Method for preparing non-covalent molecular complexes comprising hyaluronic acid and a peptide fraction, cosmetic and pharmaceutical compositions comprising same and uses thereof

Abstract

The invention relates to a method for preparing a molecular complex comprising a peptide fraction and hyaluronic acid with a molecular weight of between 1 and 10 kDa, characterized in that the complex is not bound by covalent bonds. The invention also relates to cosmetic compositions comprising such a molecular complex and to the cosmetic uses thereof for reducing the signs of skin aging. The invention also relates to compositions comprising such a molecular complex for the uses thereof in esthetic and regenerative medicine for improving healing, limiting inflammation, or for the uses thereof for repairing cartilage and treating osteoarthritis.

Description

Description Title of the invention: METHOD FOR PREPARING MOLECULAR COMPLEXES Non-covalent compounds comprising hyaluronic acid and a peptide fraction, cosmetic and pharmaceutical compositions comprising them and their uses technical field

[0001] The present invention belongs to the field of ingredients comprising hyaluronic acid. It relates more particularly to a method for preparing covalently non-bonded molecular complexes, comprising hyaluronic acid and at least one peptide fraction, and their cosmetic and pharmaceutical uses. Technical background of the invention

[0002] Hyaluronic acid, or hyaluronan, is a linear glycosaminoglycan polymer with a molar mass ranging from 100 to 10,000 kDa. It is naturally present in the extracellular matrix of epithelial, nervous, and connective tissues in vertebrates. It can form bonds with proteins, lipids, or carbohydrates to create supramolecular structures capable of trapping large quantities of water and ions.

[0003] In joints, hyaluronic acid contributes to lubrication, protects areas of friction, and maintains cartilage elasticity. In the skin, it contributes to hydration, turgor, and tissue cohesion. It also plays a role in controlling angiogenesis, inflammation, and wound healing.

[0004] These multiple properties, coupled with high biocompatibility and biodegradability, have made hyaluronic acid a widely used molecule in medicine, bioengineering and cosmetics.

[0005] However, the hyaluronic acid content decreases in the skin during aging, particularly in the epidermis, and its degradation is increased in certain inflammatory or degenerative pathologies such as osteoarthritis.

[0006] Peptides are also widely used in cosmetics, primarily in the form of short-chain peptides of synthetic or natural origin. Examples include tripeptide-1, with the sequence GHK, which may or may not be copper-bound and stimulates collagen production, and certain peptides marketed by Ashland, such as the peptide with the sequence (Gly-Pro-Gln)2, which is marketed under the name ColloxylTM, described in European patent No. 1406589, for its effectiveness in the treatment of skin and hair aging, the tripeptide with the trade name ATPeptide™ described in patent No. FR2846883), the hexapeptide with the trade name Peptide vinci™ 02 described in patent FR284178, or the dimerized dipeptide with the trade name Quintescine™ described in patent No. EPI 79 188B1.

[0007] Numerous peptide extracts have also been described for their beneficial properties on the skin. These are generally aqueous plant extracts or fermentation products that can be hydrolyzed, which improves their tolerance and bioavailability.

[0008] Many cosmetic formulas contain both peptides and hyaluronic acid, such as those described in patent documents EP1815843A2, KR2023134758 and CN1 16942584. However, nothing in these documents suggests that the formulations have been optimized to allow the formation of complexes between hyaluronic acid and peptides.

[0009] Hybrid molecules of hyaluronic acid and peptides, formed by the creation of covalent bonds between the two compounds, are also known in the prior art. These are widely used in the cosmetic industry as injectable dermal fillers or for topical application. This involves the use of crosslinking agents or the prior chemical modification of at least one of the two components. For example, document WO2017098474A1 describes a hyaluronic acid composition whose polymer chain has been modified to carry hydrophobic groups capable of covalently binding to an antimicrobial peptide.

[0010] One of the problems posed by these techniques is the toxicity, or even the carcinogenic effects, of the crosslinking agents that can be released into the body during the degradation of hyaluronic acid (Xue Y, et al. Synthesis of hyaluronic acid hydrogels by crosslinking the mixture of high-molecular-weight hyaluronic acid and low-molecular-weight hyaluronic acid with 1,4-butanediol diglycidyl ether. RSC Adv. 2020;10(12):7206-7213; Faivre J et al. Crosslinking hyaluronic acid soft-tissue fillers: current status and perspectives from an industrial point of view. Expert review of medical devices vol. 18,12 (2021): 1175-1187).

[0011] Other documents describe the chemical functionalization of hyaluronic acid molecules to enable the creation of interchain amino covalent bridges, to improve the in vivo stability of a hydrogel for aesthetic use (US1 1224566B2), or the creation of disulfide covalent bridges to prepare complexes useful in skin care (CN1 169661 14A).

[0012] Patent document EP3527194B1 describes a method for preparing composite particles comprising hyaluronic acid (high molecular weight, greater than 100 kDa) and at least one peptide that does not involve chemical reactions. In this case, the peptide must be in the form cationic, at a pH less than or equal to 5. This method makes it possible to obtain particles of a defined size (less than 500 nm) which are thought to improve skin penetration. However, this technique requires adjusting the pH to a value lower than the pKa of each peptide so that it is entirely in its cationic form.

[0013] To overcome the technical problems of the state of the art, the inventors have developed a process for preparing molecular complexes comprising at least one peptide and non-crosslinked and non-functionalized hyaluronic acid, to produce a molecular complex exhibiting satisfactory stability and biological properties superior to molecules used in isolation.

[0014] One advantage of the process described in this application is that it eliminates the need to operate under pH conditions lower than the pKa of the peptide(s) used in order to form cations capable of self-assembly with anionic hyaluronic acid polymers.

[0015] Another advantage of the process described below is that it is simple to implement, more natural and therefore more environmentally friendly than prior art processes, since it does not involve prior chemical modifications of the compounds, nor the use of potentially toxic crosslinking agents.

[0016] To the inventors' knowledge, no prior art document describes the preparation process of the present application. Summary of the invention

[0017] The invention has as its first object a process for preparing a molecular complex comprising a peptide fraction and hyaluronic acid, characterized in that the molecular complex is not linked by covalent bonds and is obtained by the following process: a) solubilize the hyaluronic acid powder of a molecular weight between 3 kDa and 10 kDa, and preferably between 3 kDa and 5 kDa, in an aqueous solvent, preferably water, at a concentration between 5000 ppm and 10,000 ppm under moderate stirring, for a time between 5 min and 30 minutes, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C;b) add the peptide fraction in powder form, comprising at least one synthetic peptide or one purified from plants, algae, fungi or yeasts, comprising 3 to 12 amino acids and preferably 3 to 9 amino acids, into the solution prepared in the previous step, at a concentration between 10 ppm and 25000 ppm, preferably between 30 ppm and 5500 ppm, under moderate stirring, at a temperature between 20°C and 50°C; preferably between 20°C and 30°C; c) Adjust the pH to a value between 2 and 3.5, preferably between 2.5 and 3; d) leave under medium stirring for a period of between 30 min and 3 hours, preferably 1 hour, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C; e) adjust the pH to a value between 4.5 and 6.5, preferably between 4.5 and 5.5.

[0018] The second object of the invention is the molecular complex obtained according to the process of the invention.

[0019] The invention has as its third object a composition comprising an effective amount of the molecular complex of the invention as an active agent and a physiologically acceptable medium.

[0020] The fourth object of the invention is the cosmetic use of the composition of the invention to reduce the signs of skin aging and in particular to firm the skin, improve its elasticity and hydration, reduce and limit the appearance of wrinkles and fine lines and also improve the homogeneity of the complexion.

[0021] The invention has as its fifth object a composition according to the invention for its use in aesthetic and regenerative medicine to improve healing, limit inflammation, or for its use to repair cartilage and treat osteoarthritis. Brief description of the figures

[0022] The following figures illustrate the advantages arising from the invention and the non-limiting embodiments presented in the description:

[0023] [Fig. 1] Comparison of the molecular structures of the molecular complexes obtained according to example 1 and at different pH by FTIR.

[0024] [Fig. 2] Evaluation of the effectiveness of the molecular complex of example 1 on the expression of different types of collagen and the synthesis of hyaluronic acid in ex vivo skin biopsies.

[0025] [Fig. 3] Demonstration of the synergistic properties of the molecular complex of example 1 on the expression of collagen I in ex vivo skin biopsies.

[0026] [Fig. 4]: Evaluation of the effect of the molecular complex obtained according to the process of example 1 compared to the mixture from process 2 according to example 2.

[0027] [Fig. 5]: Demonstration of the texturizing effect of the hybrid in example 1 formulated at 5%. a) evaluation made by consumer volunteers and b) evaluation made by experts.

[0028] [Fig. 6]: Demonstration of the hydrating properties of the hybrid of example 1 formulated at 5% in the short term and long term. Detailed description of the invention Definitions

[0029] All terms used in this description have their most widely understood meanings, unless otherwise stated. For the purposes of the invention, the following terms are defined as follows:

[0030] In this application, "hyaluronic acid" means an unbranched carbohydrate polymer formed by alternating units of glucuronic acid and N-acetylglucosamine, having a molecular weight between 3 kDa and 10 kDa.

[0031] When a range of values ​​is described, the boundaries of that range must be understood as explicitly including the upper and lower bounds of said range, as well as all intermediate values ​​within the range. For example, a range of values ​​between 1% and 10% must be understood as including 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, as well as all decimal values ​​between 1% and 10%.

[0032] Numerical values ​​in percentage and ppm (parts per million) are by weight, that is, the weight of a compound relative to the total weight of the mixture under consideration, unless otherwise specified.

[0033] The compositions described in this application may "include", "consist of" or "consist essentially of", the essential compounds or the optional ingredients.

[0034] "Consist essentially of" means that the composition or component may include additional ingredients, but only if the additional ingredients do not alter the basic characteristics or new characteristics of the composition or use described in this application.

[0035] The term "physiologically acceptable medium" means a solvent suitable for any route of administration in the body, for example oral route or injection, and more particularly for contact with the external layers of the skin, scalp or hair, without toxicity, irritation, undue allergic response and similar or intolerance reaction, and proportionate to a reasonable benefit / risk ratio, at the concentrations used.

[0036] By "effective quantity" is meant the minimum quantity of molecular complex according to the invention which is necessary to obtain at least one of the desired biological activities.

[0037] The term "Tripeptide-1" refers to the peptide with the sequence H-Gly-His-Lys-OH

[0038] "Hexapeptide-9" refers to the peptide consisting of the sequence (SEQ ID: No. 1), H-Gly-Pro-GIn-Gly-Pro-GIn-OH, marketed under the name CollaxylTM biofunctional by Ashland.

[0039] In this application, "molecular complex(s)" means one or more complexes formed of hyaluronic acid and a peptide fraction, not covalently linked, in solution, obtained by self-assembly.

[0040] In this application, "self-assembly" means the phenomenon of spontaneous association of molecules of different nature to form supramolecular structures not covalently linked to each other.

[0041] In this application, the term “peptide fraction” means at least one synthetic peptide or one purified peptide from plants, algae, fungi or yeasts, comprising from 3 to 12 amino acids and preferably from 3 to 9 amino acids,

[0042] In this application, "hybrid" means a particular formulation comprising 5% to 40% of the molecular complex of the invention, incorporated into a cosmetic vector composed of water, glycerin and 1% to 10% of a mixture of several carboxymethyl celluloses of different grades and degrees of substitution.

[0043] The term “peptide” refers to a molecule composed of 2 to 12 amino acids linked together by peptide bonds, of natural or synthetic origin, linear or cyclic, substituted or unsubstituted.

[0044] The term “texturizing effect” refers to an effect that improves the consistency and feel of a cosmetic composition. Preparation process

[0045] The present invention relates to a new method for preparing molecular complexes of hyaluronic acid and a peptide fraction, capable of self-assembling in a non-covalent manner.

[0046] The invention thus relates as its primary object a process for preparing a molecular complex comprising a peptide fraction and hyaluronic acid, characterized in that the molecular complex is not linked by covalent bonds and is obtained by the following process: a) solubilizing hyaluronic acid powder with a molecular weight between 3 kDa and 10 kDa, and preferably between 3 kDa and 5 kDa, in an aqueous solvent, preferably water, at a concentration between 5000 ppm and 10000 ppm under moderate stirring, for a time between 5 min and 30 min, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C; b) adding the peptide fraction comprising at least one synthetic peptide or one purified from plants, algae, fungi, or yeasts, comprising of 3 to 12 amino acids and preferably of 3 to 9 amino acids, in the solution prepared in the previous step, so as to obtain a concentration of between 10 ppm and 25000 ppm, preferably between 30 ppm and 5500 ppm, then leave under moderate stirring, at a temperature of between 20°C and 50°C; preferably between 20°C and 30°C; c) adjust the pH to a value of between 2 and 3.5, preferably between 2.5 and 3; d) leave under medium stirring for a time of between 30 min and 3 hours, preferably 1 hour, at a temperature of between 20°C and 50°C, preferably between 20°C and 30°C; e) adjust the pH to a value of between 4.5 and 6.5, preferably between 4.5 and 5.5.

[0047] The spatial organization of hyaluronic acid is controlled by the combination of physicochemical factors such as molecular weight, biopolymer concentration, temperature, pH, and dissolution method.

[0048] Hyaluronic acid of intermediate or high molecular weight, i.e. greater than 100 kDa, at a concentration less than 1 mg / ml is in the form of long supercoiled chains, stabilized by numerous inter-chain interactions (De Souza A. et al., Carbohydrate Polymers 222 (201), 1 15001). This spatial conformation is not very favorable to the formation of complexes by self-assembly with peptides, such as those present in the peptide fraction described in the present invention.

[0049] The inventors have shown that it is advantageous, in order to implement the process of the present invention, to use hyaluronic acid of low molecular weight, that is to say less than 10 kDa, and to place oneself in conditions which favor the spatial conformation of hyaluronic acid in a double helix.

[0050] The process of the present invention is therefore based on the rearrangement of the spatial architecture of hyaluronic acid in solution into short, antiparallel double helices. This spatial conformation of hyaluronic acid promotes its non-covalent self-assembly with peptides to form stable molecular complexes.

[0051] In step a) of the process, the hyaluronic acid is in powder form, and the solvent is an aqueous solvent that may optionally contain different types of solutes. Preferably, the solvent is pure water.

[0052] The method of stirring can include any method known to a person skilled in the art, such as magnetic stirring.

[0053] The solubilization time should be between 5 min and 30 minutes and is proportional to the molecular weight of the hyaluronic acid used as well as its concentration.

[0054] At the end of step a) the hyaluronic acid is at a concentration between 5000 ppm and 10000 ppm.

[0055] In step b) of the process, the peptide fraction is added to the hyaluronic acid solution obtained in the previous step. The peptide fraction is in powder form.

[0056] Solubilization is carried out under moderate stirring at a temperature between 20°C and 50°C, preferably between 20°C and 30°C. The stirring method may include any method known to those skilled in the art, such as magnetic stirring. This step continues until all components are completely dissolved.

[0057] To perform step b), the peptide fraction may contain one or more peptides, which may have a linear or cyclic structure and be of natural or synthetic origin. When the peptides are synthetic, they may or may not carry chemical modifications well known to those skilled in the art, such as one or more amidations, carboxylations, myristoylations, palmitoylations, or farnesylations, or any combination thereof. When the peptides are synthetic, they may or may not be associated with a vitamin, trace element, or any other compound to form associations well known to those skilled in the art.

[0058] The concentration of peptide fraction can be between 10 ppm and 25000 ppm, preferably between 30 ppm and 5500 ppm, by weight relative to the total weight of the solution obtained at the end of step b).

[0059] In a particular embodiment, the peptide fraction is composed of at least one synthetic peptide of a length of 3 to 12 amino acids, and preferably of 3 to 9 amino acids.

[0060] In one particular embodiment, the peptide fraction comprises at least tripeptide-1 (of sequence GHK).

[0061] In a particular embodiment, the peptide fraction comprises at least some of the peptide of general formula (I) comprising the sequence (Gly-Pro-Gln)n, where n is between 1 and 3 and the carboxyl end is either amidated or not. When n equals 3, then the peptide has the sequence (SEQ ID No. 2).

[0062] In one particular embodiment, the peptide fraction comprises at least hexapeptide-9 of sequence (Gly-Pro-Gln)2 (SEQ ID No 1).

[0063] In another particular embodiment, the peptide fraction comprises hexapeptide-9 (SEQ ID: No. 1) and tripeptide-1.

[0064] In this last particular embodiment, tripeptide-1 is present at a concentration between 500 ppm and 5000 ppm, preferably at a concentration between 1000 ppm and 3000 ppm, by weight relative to the total weight of solution from step b).

[0065] In this same particular embodiment, hexapeptide-9 is present at a concentration between 20 ppm and 500 ppm, preferably at a concentration between 50 ppm and 300 ppm, by weight relative to the total weight of solution from step b) (or molecular complex).

[0066] Alternatively, the process for preparing the present application can also be carried out with a peptide fraction comprising at least one peptide from plants, algae, fungi or yeasts, comprising from 3 to 12 amino acids and preferably from 3 to 9 amino acids.

[0067] The most advantageous mass ratio between hyaluronic acid and the peptide fraction is between 10 / 1 and 2 / 1, preferably between 4 / 1 and 2 / 1.

[0068] Steps a) and b) can be carried out in either the reverse order indicated above.

[0069] In step c) of the process, the pH is adjusted to a value between 2 and 3.5, preferably between 2.5 and 3, using an acid such as citric, lactic or hydrochloric acid, preferably citric acid.

[0070] Indeed, at pH levels close to 2.5 to 3, the literature reports significant structural changes in hyaluronic acid, with the formation of antiparallel double helices (Snetkov, Petr et al. “Hyaluronic Acid: The Influence of Molecular Weight on Structural, Physical, Physico-Chemical, and Degradable Properties of Biopolymer.” Polymers vol. 12,8 1800. 1 1 Aug. 2020).

[0071] However, at a pH below 2.5, hyaluronic acid is in single-strand form, which is very rapidly hydrolyzed.

[0072] The process of the invention thus exploits the ability of hyaluronic acid to form these particular structures at pH levels close to 2.5 to 3, allowing the formation of molecular complexes by self-assembly with a peptide fraction.

[0073] In step d) the stirring method may include any methods known to a person skilled in the art, such as magnetic stirring.

[0074] In step e) the pH is adjusted to a value between 4.5 and 6.5, preferably between 4.5 and 5.5, by adding a basic solution, preferably concentrated sodium hydroxide.

[0075] This increase in pH allows the peptide(s) contained in the peptide fraction to be trapped by the abrupt modification of the spatial organization of the hyaluronic acid double helices formed in step c). This step allows higher-order molecular self-assembly, and the formation of stable molecular complexes.

[0076] The inventors were thus able to demonstrate that the process of the present application leads to the formation of stable molecular complexes, which was not suggested in the state of the art.

[0077] The solution obtained in step e) constitutes the molecular complex within the meaning of the invention.

[0078] In one particular embodiment, the process can be continued by a step (f) in which the molecular complex is diluted in a cosmetic carrier such as a mixture of water, glycerin, and cellulose derivatives. The cellulose derivatives used can be sodium carboxymethyl celluloses, marketed under the name blanose™ carboxymethylcellulose (CMC) by Ashland. For example, several CMCs with different grades and degrees of substitution can be mixed. The total concentration of CMCs can be between 1% and 10%, and preferably between 1% and 3%.

[0079] This particular mixture is described as a hybrid in this application.

[0080] Advantageously, the cosmetic vector consists of 40% to 52% water, 47% to 57% glycerin and 1% to 3% of a mixture of several carboxymethyl celluloses.

[0081] The molecular complex can thus be incorporated into this cosmetic carrier at a concentration of between 5% and 40%, preferably at a concentration of between 5% and 15%, and even more preferably at a concentration of 10%, by weight relative to the total weight of the mixture.

[0082] When incorporated into the above cosmetic vector, the molecular complex of the invention forms a mixture described as a hybrid. Molecular complexes

[0083] The second object of the invention is the molecular complex that can be obtained according to steps a) to e) of the process described above.

[0084] In one particular embodiment, the molecular complex comprises a peptide fraction including hexapeptide-9 (SEQ ID: No. 1) and tripeptide-1.

[0085] In this last particular embodiment, tripeptide-1 is present at a concentration between 500 ppm and 5000 ppm, preferably at a concentration between 1000 ppm and 3000 ppm, by weight relative to the total weight of the molecular complex.

[0086] In this same particular embodiment, hexapeptide-9 is present at a concentration between 20 ppm and 500 ppm, preferably at a concentration between 50 ppm and 300 ppm, by weight relative to the total weight of the molecular complex.

[0087] Comparative analysis by FTIR (Fourier Transform Infrared Spectrometry) showed that the molecular complex of the invention has a specific structure, different from the structures obtained by comparative processes, as illustrated in Figure 1.

[0088] Stability studies have shown that the molecular complex of the invention exhibits remarkable stability after 15 days at 50 °C.

[0089] The invention further relates to the hybrid that can be obtained according to steps a) to f) of the process described above, that is to say that the molecular complex at a concentration of between 5% and 40%, preferably at a concentration of between 5% and 15%, and even more preferably at a concentration of 10%, by weight relative to the total weight of the mixture, has been incorporated into a cosmetic vector comprising 40% to 52% water, 47% to 57% glycerin and 1% to 3% of a mixture of several carboxymethyl celluloses. Compositions

[0090] A third object of the invention is a composition comprising, as an active agent, an effective amount of the molecular complex described above and a physiologically acceptable medium.

[0091] Advantageously, the molecular complex is present in the composition at a concentration of between 0.01% and 10% by weight relative to the total weight of the composition, preferably at a concentration of between 0.05% and 5% by weight relative to the total weight of the composition, and even more preferably at a concentration of between 0.1% and 2% by weight relative to the total weight of the composition.

[0092] In a particular embodiment of the invention, the molecular complex is pre-diluted to a concentration of between 5% and 40%, preferably between 5% and 15%, and even more preferably to 10% in a cosmetic vector such as a mixture of water, glycerin and cellulose derivatives to form a hybrid.

[0093] Advantageously, the cosmetic carrier comprises 40% to 52% water, 47% to 57% glycerin and 1% to 3% of a mixture of several carboxymethyl celluloses.

[0094] In this particular embodiment, the composition comprises from 1% to 10% hybrid and preferably 5% hybrid by weight relative to the total weight of the composition.

[0095] When incorporated into the above cosmetic vector, the molecular complex of the invention forms a composition described as a hybrid, which exhibits rheological parameters and a texturizing effect are particularly sought after in a cosmetic composition.

[0096] The composition of this application is formulated to be administered by any appropriate route, including injection, oral route, or external topical route, and the formulation of the compositions will be adapted by a person skilled in the art.

[0097] Preferably, the composition of the present application is in a form suitable for topical application and comprises a physiologically acceptable medium, particularly suited to this type of application.

[0098] The compositions may include, in particular, aqueous, hydroalcoholic or oily solutions or gels, oil-in-water, water-in-oil or multiple emulsions; they may also be presented as suspensions or powders, suitable for application to the skin, mucous membranes, lips and / or hair.

[0099] The composition can be more or less viscous and may also have the appearance of a cream, lotion, fluid, milk, serum, ointment, gel, paste, balm, or mousse. It can also be in solid form, such as a stick, or applied to the skin as an aerosol.

[0100] Examples of physiologically acceptable media commonly used in the intended field of application include formulation adjuvants such as solvents, thickeners, gelling agents, diluents, emulsifiers, antioxidants, colorants, sunscreens, self-tanning agents, pigments, fillers, preservatives, perfumes, odor absorbers, essential oils, vitamins, essential fatty acids, surfactants, film-forming polymers, esters, vegetable oils or butters, etc.

[0101] In all cases, a person skilled in the art will ensure that these adjuvants and their proportions are chosen in such a way as not to impair the advantageous properties sought in the composition according to the invention.

[0102] Advantageously, the composition may include, in addition to the active agent, i.e. the molecular complex, at least one other active agent having similar and / or complementary cosmetic effects to those of the invention.

[0103] Such additional active agents may also be chosen according to their chemical composition, from the group including: amino sugars, glucosamine, D-glucosamine, N-acetylglucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetylmannosamine, galactosamine, N-acetylgalactosamine, vitamin B3 and its derivatives, niacinamide, sodium dehydroacetate, dehydroacetic acid and its salts, phytosterols, salicylic acid compounds, hexamidines, dialkanoyl dihydroxyproline compounds, soy extracts and derivatives, equol, isoflavones, flavonoids, phytantriol, farnesol, geraniol, bisabolol, peptides and their derivatives, di-, tri-, tetra-, penta-, and hexapeptides and their derivatives, lys-thr-thr-lys- Ser, palmitoyl-lys-thr-thr-lys-ser, carnosine, N-acyl amino acid compounds, retinoids, retinyl propionate, retinol, retinyl palmitate, retinyl acetate, retinal, retinoic acid, water-soluble vitamins, ascorbates, vitamin C, ascorbyl glucoside, ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate, vitamins and their salts and derivatives, provitamins and their salts and derivatives, panthenol ethyl, vitamin A and its derivatives, vitamin B and its derivatives, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin E, vitamin F, vitamin K and its derivatives, pantothenic acid and its derivatives, pantothenyl ethyl ether, panthenol and its derivatives, ethyl panthenol, dexpanthenol, biotin, amino acids and their salts and derivatives, amino acids water-soluble, asparagine, alanine, indole, glutamic acid, water-insoluble vitamins, beta-ionol, cedrol, and their derivatives, water-insoluble amino acids, tyrosine, tryptamine,particulate materials, butylated hydroxytoluene, butylated hydroxyanisole, allantoin, tocopheryl nicotinate, tocopherol, tocopherol esters, palmitoylglycerol, phytosterol, hydroxy acids, glycolic acid, lactic acid, lactobionic acid, keto acids, pyruvic acid, phytic acid, lysophosphatidic acid, stilbenes, cinnamates, resveratrol, kinetin, zeatin, dimethylaminoethanol, natural peptides, soy peptides, acidic sugar salts, manganese gluconate, zinc gluconate, piroctone olamine, 3,4,4'-trichlorocarbanilide, triclocarban, zinc pyrithione, hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucoside, pyridoxine, aloe vera, terpene alcohols, allantoin, bisabolol, dipotassium glycyrrhizate, glycerol acid, sorbitol, pentaerythritol, pyrrolidone and its salts, dihydroxyacetone, erythrulose, glyceraldehyde, tartaraldehyde, clove oil, menthol, camphor,eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate, eicosene-vinylpyrrolidone copolymer, iodopropyl butylcarbamate, a polysaccharide, an essential fatty acid, a salicylate, glycyrrhetinic acid, carotenoids, ceramides and pseudoceramides, a complex lipid, oils of general natural origin such as shea butter, apricot oil, evening primrose oil, prune oil, palm oil, monoi oil, kahai oil, hydroquinone, HEPES, procysteine, O-octanoyl-6-D-maltose, disodium salt of methylglycine diacetate, steroids such as diosgenin and DHEA derivatives, DHEA dehydroepiandrosterone and / or a chemical or biological precursor or derivative, N-ethylcarbonyl-4-para-aminophenol, alpha hydroxy acids, beta hydroxy acids, moisturizers, epidermal hydrolytic enzymes, plant extracts, phytohormones, yeast extracts,metalloproteinase inhibitors, enzymes, enzyme inhibitors, enzyme inducers, coenzymes, chelating agents, plant extracts and plant derivatives, essential oils, marine extracts, agents from a biofermentation and / or biotechnology process, mineral salts, cell extracts.

[0104] As examples, we can also mention the peptides marketed by Ashland under the names Chronogen™, Laminixyl™ IS, Peptide Q10™, Collaxyl™ (patent FR2827170, Ashland), Peptide vinci™ 01 (patent FR2837098, Ashland), Peptide vinci™ 02 (patent FR2841781, Ashland), ATPeptide™ (patent FR2846883, Ashland) and Artemia salina extract, marketed under the name GP4G™ (FR2817748, Ashland). Uses

[0105] The fourth object of the invention is the cosmetic use of one of the compositions comprising the molecular complex or hybrid described above to reduce at least one sign of skin aging chosen from: firming the skin, improving its elasticity and hydration, reducing and limiting the appearance of wrinkles and fine lines and also improving the homogeneity of the complexion.

[0106] The term "cosmetic use" means that the use is intended for individuals with healthy skin, scalp, or hair.

[0107] By "signs of skin aging" we mean any change in the external appearance of the skin due to aging such as, for example, the appearance of wrinkles and fine lines at the corners of the lips and crow's feet, sagging, loss of elasticity, firmness and / or tone of the skin, the appearance of a duller and less even complexion but also any internal changes in the skin which do not systematically result in a changed external appearance such as, for example, thinning of the skin, or any internal degradation of the skin resulting from external aggressions producing free radicals, such as pollution and solar radiation including UV.

[0108] The extracellular matrix is ​​one of the primary determinants of facial morphology, therefore the molecular complex of the invention was tested on the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid and showed a positive effect on the expression of these compounds.

[0109] The invention thus relates to the cosmetic use of the composition comprising the molecular complex or hybrid described above to increase the expression of collagens I, III, V, VI, VIII, XI, XIIX, XXIV, XXVII, involved in skin aging and more generally in skin laxity and the appearance of wrinkles and fine lines and to increase the expression of collagens IV, VII, XVII present in the dermo-epidermal junction and also to increase the expression of hyaluronic acid.

[0110] The composition of the invention has been tested on the expression of collagen I and has shown a positive effect superior to the effect obtained by using each compound separately, which demonstrates a synergistic effect between the different components of the molecular complex. [01 1 1 ] The invention further relates to the cosmetic use of the hybrid described above to increase the texturizing effect of a formulation.

[0112] The invention further relates to a cosmetic treatment method for reducing at least one sign of skin aging selected from: firming the skin, improving its elasticity and hydration, reducing and limiting the appearance of wrinkles and fine lines and also improve the evenness of skin tone, including the topical application on at least one area of ​​skin to be treated, of one of the compositions described above.

[0113] The invention also relates to pharmaceutical compositions comprising such a molecular or hybrid complex which may be in the form of injectable compositions, for oral administration or for topical application, for their uses to stimulate the expression of hyaluronic acid and collagens in various applications in aesthetic and regenerative medicine.

[0114] In particular, the invention relates to a composition comprising the molecular complex for its use to improve healing, limit inflammation, or for its use to repair cartilage and treat osteoarthritis. Examples

[0115] The present invention is illustrated by means of the following non-limiting examples: Example 1: Preparation of a molecular complex comprising hyaluronic acid with a molecular weight of 3 kDa to 5 kDa and two peptides

[0116] Principle: the aim of this experiment is to obtain a molecular complex made up of hyaluronic acid with peptides whose molecular interactions are as strong as possible.

[0117] Protocol: The hyaluronic acid used is marketed under the name microHATM Super Active Hyaluronic Acid by Bloomage Biotech. It is derived from bacterial fermentation, is in powder form, and has a molecular weight between 3 kDa and 5 kDa.

[0118] a) Hyaluronic acid in powder form is solubilized in 1 liter of osmosis water at a concentration of 6000 ppm, at room temperature (20 °C) under medium intensity magnetic stirring for 5 minutes.

[0119] b) Tripeptide-1 at a concentration of 2000 ppm and hexapeptide-9 at a concentration of 200 ppm, in powder form, are solubilized at room temperature under medium stirring for 20 minutes in the solution obtained in step a). The initial pH of this solution is 7.2.

[0120] c) The pH is adjusted by adding 50% diluted citric acid to reach a pH value of 2.8.

[0121] d) The solution is maintained for 1 hour under medium magnetic stirring, at room temperature, at acidic pH 2.8.

[0122] e) The pH is readjusted to a value of 5.0 by the addition of concentrated sodium hydroxide.

[0123] Results: the solution containing the molecular complex obtained is colorless and clear.

[0124] Optionally, the molecular complex obtained above can be diluted to a concentration of 10% in a cosmetic carrier composed of 40% to 52% water, 47% to 57% glycerin, and 1% to 3% of a mixture of several carboxymethyl celluloses of varying grades and degrees of substitution. This particular mixture has the advantage of being easily incorporated into a finished cosmetic formulation and is described in this application as a "hybrid." Example 2: FTIR characterization of the molecular complex obtained according to process 1 of Example 1, in comparison with mixtures obtained at different pH levels

[0125] Principle: The aim of this experiment is to validate the interest of the process of example 1, and in particular the role of pH adjustments, by comparing the molecular structures of the different mixtures of hyaluronic acid and peptides obtained according to different preparation processes, by FTIR (Fourier Transform Infrared Spectrometry).

[0126] The infrared spectrum obtained is between 600 and 4000 cm-1 and can be divided into two:

[0127] The part of the "functional groups" from -4000 cm-1 to 1500 cm-1 is a region of the spectrum that is easily analyzable.

[0128] The "footprint region" ranges from 1500 cm-1 to 600 cm-1. This is a complex area to interpret, having only comparative value.

[0129] Protocol: The three processes carried out are presented below in Table 1:

[0130] [Table 1]

[0131] The solutions obtained according to the 3 processes above are frozen at -80°C and then lyophilized to obtain a powder that can be analyzed by FTIR spectrometry on a Perkin Elmer Spectrum 100 FT-IR spectrometer equipped with a diamond ATR module which allows the analysis of powders without special preparation.

[0132] The powders are deposited so as to completely cover the diamond of the FT-IR spectrometer. The samples are then pressed with a pressure force of 80 using a gauge, in order to remove the air trapped between the grains.

[0133] Results: The spectra of the three molecular complexes obtained by FTIR, presented in Figure 1, show different profiles in both the functional group region and the imprint region. The hyaluronic acid and peptide mixtures obtained using the three methods have different molecular structures and also possess a specific imprint region.

[0134] Measuring the correlation factors between the different spectra allows us to appreciate the difference in structure between the different mixtures obtained.

[0135] Indeed, identical spectra have a correlation of 1, but the lower the correlation factor, the more different the spectra, and therefore the molecular structures, are.

[0136] Thus, the correlation factor between the spectra of the mixtures obtained by processes 1 and 3 is low (0.163234), demonstrating significant differences in their structure. Conclusion: pH adjustments cause significant changes in the structure of the resulting molecular complexes. Example 3: Evaluation of the stability of molecular complexes by spectrophotometry and colorimetry

[0137] Principle: The aim of this experiment is to evaluate the stability of the molecular complex obtained according to example 1 (process 1) and the mixture obtained according to process 2 of example 2 described in Table 1, and in particular to evaluate the impact of pH changes on stability.

[0138] Protocol: The solutions obtained by processes 1 and 2 (described in Table 1) were placed at 50°C for 15 days. The differences in appearance of the solutions were then analyzed by spectrophotometry by measuring the parameters L*, a*, and b*. L* represents the luminance of the solution, a* and b* express the difference in color compared to a colorless solution, with a* representing the value on a green-red axis and b* on a blue-yellow axis.

[0139] The color of the solutions was measured according to the Gardner scale, which allows the colors of liquids to be compared on a scale from 0 to 18; 0 being a colorless liquid and 18 being a dark brown liquid.

[0140] Result: On day 0, both solutions are clear and colorless. After 15 days at 50°C, the molecular complex from Example 1 (Process 1) has colorimetric parameters similar to those on day 0, whereas the molecular complex obtained by Process 2, i.e., without pH adjustment, turned yellow. The results are presented in Table 2.

[0141] [Table 2] stable molecular complex after 15 days at 50 °C, unlike the molecular complex obtained without pH adjustment. Example 4: Evaluation of the effect of the hybrid comprising the molecular complex on the expression of different types of collagen and hyaluronic acid

[0143] Principle: The aim of this experiment is to demonstrate an effect of the hybrid obtained according to example 1 and therefore comprising 10% of molecular complex on the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid in ex vivo human skin biopsies.

[0144] Protocol: The expression of different collagens and hyaluronic acid is evaluated by indirect immunofluorescence on skin biopsies, pre-treated by topical application of the hybrid obtained according to Example 1 at a concentration of 5% diluted in PBS, for 48 hours (twice a day). Control biopsies incubated in parallel under the same conditions receive Phosphate Buffer Saline (PBS). At the end of incubation, the biopsies are fixed and embedded in paraffin and cryostat mounting medium for histological sectioning.

[0145] The detection of different collagens and hyaluronic acid is performed by incubation with antibodies against Collagen I (Proteintech), Collagen III (Proteintech), Collagen IV (Merck Millipore), Collagen V (Proteintech), Collagen VI (Proteintech), Collagen VII (Sigma-Aldrich), Collagen XVII (Abeam), and a biotinylated HABP (hyaluronan-binding protein) probe (Merck Millipore). After two hours of incubation followed by rinsing, the sections are incubated with a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagens I, III, V, VI, and XVII. The sections are incubated in the presence of the anti-mouse secondary antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagens IV and VIL. A biotin / streptavidin system coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) is used to detect hyaluronic acid.The sections are then examined using an epifluorescence microscope (Nikon Eclipse Ni), controlled by NIS-Element software (Nikon). Expression levels are quantified by image analysis (Volocity® software, Improvision).

[0146] Results: When biopsies were treated with the 5% hybrid, the expression of collagens I, III, IV, V, VI, VII, XVII and hyaluronic acid increased significantly after 48 hours of treatment compared to control samples. The results are shown in Figure 2.

[0147] Conclusion: The hybrid obtained according to example 1, comprising 10% of molecular complex, showed a positive effect on the expression of collagens I, III, IV, V, VI, VII, XVII and the synthesis of hyaluronic acid in ex vivo human skin biopsies. Example 5: Evaluation of the synergistic effect of the hybrid including the molecular complex on the expression of collagen I

[0148] Principle: The aim of this experiment is to demonstrate a synergistic effect of the hybrid obtained according to example 1 comprising 10% molecular complex on the expression of collagen I in ex vivo human skin biopsies.

[0149] Protocol: The hybrid processing of example 1 is carried out as in example 4.

[0150] The treatments under controlled conditions are carried out as follows:

[0151] Stock solutions of hyaluronic acid at 600 ppm, hexapeptide-9 at 20 ppm and tripeptide-1 at 200 ppm, were diluted to 5% in PBS.

[0152] Collagen I detection is performed by incubation with anti-Collagen I antibody (Proteintech). After two hours of incubation followed by rinsing, the sections are incubated with a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagen I. The sections are then examined using an epifluorescence microscope (Nikon Eclipse Ni), controlled by NIS-Element software (Nikon). Finally, expression levels are quantified by image analysis (Volocity® software, Improvision).

[0153] Results: When biopsies were treated with the 5% hybrid, collagen I expression increased more significantly than when biopsies were treated separately with 5% tripeptide-1, 5% hexapeptide-9, or 5% hyaluronic acid, as shown in Figure 3.

[0154] Conclusion: The hybrid comprising 10% of the molecular complex from Example 1 showed a synergistic effect on collagen I expression, since the effects of the hybrid are greater than the average increase in collagen I expression specific to each of the compounds taken separately. Example 6: Evaluation of the effect of the molecular complex obtained according to the method of Example 1 compared to method 2 of Example 2 on the expression of collagens I and IV

[0155] Principle: The aim of this experiment is to compare the effect of the molecular complex obtained according to process 1 of example 1 versus the effect of the mixture obtained according to method 2 of example 2 on the expression of collagens I and IV in ex vivo human skin biopsies.

[0156] Protocol: The expression of these two collagens is evaluated by indirect immunofluorescence on skin biopsies pre-treated by topical application of the molecular complex obtained according to procedure 1 at a concentration of 0.5% diluted in PBS for 48 hours (twice a day). In parallel, skin biopsies are treated by topical application of the mixture from procedure 2 of Example 2 at a concentration of 0.5% diluted in PBS for 48 hours (twice a day). Control biopsies incubated in parallel under the same conditions receive Phosphate Buffer Saline (PBS). At the end of incubation, the biopsies are fixed and embedded in paraffin for histological sectioning.

[0157] The detection of collagens I and IV is performed by incubation with anti-Collagen I (Proteintech) and anti-Collagen IV (Merck Millipore) antibodies. After two hours of incubation followed by rinsing, the sections are incubated with a secondary anti-rabbit antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagen I. The sections are then incubated with a secondary anti-mouse antibody coupled to a fluorophore (Alexa Fluor® 488, Invitrogen) to detect collagen IV. The sections are then examined using an epifluorescence microscope (Nikon Eclipse Ni), controlled by NIS-Element software (Nikon). Expression levels are quantified by image analysis (Volocity® software, Improvision).

[0158] Results: When biopsies were treated with the molecular complex obtained according to method 1 and with the mixture obtained according to method 2 at 0.5%, the expression of collagens I and IV increased significantly after 48 hours of treatment compared to control samples. The increase in collagen I and IV expression observed in biopsies treated with the molecular complex obtained according to method 1 was significantly greater than the increase observed following treatment of biopsies with the mixture obtained according to method 2. The results are presented in Figure 4.

[0159] Conclusion: The molecular complex obtained according to process 1 of example 1 showed a superior effect on the expression of collagens I and IV, compared to a mixture obtained according to process 2. Example 7: Formulation of the hybrid from Example 1 in an aqueous gel

[0160] [Table 3]: List of Ingredients for the formulation of an aqueous gel [ref.. # 12951 ]

[0161] Description: transparent gel

[0162] pH: 5.5 - 6.5

[0163] viscosity: 3.000 - 6.000 Example 8: Evaluation of the effect of the hybrid according to example 1 on the texture of a cream

[0164] Principle: evaluate the texturizing effect of the hybrid according to example 1, formulated at 5%, and compare its texture to a cream containing 5% silicone

[0165] Protocol: comparative double-blind study against a cream containing 5% silicone, conducted on the back of the hand of 8 expert volunteers and 10 consumer volunteers.

[0166] [Table 4]: Care formula containing the hybrid of example 1 at 5% used in the texturizing test.

[0167] [Table 5]: Care formula containing 5% silicone used in the texturizing test.

[0168] The 8 expert volunteers compared the texturizing effect of the 2 creams according to the questionnaire in Table 6 and the 10 consumer volunteers according to the questionnaire in Table 7.

[0169] [Table 6]: Questionnaire for expert volunteers.

[0170] [Table 7]: Questionnaire for consumer volunteers.

[0171] The measurements were taken at 21°C + / -1 and with a relative humidity of 50% + / - 5.

[0172] The results show that expert volunteers and consumers gave comparable scores to both creams, finding the texture of the two creams similar as illustrated in Figure 5.

[0173] Conclusion of the test: the above results demonstrate that the hybrid according to example 1 formulated at 5% provides a texturizing effect to the cream. Example 9: In vivo (clinical) evaluation of the hybrid according to example 1 formulated at 5% on the appearance of wrinkles as well as on skin hydration and elasticity. Short-term and long-term effect.

[0174] Principle: To study the in vivo efficacy of the hybrid according to example 1, formulated at 5%, and to link it with the composition of the hybrid and the in vitro results obtained on the different collagens.

[0175] Protocol: Two comparative, double-blind, placebo-controlled studies were conducted on the faces of 30 volunteers of Asian descent (aged 40 to 63 years) and on the faces of 34 volunteers of Caucasian descent (aged 37 to 69 years). Volunteers of Asian descent were randomly assigned to one side of a mask containing the hybrid and the other side a placebo for 25 minutes. Volunteers of Caucasian descent were randomly divided into two groups of 17 volunteers, homogeneous in age and gender, and were assigned to either the cream containing the 5% hybrid or the placebo.

[0176] [Table 8]: Skincare formula containing the hybrid of example 1 at 5% used for the clinical test on Asian skin.

[0177] The placebo group received the same treatment formula but without the hybrid.

[0178] [Table 9]: Skincare formula containing the hybrid of example 1 at 5% used in the clinical test on Caucasian skin.

[0179] The placebo group received the same treatment formula but without the hybrid.

[0180] Study duration: 1 day for the Asian skin study and 28 days for the Caucasian skin study.

[0181] Follow-up visits: at T0 (measurement before application), T30min and T4h for the study on Asian skin

[0182] at D0 (first day of the study), D14 (14 days after application) and D28 (28 days after application) for the study on Caucasian skin.

[0183] Measures :

[0184] - Measuring skin hydration with the Corneometer® CM 825 (Courage & Khazaka®)

[0185] - Acquisition of crow's feet skin topography using silicone replicas and analysis with VL650 software and by fringe projection and stereometry associated with the AEAVA-HE® system (Eotech®). The various parameters of crow's feet wrinkles (circumference, depth, length, etc.) were measured, and 2D and 3D representations of the skin topography were created.

[0186] - Colour photos of the face with the Visia-CR® and the HeadScan* V03 bench (Orion Concept*).

[0187] - Measurement of skin elasticity with the cutometer® dual MPA 580 (Courage & Khazaka®)

[0188] - Evaluation on a scale of 0 to 100 by an expert and by volunteers of the condition of facial skin by touch and according to the visual appearance of the following parameters:

[0189] - skin hydration: 0 corresponds to dry and dehydrated skin and 100 to very well hydrated skin.

[0010] - Crow's feet wrinkle: 0 corresponding to smooth skin without wrinkles and 100 to skin with marked and very visible wrinkles.

[0191] - Skin tone: 0 corresponds to sagging skin with no tone, and 100 to very firm skin with very good tone.

[0192] The measurements were carried out on the subject after 15 minutes of rest in a room at 21°C + / -1 and with a relative humidity of 50% + / - 5.

[0193] Results :

[0194] As illustrated in Figure 6, the hydration measurement results show an increase in hydration after 30 minutes and 4 hours of application on Asian skin, as well as after 14 and 28 days of application on Caucasian skin. These results support the benefit of diluting our molecular complex to a concentration of 10% in 90% of a cosmetic carrier known for its hydrating properties.

[0195] The results of skin topography measurements at the crow's feet area show a reduction in various wrinkle parameters after application of the cream containing the hybrid compared to the placebo. On Asian skin, a decrease in the length, depth, area, and number of wrinkles was observed 4 hours after mask removal. Regarding the study on Caucasian skin, a decrease was observed after 1 month of application for the Circumference and wrinkle area for the group that applied the cream containing the 5% hybrid compared to the placebo. The results are summarized in Table 10.

[0196] [Table 10]: Results of skin topography measurements at crow's feet during the Asian skin study and during the Caucasian skin study. *: significant; ***: highly significant; ****: extremely significant (with Student's t-test)

[0197] An improvement in skin elasticity was observed after application of the cream containing the hybrid compared to the placebo in both the Asian and Caucasian studies.

[0018] These results are consistent with the in vitro results obtained on the various collagens, but also demonstrate the benefit of diluting our molecular complex to a concentration of 10% in 90% of a cosmetic carrier. Indeed, the cosmetic carrier provides a rapid effect after application by delivering good hydration to the skin, thus improving its microrelief and elasticity. The long-term effect is achieved by the molecular complex, which, by acting on several collagen families, reduces wrinkles and improves elasticity.

[0199] These results are confirmed by observation of color photos of the face and evaluation by the expert and the volunteers.

[0200] Conclusion of the tests: the above results demonstrate that the hybrid according to example 1 formulated at 5% allows in the short term and long term to reduce and limit the appearance of wrinkles and fine lines, to improve the elasticity of the skin but also hydration.

Claims

Demands

1. A process for preparing a molecular complex comprising a peptide fraction and hyaluronic acid, characterized in that the molecular complex is not linked by covalent bonds and is obtained by the following process: a) solubilizing hyaluronic acid powder of a molecular weight between 3 kDa and 10 kDa, and preferably between 3 kDa and 5 kDa, in an aqueous solvent, preferably water, at a concentration between 5,000 ppm and 10,000 ppm under moderate stirring, for a time between 5 min and 30 minutes, at a temperature between 20°C and 50°C, preferably between 20°C and 30°C;b) add the peptide fraction in powder form, comprising at least one synthetic peptide or one purified from plants, algae, fungi or yeasts, comprising 3 to 12 amino acids and preferably 3 to 9 amino acids, into the solution prepared in the previous step, at a concentration of between 10 ppm and 25,000 ppm, preferably between 30 ppm and 5,500 ppm, under moderate stirring, at a temperature of between 20°C and 50°C; preferably between 20°C and 30°C; c) adjust the pH to a value of between 2 and 3.5, preferably between 2.5 and 3; d) leave under medium stirring for a time of between 30 min and 3 hours, preferably 1 hour, at a temperature of between 20°C and 50°C, preferably between 20°C and 30°C; e) adjust the pH to a value between 4.5 and 6.5, preferably between 4.5 and 5.5;

2. The process of claim 1, wherein the peptide fraction of step b) consists of a mixture of tripeptide-1 at a concentration of between 500 ppm and 5000 ppm, preferably between 1000 ppm and 3000 ppm, and sequence hexapeptide-9 (SEQ ID: No. 1) at a concentration of between 20 ppm and 500 ppm, preferably between 50 ppm and 300 ppm, in the hyaluronic acid solution obtained in the previous step.

3. A method of any one of claims 1 or 2, wherein the mass ratio between hyaluronic acid and the peptide fraction is between 10 / 1 and 2 / 1, and preferably between 4 / 1 and 2 / 1.

4. A method of any one of claims 1 to 3, further comprising a step f) wherein 5% to 40%, preferably 5% to 15% and even more preferably 10% of the molecular complex obtained in step e) is diluted in a cosmetic vector comprising water, glycerin and a mixture of several carboxymethyl celluloses of different grades and degrees of substitution, to obtain a hybrid.

5. The process of claim 4, wherein the cosmetic vector comprises 40% to 52% water, 47% to 57% glycerin and 1% to 3% of a mixture of carboxymethyl celluloses.

6. Molecular complex obtained by the process of any one of claims 1 to 3.

7. Hybrid obtained by the process of one of claims 4 or 5.

8. Composition comprising an effective amount of the molecular complex of claim 6 as the active agent and a physiologically acceptable medium.

9. Composition of claim 8, characterized in that it comprises between 0.01% and 10%, preferably between 0.05% and 5% and even more preferably between 0.1% and 2% of the molecular complex, by weight relative to the total weight of the composition.

10. Composition comprising an effective amount of the hybrid of claim 7 as the active agent and a physiologically acceptable medium. [Claim 1 1 ] Composition of claim 10, characterized in that it comprises between 1% and 10% and preferably 5% of hybrid, by weight relative to the total weight of the composition.

12. Cosmetic use of the composition of any one of claims 8 to 11 to reduce at least one sign of skin aging selected from: firming the skin, improving its elasticity and hydration, reducing and limiting the appearance of wrinkles and fine lines and also improving the evenness of the complexion.

13. Cosmetic use according to claim 12, wherein the expression of collagens I, III, IV, V, VI, VII, XXVII, XVII and XXIV, as well as the expression of hyaluronic acid are increased.

14. Composition of any one of claims 8 to 1 1 for its use in aesthetic and regenerative medicine to improve wound healing, limit inflammation, or for its use to repair cartilage and treat osteoarthritis.

15. Use of the hybrid of claim 7 to increase the texturizing effect of a formulation.

Images