamin™, Sigma), Pal-GHK, Lipospondin (N-transoleoyl-KFK) and its conservatively substituted analogs, N-acetyl-RKR-NH2 (peptide CK+), Pal-KavaK, Pal-KβAlaK, Pal-KabuK, Pal-KacaK, Pal-KMO2K, N-Biot-GHK (Sederma), and its derivatives.

[0227] Here we can also mention the general formula X–Pro described in WO2015181688. –Pro –Xaa–Y is an anti-aging tripeptide, where Xaa is selected from Leu, Arg, Lys, Ala, Ser, and Asp, and X at the N-terminus is selected from H, -CO-R. 1 and -SO2-R 1 Furthermore, at the C-end, Y is selected from OH and OR. 1 NH2, NHR 1 or NR 1 R 2 R 1 and R 2 The groups are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, and aryloxy groups, which can be straight-chain, branched, cyclic, polycyclic, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfided. These groups may have heteroatoms in their main chain, particularly O, S, and / or N, and Pro Corresponding to proline, its analogues or derivatives; including, for example, Myr-PPL-OH and Myr-PPR-OH.

[0228] The general formula X–(Xaa1)n–Pro, published in WO2014 / 080376, can also be cited here. –Xaa2–Y is a staining-promoting and / or pro-MEC dipeptide and tripeptide, where n = 0, 1, or 2; Xaa1 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, Pro, and its analogues and derivatives; or a polar amino acid selected from Ser, Thr, Tyr, Asp, Glu, and its analogues and derivatives; and when n = 2, the two amino acids Xaa1 are the same or different; Xaa2 is a hydrophobic amino acid selected from Ala, Val, Met, Leu, Iso, Phe, and its analogues and derivatives. The compound, or a basic amino acid selected from Arg, Lys, His, and its analogues and derivatives; at the N-terminus, X is selected from H, -CO-R1, and -SO2-R1; at the C-terminus, Y is selected from OH, OR1, NH2, NHR1, or NR1R2; R1 and R2 are independently selected from alkyl, aryl, aralkyl, alkylaryl, alkoxy, and aryloxy groups, which can be straight-chain, branched, cyclic, polycyclic, saturated, unsaturated, hydroxylated, carbonylated, phosphorylated, and / or sulfided, said groups having or not having O, S, and / or N heteroatoms in their skeleton, and Pro Corresponding to proline, its analogues or derivatives; including, for example, the following peptides: Pal-SPR-OH, Pal-PPR-OH, Pal-QPA-OH, Pal-LPA-OH, Myr-SPA-OH, Pal-PM-OH, Pal-PA-OH and Pal-PP-OH.

[0229] Suitable tetrapeptide derivatives for use as other peptides according to the invention include, but are not limited to, Ela-KTAK (SEQ ID NO: 23), Ela-KAYK (SEQ ID NO: 24), Ela-KFYK (SEQ ID NO: 25), Pal-GQPR (SEQ ID NO: 3) or Pal-KTFK (SEQ ID NO: 5).

[0230] Suitable pentapeptide derivatives for use as other peptides in this article include, but are not limited to, Pal-KTTKS (SEQ ID NO: 2), Pal-KTSKS (SEQ ID NO: 4), and Pal-YGGFXaa (SEQ ID NO: 6), wherein Xaa is Leu or Pro.

[0231] Suitable hexapeptide derivatives for use as other peptides herein include, but are not limited to, Pal-HLDIIXaa (SEQ ID NO: 26) (where Xaa is Trp, Phe, Tyr, Tic, 7-hydroxy-Tic or Tpi, or a mixture thereof), Pal-GKTTKS (SEQ ID NO: 27), and Pal-VGVAPG (SEQ ID NO: 1).

[0232] Preferred compositions that are commercially available and sold by Sederma:

[0233] - Tripeptides or derivatives including Biopeptide-CL™, Maxi Lip™ or Procapil™ containing GHK;

[0234] - Tetrapeptides or derivatives include RIGIN™ and Eyeliss™ containing Pal-GQPR (SEQ ID NO: 3) and excipients;

[0235] - Pentapeptides or derivatives, such as Matrixyl™ containing Pal-KTTKS (SEQ ID NO: 2).

[0236] You can also mention:

[0237] - A mixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 3) (Matrixyl™ 3000), and

[0238] - A mixture of Pal-GHK and Pal-VGVAPG (SEQ ID NO: 1) (Biobustyl™)

[0239] The following peptides and other active ingredients may be mentioned:

[0240] - Vialox™, Syn-ake™ (β-Ala-Pro-Dab-NH-Bzl), or Syn-Coll™ (Pal-Lys-Val-Lys-OH) sold by Pentapharm;

[0241] - Argireline™ (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2 (INCI name = Acetyl Hexapeptide-3) (SEQ ID NO: 28), Leuphasyl™ (Tyr-D-Ala-Gly-Phe-Leu) (SEQ ID NO: 29), Aldenine™ (Gly-His-Lys), and Trylagen, marketed by Lipotec. TM (INCI name = Pseudomonas aeruginosa fermentation product extract, hydrolyzed wheat protein, hydrolyzed soybean protein, tripeptide-10 citrulline (reaction product of citrulline and tripeptide-10 (a synthetic peptide composed of aspartic acid, isoleucine and lysine)), tripeptide-1), Eyeseryl™ (Ac-β-Ala-His-Ser-His) (SEQ ID NO: 30), Serilesine™ (Ser-Ile-Lys-Val-Ala-Val) (SEQ ID NO: 31) or Decorinyl™ (INCI name: tripeptide-10 citrulline = reaction product of citrulline and tripeptide-10 (a synthetic peptide composed of aspartic acid, isoleucine and lysine);

[0242] - Collaxyl™ (Gly-Pro-Gln-Gly-Pro-Gln (SEQ ID NO:32)) or Quintescine™ (Cys-Gly) sold by Vincience;

[0243] - Cytokinol™ LS (casein hydrolysate) sold by Les Laboratoires Serobiologiques / Cognis;

[0244] - Kollaren™ (Gly-His-Lys), IP2000™ (Pal-Val-Tyr-Val), or Meliprene™ (INCI name = monofluoroheptapeptide-1: a reaction product of acetic acid and a synthetic peptide containing arginine, glycine, glutamic acid, histidine, leucine, p-fluorophenylalanine, and tryptophan) sold by l'institut Européen de Biologie Cellulaire;

[0245] - Neutrazen™ (Pal-His-D-Phe-Arg-NH2) sold by Innovations; or

[0246] - BONT-L-Peptide™, Timp-Peptide™, or ECMModuline™ sold by lnfinitec Activos.

[0247] It is also conceivable to combine the present invention with one or more cyclic peptides, particularly the cyclic peptides extracted from flaxseed oil described in the applicant’s patent application WO2019 / 149450.

[0248] Essence Form

[0249] [Table 1]

[0250]

[0251] plan:

[0252] Weigh and mix A. Weigh and mix B. While stirring, add B to A and mix. Weigh and mix C. While stirring, add C to B+A and mix. Add D to B+A+C and mix.

[0253] Milky fog form

[0254] [Table 2]

[0255]

[0256] plan:

[0257] Weigh and mix A. Weigh and mix B. While stirring, add B to A and mix. Activate A+B by vigorously mixing them. Weigh and mix C. While stirring, add C to B+A and mix. Add D to B+A+C and homogenize.

[0258] 5. Results of in vitro testing

[0259] The mixture of oligosaccharides obtained in Part 3 above was used for the following in vitro tests, denoted as "the active ingredient according to the invention".

[0260] 5.1. Protect against loss of radiance and the appearance of signs of skin aging.

[0261] 5.1.1 Resistance to oxidation

[0262] 5.1.1.1 Effects on reactive oxygen species

[0263] plan

[0264] Human keratinocytes (HK) were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 28 hours and subsequently subjected to intracellularly generated fluorescent probes designed to label ROS. Following incorporation, the cells became sensitive to oxidants. The cells were then either given or not given an agent designed to generate ROS (oxidative stress). Fluorescence readings (excitation (ex): 490 nm / emission (em): 520 nm) allowed for estimation of the amount of intracellular ROS. Cell numbers were estimated using the Hoechst method (DNA staining) to weight the obtained data.

[0265] result

[0266] Changes in ROS production in HK. The effect of 2% of the active ingredient according to the invention (n=6) compared to the control:

[0267] [Table 3]

[0268]

[0269] The results showed that 2% of the active ingredient according to the present invention significantly reduced ROS generation by -39.6% (p<0.01).

[0270] 5.1.1.2. Effects on protein Nrf2

[0271] plan

[0272] HK cells were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 2 hours or 24 hours. Subsequently, immunocytochemical labeling of the protein Nrf2 was performed, and its transfer from the cytoplasm to the nucleus was monitored by image analysis.

[0273] result

[0274] Changes in Nrf2 translocation over time in HK cultures. The effect of 3% of the active ingredient according to the invention (n=4) relative to the control:

[0275] [Table 4]

[0276]

[0277] The results showed that 3% of the active ingredient according to the invention significantly increased the translocation of protein Nrf2 by +41% (p<0.01) after 2 hours of contact and by +36% (p<0.05) after 24 hours of contact.

[0278] 5.1.1.3. NQO-1

[0279] plan

[0280] HK cells were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 6 hours. The HK carpet was then rinsed, and the cells were milled to extract their mRNA. These mRNAs were then converted into small DNA sequences, which were analyzed after being applied to a DNA microarray and amplified using a method closely related to qRT-PCR. Changes in mRNA due to the components according to the invention were compared with controls.

[0281] result

[0282] The results showed that 3% of the active ingredient according to the present invention significantly increased the production of protein NQO-1 (+67%). p <0.01 ).

[0283] 5.1.1.4. HMOX-1

[0284] plan

[0285] In the first stage, HK cells were cultured in their medium until confluence. The cells were then exposed to the components according to the invention for 54 hours. The cells were washed, dried, and cryopreserved. Following extraction, the amount of HMOX-1 was measured by ELISA.

[0286] After extraction, the amount of total protein was evaluated using the bicinchoninic acid method to weight the obtained data.

[0287] In the second stage, cells are contacted with 500 µM Trolox instead of the components according to the invention. Trolox is a close hydrophilic analog of vitamin E, known for its antioxidant activity and used to limit damage associated with oxidative stress.

[0288] result

[0289] Changes in the stimulation of HMOX-1 production. The effect of 3% of the active ingredient according to the invention at 54 hours (n=4) compared to the control:

[0290] [Table 5]

[0291]

[0292] The results showed that in both cases, the stimulation of HMOX-1 production was significantly increased, but 3% of the active ingredient according to the invention stimulated more HMOX-1 production (+65%; p<0.01 compared to the control) compared to 500 µMTrolox (+24%; p<0.01).

[0293] 5.1.2. Resistance to cellular hypoxia

[0294] 5.1.2.1. Mitochondrial potential ΔΨ, driving force

[0295] plan

[0296] HK cells were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 28 hours and then labeled with a fluorescent probe.

[0297] These probes were used to measure both forms and establish the monomer / polymer ratio (520 nm / 590 nm) by fluorescence reading. Higher values ​​of this ratio were less favorable for cell survival than lower values.

[0298] Cell counts were estimated using the Hoechst method (DNA staining) to weight the obtained data.

[0299] result

[0300] Changes in mitochondrial potential ΔΨ in HK. The effect of 3% of the active ingredient according to the invention (n=6) relative to the control:

[0301] [Table 6]

[0302]

[0303] The results showed that 3% of the active ingredient according to the present invention significantly increased the mitochondrial potential ΔΨ by +33.2% (p<0.01).

[0304] 5.1.2.2. Respiratory protection and oxygen consumption

[0305] plan

[0306] HK cells were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 24 hours. The HK cells were then detached from their support and placed in equal volumes in the wells of a plate equipped with sensors that measured the oxygen consumption of these cells over time.

[0307] result

[0308] Changes in oxygen consumption (oxygen pressure / min: pO2) in HK. The effect of 3% of the active ingredient according to the invention (n=6) relative to the control:

[0309] [Table 7]

[0310]

[0311] The results showed that 3% of the active ingredient according to the invention had a beneficial effect on cellular respiration, stimulating it by +23.6% (p<0.05).

[0312] 5.1.2.3. ATP Synthesis

[0313] plan

[0314] HK cells were cultured in their culture medium until confluence. The cells were then exposed to the components according to the invention for 26 hours. Intracellular ATP was extracted and measured by bioluminescence; the obtained signal was proportional to the amount of ATP in the cells.

[0315] Cell counts were estimated using a propidium iodide fluorescent probe to weight the obtained data.

[0316] result

[0317] Changes in ATP synthesis in HK. The effect of 3% of the active ingredient according to the invention (n=6) compared to the control:

[0318] [Table 8]

[0319]

[0320] The results showed that, compared with the control, 3% of the active ingredient according to the invention significantly increased ATP synthesis by +84% in a dose-dependent manner (p<0.01).

[0321] 5.1.3. Enhance barrier function

[0322] 5.1.3.1. Thickness of the stratum corneum

[0323] plan

[0324] An equivalent skin sample was prepared and contacted with the ingredients according to the invention for 2 days. The skin was then labeled with hematoxylin and eosin to visualize the macroscopic structure of the individual skin layers. This labeling was monitored by image analysis.

[0325] result

[0326] Changes in stratum corneum thickness. The effect of 3% of the active ingredient according to the invention (n=6) compared to the control:

[0327] [Table 9]

[0328]

[0329] The results showed that 3% of the active ingredient according to the present invention significantly increased the thickness of the stratum corneum by +43.4% (p<0.01).

[0330] 5.1.3.2. Inner lining protein and keratin desmosome

[0331] plan

[0332] An equivalent skin sample was prepared and exposed to the ingredients according to the invention for 2 days. The skin was then labeled with antibodies against inner lining protein and keratin desmosome. This labeling was monitored by image analysis (N = 57 to 60 photographs per case).

[0333] Cell counts were estimated using the Hoechst method (DNA staining) to weight the obtained data.

[0334] result

[0335] Changes in the synthesis of keratin and inner laminarin. The effect of 3% of the active ingredient according to the invention (n=5) relative to the control:

[0336] [Table 10]

[0337]

[0338] The results showed that 3% of the active ingredient according to the present invention significantly increased the production of inner lining protein (+42%; p<0.01) and keratinized desmosome (+46%; p<0.01). Therefore, the quality of barrier function was improved.

[0339] 5.1.4. Enhance moisturizing function

[0340] plan

[0341] Skin explants were prepared and the ingredients according to the invention were placed on their surfaces for 3 days. Subsequently, the explants were cut into 7 µm slices and labeled using antibodies against moisturizing markers: polyfilamentin and aquaporin-3. This labeling was monitored by image analysis (N = 41 to 45 images per case).

[0342] result

[0343] Changes in the synthesis of fimbriaein and aquaporin-3 in skin explants. The effect of 3% of the active ingredient according to the invention (n=3) relative to the control:

[0344] [Table 11]

[0345]

[0346] The results showed that 3% of the active ingredient according to the invention significantly increased both moisturizing markers.

[0347] 5.1.5. Enhance the function of the dermal-epidermal junction (DEJ)

[0348] plan

[0349] The same protocol was used for fibroin and aquaporin-3 for sealing protein-1, collagen IV, and collagen VII. Labeling was monitored via image analysis (N = 41 to 45 images per case).

[0350] result

[0351] Changes in the synthesis of sealing protein-1, collagen IV, and collagen VII in skin explants. The effect of 3% of the active ingredient according to the invention (n=3) relative to the control:

[0352] [Table 12]

[0353]

[0354] The results showed that 3% of the active ingredient according to the invention significantly increased the markers of DEJ (collagen IV and collagen VII) and the marker of tight junctions (sealing protein-1).

[0355] 5.1.6. Enhance microbial function

[0356] 5.1.6.1. Growth of Staphylococcus epidermidis

[0357] plan

[0358] The components according to the invention were mixed with a culture of Staphylococcus epidermidis (ATCC 14990) at a concentration of 4.10. 5 CFU The entire sample was contacted at 37°C in a culture medium that allowed the bacteria to grow. Growth kinetics were monitored by measuring optical density (OD) at 600 nm.

[0359] result

[0360] Growth kinetics of Staphylococcus epidermidis over time. Effect of 3% of the active ingredient according to the invention (n=3) relative to the control:

[0361] [Table 13]

[0362]

[0363] The results showed that 3% of the active ingredient according to the present invention significantly increased the growth of Staphylococcus epidermidis.

[0364] 5.1.6.2. Ceramide Synthesis in the Skin

[0365] plan

[0366] The keratinocytes in the culture were contacted with the components according to the invention for 24 hours. At the end of this contact, the cells were detached from the culture medium. The ceramide was quantified by an ELISA method. The cell number was standardized to harmonize the results.

[0367] result

[0368] Changes in the production of ceramides in the skin. The effect of 3% of the active ingredient according to the invention (n=4) compared to the control:

[0369] [Table 14]

[0370]

[0371] The results showed that 3% of the active ingredient according to the invention significantly increased the synthesis of ceramides in the skin.

[0372] 5.1.6.3. Ceramide Synthesis in Staphylococcus epidermidis

[0373] plan

[0374] Staphylococcus epidermidis was exposed to the component according to the invention for 24 hours. RNA was extracted and sphingomyelinase was quantified by RT-qPCR. sph Gene expression.

[0375] result

[0376] Sphingomyelinase sph Changes in gene expression. The effect of 3% of the active ingredient according to the invention (n=4) compared to the control:

[0377] [Table 15]

[0378]

[0379] The results showed that 3% of the active ingredient according to the present invention enabled sphingomyelinase to... sph Gene expression is significantly increased, and the sphingomyelinase of Staphylococcus epidermidis is able to convert sphingomyelin into ceramide and phosphocholine.

[0380] 5.1.6.4. Conclusion

[0381] All the in vitro results presented above demonstrate that the oligosaccharide mixture according to the invention protects against the growth of Staphylococcus epidermidis and the increased production of ceramides by Staphylococcus aureus and Staphylococcus epidermidis. Skin microbe function .

[0382] 5.2. Protection against pollution

[0383] The tests were conducted in the presence of atmospheric UMP to simulate the effects of pollution.

[0384] 5.2.1. Resistance to oxidation

[0385] 5.2.1.1. Effects on reactive oxygen species

[0386] plan

[0387] The effects of atmospheric pollutants on ROS formation were evaluated. The same protocol as described in Section 5.1.1.1 was used, and the UMP solution was contacted with and labeled as previously.

[0388] result

[0389] The generation of ROS in HK varies with the influence of atmospheric pollutants (n=6). In the presence of UMP, the effect of 2% of the active ingredient according to the invention relative to the control (n=6):

[0390] [Table 16]

[0391]

[0392] The results showed that pollutant microparticles increased ROS production by +26.9% (p<0.01), and 2% of the active ingredient according to the present invention could prevent the effects of pollution by significantly reducing the production of oxidative free radicals by -41.9% (p<0.01).

[0393] 5.2.1.2. Protein Nrf2

[0394] plan

[0395] The effects of atmospheric pollutants on the synthesis of the protein Nrf2 were evaluated. The same protocol was used as described in Section 5.1.1.2, and the cultured cells were contacted with and labeled with a solution of UMP or benzo[a]pyrene as previously described.

[0396] result

[0397] Changes in Nrf2 translocation over time in HK cultures. Effects of 3% of the active ingredient according to the invention, atmospheric UMP solution, and benzo[a]pyrene (B[a]P):

[0398] [Table 17]

[0399]

[0400] The results showed that, under all three conditions, the translocation of protein Nrf2 from the cytoplasm to the nucleus was significantly increased, but there were large differences in both time and intensity.

[0401] Regarding atmospheric UMP, the results showed a significant increase in translocation from 2 hours after exposure (+136%; p<0.01, relative to the control) and a very significant increase in translocation after 24 hours of exposure (+401%; p<0.01).

[0402] Regarding benzo[a]pyrene, one of the most well-known air pollutants and carried by UMP, the results showed a more moderate increase in translocation by +58% (p<0.05) after 2 hours of exposure and an exponential increase (+199%; p<0.01) after 24 hours of exposure.

[0403] Regarding contact with 3% of the active ingredient according to the invention, the results showed an increase of +41% (p<0.01) in the translocation of protein Nrf2 after 2 hours of contact and an increase of +36% (p<0.05) after 24 hours of contact.

[0404] These results were moderate compared to the other two cases (UMP and B[a]P), but significant relative to the control. This highlights the controlled “activation” of the cell’s detoxification system for the purpose of defending against contaminants.

[0405] 5.2.2. Resistance to cellular hypoxia

[0406] 5.2.2.1. Mitochondrial potential ΔΨ, driving force

[0407] plan

[0408] The effects of atmospheric pollutants on mitochondrial potential ΔΨ were evaluated. The protocol used was the same as in Section 5.1.2.1, and the atmospheric UMP solution was contacted with and labeled as previously.

[0409] result

[0410] Changes in mitochondrial potential ΔΨ in HK. The effect of 3% of the active ingredient according to the invention (n=6) relative to the control, with or without UMP:

[0411] [Table 18]

[0412]

[0413] The results showed that the pollutant microparticles reduced the mitochondrial potential ΔΨ by -72% (p<0.01), and 3% of the active ingredient according to the invention could prevent the effects of contamination by significantly increasing the mitochondrial potential ΔΨ by +49.6% (p<0.01).

[0414] 5.2.2.2. Respiratory protection and oxygen consumption

[0415] plan

[0416] The effects of atmospheric pollutants on oxygen depletion in HK were evaluated. The protocol used was the same as in Section 5.1.2.2, and the atmospheric UMP solution was contacted with the cultured cells as previously described.

[0417] result

[0418] Changes in oxygen consumption (oxygen pressure / min: pO2) in HK. The effect of 3% of the active ingredient according to the invention (n=6) relative to the control, with or without UMP:

[0419] [Table 19]

[0420]

[0421] The results showed that pollutant microparticles reduced cellular oxygen consumption by -29.8% (p<0.01), and 3% of the active ingredient according to the invention could prevent the effects of pollution by significantly increasing cellular respiration by +24.2% (p<0.01).

[0422] 5.2.2.3. Protection against ATP production

[0423] plan

[0424] The effects of atmospheric pollutants on ATP synthesis in HK were evaluated. The protocol used was the same as in Section 5.1.2.3, and the atmospheric UMP solution was contacted with the cultured cells as previously described.

[0425] result

[0426] Changes in ATP synthesis in HK. The effect of the 3% oligosaccharide mixture relative to the control, with or without UMP:

[0427] [Table 20]

[0428]

[0429] The results showed that the pollutant microparticles reduced ATP production by -21% (p<0.01), and 3% of the active ingredient according to the invention could prevent the effects of pollution by significantly increasing ATP synthesis by +74% (p<0.01).

[0430] 5.2.2.4. Conclusion

[0431] All the in vitro results presented above indicate that the mixture of oligosaccharides according to the present invention protects cells against oxidation and hypoxia, with the aim of protecting cellular detoxification from micropollutants.

[0432] 5.2.3. Enhance the skin barrier

[0433] 5.2.3.1. Thickness of the stratum corneum

[0434] plan

[0435] The effects of atmospheric pollutants on the thickness of the stratum corneum were evaluated. The same protocol as in Section 5.1.3.1 was used, and the atmospheric UMP solution was brought into contact with the equivalent skin.

[0436] result

[0437] Changes in the thickness of the stratum corneum. Effects of 3% of the active ingredient according to the invention (n=4):

[0438] [Table 21]

[0439]

[0440] The results showed that pollutant microparticles reduced the thickness of the stratum corneum by -61.3% (p<0.01), and that 3% of the active ingredient according to the invention could prevent the effects of pollution by significantly increasing the thickness of the stratum corneum by +136.4% (p<0.01). This demonstrates the positive effect of the ingredients according to the invention on combating the harmful effects of pollution.

[0441] 5.3. Comparison Results

[0442] The monosaccharides and monosaccharide acids (i.e., N-acetylquinolones, galacturonic acid, glucuronic acid, and rhamnose) in the oligosaccharide mixture were tested individually and as a mixture to allow for comparison with the active ingredient according to the invention. The amounts tested were:

[0443] - 50 ppm rhamnose;

[0444] - 50 ppm of N-acetylquinolones;

[0445] - 50 ppm galacturonic acid;

[0446] - 50 ppm glucuronic acid;

[0447] - In a mixture of three sugars: 50 ppm N-acetylquinoline, 50 ppm galacturonic acid, and 50 ppm glucuronic acid;

[0448] - In a mixture of four sugars: 50 ppm N-acetylquinoline, 50 ppm rhamnose, 50 ppm galacturonic acid and 50 ppm glucuronic acid.

[0449] 5.3.1. ATP Synthesis

[0450] plan

[0451] The scheme used is the same as that in section 5.1.2.3.

[0452] result

[0453] Changes in ATP synthesis in HK. Comparison of the effects of sugar alone or as a mixture with 3% of the oligosaccharide according to the invention (n=8) relative to the control:

[0454] [Table 22]

[0455]

[0456] The results showed that, compared with the active ingredient according to the invention, monosaccharides (alone or in combination) had no effect on ATP production. Therefore, the oligosaccharides according to the invention, i.e., monosaccharides bonded together, play a role in ATP production.

[0457] 5.3.2. Kinetics of Staphylococcus epidermidis

[0458] plan

[0459] The scheme used is the same as that in section 5.1.6.

[0460] result

[0461] Growth kinetics of Staphylococcus epidermidis over time. Comparison of the effects of sugar alone or as a mixture of sugar and 3% oligosaccharides (n=7) relative to the control:

[0462] [Table 23]

[0463]

[0464] The results showed that, compared with the active ingredient according to the present invention, monosaccharides (alone or in combination) had no effect on the production of Staphylococcus epidermidis. 。Therefore, it is undeniable that the oligosaccharides according to the present invention, i.e., monosaccharides bonded to each other, have an effect on the growth of Staphylococcus epidermidis.

[0465] 6. Results of in vivo testing

[0466] Test product

[0467] The serums described in Table 1 above are compared to placebo serums that have the same base as the serums but do not contain the active ingredients according to the invention.

[0468] principle

[0469] In two supplemental studies, the efficacy of the cosmetic treatment according to the invention was evaluated in a total of 181 volunteers: one group of Caucasians and one group of Chinese. Group members were required to have dull skin tone and irregular skin texture.

[0470] Measurement methods are evaluated through the use of expert analysis (judgment) and self-evaluation (perceived effects).

[0471] plan

[0472] The study of Caucasian volunteers was conducted in France, with a maximum of 54 volunteers, who were women with an average age of 38 (20–50 years old).

[0473] The study of Chinese volunteers was conducted in China, with a maximum of 127 volunteers, men and women with an average age of 35 (18–50 years).

[0474] Study type, duration and application

[0475] The volunteers were unaware of the type of product (the active ingredient or placebo according to the invention).

[0476] According to Table 25 below, a group of 54 Caucasian women was divided into two groups and various measurements and data were collected before and after administration:

[0477] [Table 24]

[0478]

[0479] A group of 127 Chinese volunteers applied a sheet mask infused with the essence according to the invention four times over 14 days, for 15 minutes each time. After the first application and after the fourth and final application, participants evaluated their perceived effectiveness of the essence according to the invention in the mask using a self-evaluation questionnaire.

[0480] statistics

[0481] For instrument parameters, statistical calculations are performed using Student...t The test is performed, or, if necessary, using a nonparametric Wilcoxon or Mann-Whitney test. A one-tailed test is performed on unpaired series.

[0482] For the values ​​in the questionnaire, statistical calculations were performed using the χ² test. 2 The study proceeded. A level of 0.05 was chosen as the minimum acceptance point for statistical significance.

[0483] 6.1. Evaluation of skin tone luster

[0484] The Skin Luminosity Index (SRI) is based on a calculation that takes into account four areas of the face—the forehead, nose, area under the eyes, and cheeks—and three colorimetric components, namely L... (This represents lightness or brightness), H (which represents the hue of a color), and IWA (Individual White Angle) (an index developed by Newtone Technologies to evaluate skin whiteness, taking into account the parameter L). a and b (CIELAB or CIELCH color space).

[0485] plan

[0486] Using Headscan V05 TM The camera station (Orion Concept, France) captures facial photographs using a cross-polarization mode to eliminate natural light.

[0487] For this evaluation, only 52 Caucasian volunteers participated: 26 received the serum containing the active ingredient according to the invention, and 26 received a placebo serum. The following results are averages of measurements taken on various parts of the face.

[0488] result

[0489] Changes in skin radiance index after 28 days of application. Effect of 3% of the active ingredient according to the invention relative to placebo (N=52):

[0490] [Table 25]

[0491]

[0492] DNS: Data not significant

[0493] The results showed that, in the case of the essence according to the invention, the skin radiance index increased significantly (+3.3%; p<0.05, compared to placebo). This indicates that the ingredients according to the invention can improve skin radiance.

[0494] 6.2. Evaluation of micro-protrusions

[0495] Multiple parameters were studied to demonstrate the advantages of the active ingredients according to the invention for treating skin microprotrusions.

[0496] The first parameter is called complexity. It represents the ratio between the actual surface and the ideal apparent surface.

[0497] The second parameter is called the maximum amplitude. It corresponds to the average height of the highest protrusion. It conveys changes in the skin.

[0498] The third parameter is called roughness. It corresponds to the average roughness of facial skin. The greater the roughness, the less smooth the skin.

[0499] plan

[0500] This technique projects lines and shadows (stripes) onto the face and measures the corresponding deformations associated with skin protrusions using triangulation. This allows for the reconstruction of protrusions in three dimensions.

[0501] For this evaluation, only 53 Caucasian volunteers were included: 26 received the serum containing the active ingredient according to the invention, and 27 received the placebo serum.

[0502] Measurements were performed on the cheek, and surface roughness was analyzed based on the acquisition results to qualitatively characterize the smoother or rougher appearance of skin texture (micro-protrusions, pores, fine lines). The following results are averages of the various measurements performed.

[0503] result

[0504] Changes in complexity, maximum amplitude, and roughness after 28 days of application. Effect of 3% of the active ingredient according to the invention relative to placebo (N=53):

[0505] [Table 26]

[0506]

[0507] The results showed that the essence according to the invention significantly reduced skin complexity, maximum amplitude, and roughness after 28 days of application. This reduction in these three parameters resulted in smoother, brighter, and more radiant facial skin.

[0508] 6.3. Evaluation of moisturizing effect

[0509] Skin hydration involves the formation and maintenance of an effective skin barrier to fight off external aggressors, protect against allergens, limit moisture loss, make skin stronger, smoother and firmer, and contribute to skin radiance.

[0510] plan

[0511] MoistureMeter-D™ is a device for measuring skin hydration using the principle of impedance measurement.

[0512] In this study, six regions were examined in parallel: forehead, temples, cheekbones, cheeks, jawline, and chin. The following results are averages of the various measurements performed and are used to represent the overall hydration of the volunteers' faces. Measurements were taken at least 12 hours after the last application.

[0513] For this evaluation, 54 Caucasian volunteers were included: 27 received the serum containing the active ingredient according to the invention, and 27 received a placebo serum. Of the 54 volunteers, 24 had dry skin: 13 received the serum containing the active ingredient according to the invention, and 11 received a placebo serum.

[0514] result

[0515] Changes in hydration after 28 and 56 days of application. Effect of 3% of the active ingredient according to the invention relative to placebo:

[0516] [Table 27]

[0517]

[0518] The results showed that, compared to the placebo, the essence according to the invention significantly increased skin hydration after 28 and 56 days of application. Furthermore, extraction of "dry skin" indicated a significant increase in long-term hydration from the essence according to the invention.

[0519] Because the measurements were taken at least 12 hours after the last application of the product to the skin, the results demonstrate the truly beneficial effects of long-term skin hydration.

[0520] 6.4. Evaluation of Skin Firmness

[0521] Skin firmness is one of the most closely watched parameters because it is related to youth, skin strength, and its elasticity.

[0522] plan

[0523] The Cutometer® is used to measure the viscoelastic parameters of the skin. This instrument measures the deformation and recovery capacity of skin areas subjected to repeated mechanical stress. The instrument provides graphs showing the change in skin deformation over time, such as […]. Figure 1As shown in the image. Figure 1 The text indicates parameters Uf and R10, representing the skin's elasticity and "fatigue susceptibility," respectively.

[0524] In this study, three measurements were performed on the right and left sides of the face. The results for stretchability are the average of the various measurements performed, and the results for resilience are the average of the last measurements performed on the right and left sides of the face.

[0525] For this evaluation, 54 Caucasian volunteers were included: 27 received the serum containing the active ingredient according to the invention, and 27 received the placebo serum.

[0526] result

[0527] Changes in stretchability (Uf) and resilience (R10) after 56 days of application (N=54). Effect of 3% of the active ingredient according to the invention relative to placebo:

[0528] [Table 28]

[0529]

[0530] The results showed that the essence according to the invention significantly reduced stretchability and skin fatigue, thereby indicating an increase in skin firmness.

[0531] 6.5. Perception of the effects of the components according to the present invention

[0532] 6.5.1. In the Caucasian group

[0533] plan

[0534] Volunteers’ opinions were collected using a self-evaluation questionnaire 56 days after application of the serum or placebo serum containing the active ingredient according to the invention.

[0535] result

[0536] Number of favorable opinions for each statement. Effect of 3% of the active ingredient according to the invention relative to placebo:

[0537] [Table 29]

[0538]

[0539] The results showed that, compared to the placebo serum, the serum according to the invention had a significantly more favorable perceived effect. Team members found that their skin was immediately and lastingly more oxygenated, purified, and moisturized, and looked more beautiful.

[0540] 6.5.2. In the Chinese group

[0541] plan

[0542] Volunteers’ opinions were collected using a self-evaluation questionnaire 56 days after applying a sheet mask soaked in an essence or placebo essence containing the active ingredients according to the present invention.

[0543] result

[0544] Number of advantageous opinions regarding the essence of the invention:

[0545] [Table 30]

[0546]

[0547] The results showed that after four applications of a cloth mask infused with the essence of the present invention at short time intervals, the perceived quality of the skin was improved.

[0548] 6.6. Conclusion

[0549] Overall, the in vivo results presented above indicate that the treatment according to the invention advantageously enhances the skin's radiance, making it appear brighter and more revitalized, moisturizing it, smoothing bumps, increasing its firmness, and reducing skin fatigue.

Claims

1. An extracellular polysaccharide, characterized in that, It contains at least one monosaccharide unit that is an N-acetylquinolamine.

2. The extracellular polysaccharide according to claim 1, characterized in that, It also contains at least one monosaccharide acid unit selected from glucuronic acid and galacturonic acid.

3. The extracellular polysaccharide according to claim 1 or 2, characterized in that, It also contains at least one monosaccharide unit as rhamnose.

4. The extracellular polysaccharide according to any one of claims 1 to 3, comprising 15% to 55% by weight of N-acetylquinolamine relative to the total weight of the extracellular polysaccharide.

5. The extracellular polysaccharide according to any one of claims 1 to 4, comprising 15% to 55% by weight of glucuronic acid and / or 15% to 55% by weight of galacturonic acid relative to the total weight of the extracellular polysaccharide.

6. The extracellular polysaccharide according to any one of claims 1 to 5, comprising 15% to 55% rhamnose relative to the total weight of the extracellular polysaccharide.

7. The extracellular polysaccharide according to any one of claims 1 to 6, comprising an equimolar mixture of N-acetylquinoline, glucuronic acid, galacturonic acid and rhamnose.

8. The extracellular polysaccharide according to any one of claims 1 to 7, characterized in that, It comprises a link of three units, which contain N-acetylquinolamine and two monosaccharide acid units selected from glucuronic acid and galacturonic acid.

9. The extracellular polysaccharide according to claim 8, characterized in that, It also contains rhamnose.

10. The extracellular polysaccharide according to any one of the preceding claims, characterized in that, It has a molecular weight of 260,000 Da or greater.

11. The extracellular polysaccharide according to any one of the preceding claims, characterized in that, It was obtained from a bacterial strain of the genus *Pseudomonas* deposited at the National Center for Microbial Collections (CNCM) in Paris under the number I-5893. Pseudoalteromonas sp .).

12. An oligosaccharide, which can be obtained, particularly, by cleaving an extracellular polysaccharide according to any one of the preceding claims, characterized in that, It contains N-acetylquinolamine.

13. The oligosaccharide according to claim 12, characterized in that, It has a degree of polymerization of 2 to 12.

14. The oligosaccharide according to claim 12 or 13, characterized in that, It also contains at least two monosaccharide acid units selected from glucuronic acid and galacturonic acid.

15. The oligosaccharide according to any one of claims 12 to 14, characterized in that, It is composed of N-acetylquinoline, glucuronic acid, and galacturonic acid.

16. The oligosaccharide according to any one of claims 12 to 15, characterized in that, It contains glucuronic acid, galacturonic acid and N-acetylquinolamine.

17. A method for obtaining an extracellular polysaccharide according to any one of claims 1 to 11, comprising the following steps: - The first step of culturing a bacterial strain of the genus *Pseudomonas*, deposited at the National Center for Microbial Collections (CNCM) in Paris with the number I-5893, in a culture medium suitable for bacteria that secrete the extracellular polysaccharides in the culture medium. - A second step to recover the extracellular polysaccharides isolated from the culture medium.

18. The method according to claim 17, characterized in that, The extracellular polysaccharide present in the culture medium is separated from the solid biomass by a method that can separate the liquid phase from the solid phase.

19. The method according to claim 18, characterized in that, The extracellular polysaccharide was separated from the culture medium by tangential filtration.

20. The method according to any one of claims 17 to 19, characterized in that, The additional step of cleaving the extracellular polysaccharide into at least one oligosaccharide according to any one of claims 12 to 16.

21. The method according to claim 20, characterized in that, The pyrolysis is carried out by acid hydrolysis.

22. An oligosaccharide mixture, which can be obtained by the method according to claim 20 or 21, and is mainly composed of trisaccharides and / or heptasaccharides.

23. The oligosaccharide mixture according to claim 22, characterized in that, The trisaccharide has a linkage of glucuronic acid, galacturonic acid, and N-acetylquinolamine (Glu-Gal-NAcQ).

24. The oligosaccharide mixture according to claim 22, characterized in that, The seven sugars are linked by: glucuronic acid, galacturonic acid, N-acetylquinoline, rhamnose, glucuronic acid, galacturonic acid, and N-acetylquinoline (Glu-Gal-NAcQ-Rham-Glu-Gal-NAcQ).

25. A bacterial strain of the genus *Pseudomonas* deposited at the National Center for Microbial Collections (CNCM) in Paris, numbered I-5893.

26. A cosmetic or dermatological composition comprising an extracellular polysaccharide according to any one of claims 1 to 11 and a physiologically acceptable medium.

27. A cosmetic or dermatological composition comprising at least one oligosaccharide according to any one of claims 12 to 16 or a mixture of oligosaccharides according to any one of claims 22 to 24 and a physiologically acceptable medium.

28. Use of the extracellular polysaccharide according to any one of claims 1 to 11 or the composition containing the polysaccharide according to claim 26 in industrial sectors such as chemical, food, cosmetic, pharmaceutical, veterinary, agricultural, environmental, or paint and coating industries.

29. Use of at least one oligosaccharide according to any one of claims 12 to 16, or a mixture of oligosaccharides according to any one of claims 22 to 24, or a composition containing the oligosaccharide according to claim 27, for non-therapeutic cosmetic treatment of the skin and its appendages.

30. The use according to claim 28 or 29, characterized in that, The cosmetic treatment is localized.

31. The use according to claim 29 or 30, characterized in that, The cosmetic treatment is suitable for: - Improves skin radiance; - Improves the bumps on the skin; - Improves the firmness of the skin; and / or - Improves the skin's hydration.

32. The use according to any one of claims 29 to 31, characterized in that, The cosmetic treatment is a process to protect the skin from aging and / or pollution, particularly air pollution.

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