Method for analyzing the repairing efficacy of a cosmetic or dermatological ingredient on keratinous materials

Abstract

The Applicant has advantageously highlighted the correlation between the total quantity of water, water mobility and water compartmentalization with the repairing action of cosmetic or dermatological ingredients on normal and damaged hair as well as on skin explants.Thus, the present application concerns a method for analyzing the repairing action of an ingredient on a sample of keratinous materials, comprising the steps of: - applying said ingredient to a sample of keratinous materials; - measuring, by low-field Nuclear Magnetic Resonance, in said sample of keratinous materials, at least one parameter chosen from among the total amount of water, water mobility, and water compartmentalization, the total amount of water, water mobility, and water compartmentalization being correlated with the repairing action of the ingredient, the repairing action being chosen from among a restructuring action, a structuring action, and a hydrating action. Abstract: Figure **.

Description

Title of the invention: Method for analyzing the repairing efficacy of a cosmetic or dermatological ingredient on keratinous materials. Technical field

[0001] This disclosure relates to the field of cosmetics or dermatology and to the measurement of the restorative efficacy of a cosmetic or dermatological ingredient. Prior art

[0002] The cosmetics industry is particularly interested in finding indicators of skin hydration status. The methods used to date to measure hydration are essentially "in vivo" methods, such as transepidermal water loss (TEWL) measurement (Pinnagoda & Tupker 1995). This measurement is used to assess, in a non-invasive way, the barrier function of the stratum comeum. It reflects a mixed phenomenon composed of passive diffusion and insensible perspiration, leading to water evaporation. When the skin is damaged, transepidermal water flow is increased. Conversely, it returns to normal baseline values ​​when the skin barrier is restored. Various types of devices are currently available for performing these measurements (Barel & Clarys, 1995).Other methods can be used such as corneometry, based on the principles of electrical conductivity of the skin, which allows the hydration status of the upper layers of the epidermis to be determined (Hester et al, 2004).

[0003] However, few methods allow for the study of hydration "ex vivo", on isolated models such as proteins or skin models. Confocal Raman spectroscopy methods that assess skin hydration in vivo (Tellez-Soto et al., 2021) also find applications on isolated models such as the stratum comeum (Galliano et al., 2021).

[0004] However, these methods present their own specific analytical difficulties and provide only part of the information. Indeed, among the methods for assessing skin hydration status, there are few methods that allow for a qualitative and quantitative understanding of water behavior in a model such as skin expiants.

[0005] Thus, it remains necessary to develop new methods allowing access to quantitative and qualitative information not only on the hydration state of the model in question but also on the behavior of water within this model (distribution of very bound / bound / free water and binding forces of water within the model).

[0006] Like skin, hair hydration is a crucial aspect for consumers of hair products. Dry and damaged hair is often associated with a dull and unattractive appearance. The cosmetics industry needs information and indicators on hair hydration to develop effective hair products that meet consumer needs. Hydration indicators help hair product manufacturers understand the physical properties of hair and formulate products that deeply hydrate it. Information on hair hydration also allows hair product manufacturers to create products that meet the specific needs of different hair types.

[0007] The theoretical basis of the work carried out by low-field NMR (20 MHz) concerns the behavior of water molecules in biological systems, and more specifically the exchanges between "free water" and "associated water" with molecular species with restricted movement (proteins, membranes, etc.). It is therefore possible to monitor a change in the behavior of water in a given environment (around a protein, a skin expiant, an active ingredient, etc.) by quantifying the total water present in this environment, but also by studying its compartmentalization (percentages of tightly bound water, bound water, and free water).

[0008] Although NMR methods have already been developed to study the behavior of water molecules in biological systems, it is necessary to identify the parameters of interest for the model in question. Indeed, the total amount of water, its compartmentalization, and its mobility in a meat product, in fruits and vegetables, or in fish obviously do not allow for the same conclusions to be reached. The identification of suitable parameters for each of the systems is necessary. Furthermore, skin and hair are complex systems to study, particularly for studying the behavior of water molecules. In fact, a hair fiber contains a small amount of water (on the order of 10%) and a very rich macromolecular structure (keratin). Consequently, the water, which is already relatively small, is also highly structured, relatively immobile, and therefore very difficult to quantify.Thus, an NMR assessment of water and its state in a water-rich model with highly mobile and readily available water cannot be directly transposed to the study of water in hair. Similarly, a skin sample contains a greater quantity of water than a hair. However, this water is distributed throughout the different layers of the skin (epidermis, dermis, and hypodermis), making it difficult to assess the overall behavior of water throughout the entire sample. Therefore, in this context as well, methodological developments are necessary.

[0009] Furthermore, there are currently no ex vivo methods that allow for kinetic monitoring of skin hydration status. The challenge surrounding these methodological developments, aside from NMR measurements, was also to develop models that allow for monitoring the hydration of the expiratory subjects and the water content within the expiratory subjects over a 72-hour period. Summary

[0010] The Applicant has advantageously highlighted the correlation between the total quantity of water, the mobility of water and the compartmentalization of water with the repairing action of cosmetic or dermatological ingredients on normal and damaged hair as well as on skin expiants.

[0011] The advantages of this method lie in its ease of implementation. Indeed, the keratin sample (hair strands or skin excipient) is analyzed as is, without any preparation or sample extraction other than the application of the treatment to be tested. The method remains simple, involving the insertion of the keratin samples into an NMR tube, which is itself inserted into the spectrometer. The methods developed prior to the NMR analysis also allow for great flexibility of the sample, in that different types of treatment can be applied to the same sample, or the analyses of the same sample can be repeated after different waiting times to monitor the kinetic activity of the repair process.These kinetic monitoring studies of skin expiant hydration nevertheless require the implementation of experimental protocols allowing the expiants to survive over a relatively long period without altering the hydration characteristics induced by the treatment being tested.

[0012] Thus, the present application relates to a method for analyzing the repairing action of an ingredient on a sample of keratinous materials, comprising the steps: - application of said ingredient on a sample of keratinous materials - measurement by low-field Nuclear Magnetic Resonance, in said sample of keratinous materials, of at least one parameter chosen from among the total quantity of water, the mobility of water and the compartmentalization of water, the total quantity of water, the mobility of water and the compartmentalization of water being correlated with the repairing action of the ingredient, the repairing action being chosen from among a restructuring action, a structuring action and a hydrating action.

[0013] On hair, this measurement of repair efficacy is possible by measuring specific NMR parameters allowing assessment of: - Hydrating efficacy through the total quantity of protons, preferably combined with the distribution of protons through the respective quantities of highly bound protons and bound protons - The restructuring effect through proton mobility.

[0014] The Applicant has also advantageously established that it is possible to assess the effectiveness of an ingredient on a skin expiant. Advantageously, the Applicant has demonstrated that manually synthesized skin, bioprinted skin, and a skin expiant taken from a biopsy give a relatively similar NMR response.

[0015] The Applicant has demonstrated that measuring the repairing efficacy of an ingredient on a skin excipient is possible by measuring specific NMR parameters that allow for the assessment of: - Hydrating efficacy through the total quantity of protons, preferably combined with proton mobility, - Structuring efficiency through the quantities of free protons and / or bound protons, preferentially combined with proton mobility. Brief description of the drawings

[0016] Other features, details and advantages will become apparent from reading the detailed description below and from analyzing the accompanying drawings, in which: Fig. 1

[0017] [Fig.1] shows the compartmentalization of water in the hair. Fig. 2

[0018] [Fig.2] shows an example of a treatment applied to normal and damaged hair. Fig. 3

[0019] [Fig. 3] Figure 3A shows the total quantity of protons visible by low-field NMR in the different hair strands. Figure 3B shows the percentage change in the total quantity of protons in the different hair strands compared to the controls (Fig. 4).

[0020] [Fig. 4] Figure 4A shows the average proton mobility measured by low-field NMR in the different hair strands. Figure 4B shows the percentage change in proton mobility in the different hair strands compared to the controls (Fig. 5).

[0021] [Fig.5] shows the distribution of water and associated mobility in the different hair strands analyzed. Fig. 6

[0022] [Fig.6] shows the total quantity of protons as a function of the average mobility of the protons in the different strands of hair analyzed. Fig. 7

[0023] [Fig.7] represents the distribution of water in a biological tissue such as skin. Fig. 8

[0024] [Fig.8] shows for each experimental condition the evolution of the total quantity of protons in skin expiants between the NMR measurement performed before application and the measurement performed after application of the active ingredient for each kinetic point. Fig. 9

[0025] [Fig.9] shows for each experimental condition the evolution of the mobility of protons in skin expiants between the NMR measurement performed before application and the measurement performed after application of the active ingredient for each kinetic point Fig. 10

[0026] [Fig. 10] shows the evolution of the fraction of bound protons of dermatome expiants over time. Detailed description

[0027] The present application relates to a method for analyzing the repairing action of an ingredient on a sample of keratinous materials, comprising the steps of: - applying said ingredient to a sample of keratinous materials - measuring by low-field Nuclear Magnetic Resonance, in said sample of keratinous materials, at least one parameter chosen from the total quantity of water, water mobility and water compartmentalization, the total quantity of water, water mobility and water compartmentalization being correlated with the repairing action of the ingredient, the repairing action being chosen from a restructuring action, a structuring action and a hydrating action.

[0028] The Applicant has advantageously developed an analytical method using and combining different NMR parameters to evaluate and measure the restorative action of a cosmetic or dermatological ingredient. Indeed, the Applicant has demonstrated that the restorative action of an ingredient is correlated with the total amount of water, water mobility, and water compartmentalization.

[0029] Thus, by measuring the proton content, the distribution of tightly bound and bound protons, and the proton mobility in hair, as well as the proton content, proton mobility, and quantity of free and / or bound protons in a skin expansive, it is possible to determine the repairing action of a cosmetic or dermatological ingredient on keratinous materials.

[0030] Advantageously also, the Applicant has developed an ex vivo method which allows for kinetic monitoring of the skin's hydration status over a period of 24 hours, preferably 72 hours.

[0031] Repair action

[0032] In cosmetics or dermatology, "repairing action" refers to the ability of an ingredient to improve the condition and appearance of the skin or hair, possibly after external or internal aggression. It covers three aspects: moisturizing action, structuring action, and restructuring action.

[0033] Ingredient

[0034] The term "ingredient" refers to any type of substance used in cosmetics or dermatology, known for its restorative or non-restorative properties. These ingredients may be of natural or synthetic origin and may be, but are not limited to, in the form of molecules, proteins, or extracts derived from natural products such as plants, algae, vegetables, and fruits. The purpose of this method is to determine whether the ingredient has a restorative effect on keratinous materials.

[0035] According to one embodiment, the ingredient may be incorporated into finished cosmetic or dermatological product formulas. Preferably, the ingredient is incorporated into these formulas at a concentration equivalent to the concentration at which it will be present in the finished product.

[0036] Examples of finished cosmetic or dermatological products include, by way of illustration, creams, lotions, serums, oils, shampoos, conditioners, masks (for skin or hair), balms.

[0037] The Applicant has indeed demonstrated that it is possible, by means of the method according to the present invention, to evaluate the repairing action of an ingredient as such or when incorporated into a cosmetic or dermatological formula, and this in a concentration equivalent to that in which it will be in the finished product.

[0038] Thus, the present method relates to a method for analyzing the repairing action of an ingredient characterized in that the ingredient is in the form of a cream, lotion, serum, oil, shampoo, conditioner, mask or balm.

[0039] Keratinous materials

[0040] Typically, the keratinous materials will be the skin or a skin expansive and the hair.

[0041] Thus, according to one embodiment, the keratinous materials will be chosen from the skin and the hair.

[0042] The term “sample of keratinous material” means a strand of hair, several strands of hair, a skin expiant from a biopsy, a sample of reconstituted skin (manually or via a 3D printer), a sample of dermis, epidermis or stratum corneum.

[0043] Thus, according to one embodiment, the method for analyzing the repairing action of an ingredient according to the invention is characterized in that the sample of keratinous materials is a hair sample or a skin sample.

[0044] According to one embodiment, the sample of keratinous materials is a hair sample.

[0045] The term “hair sample” means a strand of hair or several strands of hair.

[0046] According to one embodiment, the sample of keratinous materials is a skin sample.

[0047] The term "skin sample" means a skin expiant from a biopsy or a reconstituted skin sample (manually or via a 3D printer), from the dermis, epidermis or stratum cormeum.

[0048] Low-field NMR

[0049] Low-field NMR is known to those skilled in the art.

[0050] It is thus possible by measuring the magnetic resonance signals generated by the nuclei ('H) in response to radio frequency pulses to measure the distribution of water as well as its mobility by, respectively, measuring the distribution of protons and their average mobility in the sample.

[0051] A person skilled in the art may use any instrument enabling the acquisition of measurements by low-field NMR. Typically, low-field NMR measurements may be performed on a Bruker Minispec mq20 operating at 20 MHz.

[0052] Thus, the low field NMR technique will be used to measure, in a hair sample, the total quantity of protons, the mobility of protons and the distribution of protons through the respective quantities of very bound protons and bound protons and thus determine the total water content, the mobility of water and the contents of very bound water and bound water.

[0053] In a skin sample, the low field NMR technique will be used to measure the total quantity of protons present in the sample, the mobility of the protons and the distribution of protons within the sample (very bound protons / bound protons / very free protons) in order to determine the total water content, the mobility of the water and the distribution or compartmentalization of the water (very bound water / bound water / very free water).

[0054] Thus, "compartmentalization of water" means the distribution of the fractions of very bound water, bound water and very free water measured by the fractions of very bound protons, bound protons and very free protons.

[0055] According to one embodiment, the different low-field NMR sequences used are as follows: - FID (Free Induction Decay) sequence; - CPMG (Carr-Purcell-Meiboom-Gill sequence) signal processing in monoexponential mode; - CPMG (Carr-Purcell-Meiboom-Gill sequence) with bi-exponential signal processing;

[0056] FID Sequence

[0057] The FID sequence allows for the retrieval of information on the total quantity of visible protons in the sample (FID). According to the present invention, "FID" refers to the total quantity of visible protons in the keratinous material. Those skilled in the art know how to determine the acquisition conditions to obtain a usable signal on keratinous materials. The amplitude of the signal measured through the FID sequence is normalized to the mass (P) in grams of the analyzed sample.

[0058] CPMG sequence with monoexponential signal processing

[0059] This sequence allows us to recover on the one hand the average value of the relaxation time T2 and the amplitude of the signal at the origin (Ampl).

[0060] The acquisition conditions for obtaining a usable signal on keratinous materials are also known to those skilled in the art. The amplitude of the signal measured through the CPMG sequence is normalized to the mass (P) in grams of the analyzed sample.

[0061] CPMG sequence with bi-exponential signal processing

[0062] This sequence allows us to recover on the one hand the values ​​of the relaxation times T2(l) and T2(2), as well as the amplitudes of the corresponding original signals (A(l) and A(2)).

[0063] The acquisition conditions for obtaining a usable signal on keratinous materials are also known to those skilled in the art. The amplitudes of the signals measured through the different NMR sequences are normalized to the mass (P) in grams of the sample analyzed.

[0064] According to one embodiment, the FID sequence and the CPMG sequence with monoexponential signal processing will be used when the method according to the invention is applied to hair.

[0065] According to one embodiment, the FID sequence, the CPMG sequence with monoexponential signal processing and the CPMG sequence with signal processing in Biexponential mode will be used when the method according to the invention is applied to a skin expiant.

[0066] NMR parameter s

[0067] Thus, and according to one embodiment, the low-field NMR parameters measured according to the method according to the invention are as follows: FID / P corresponding to the total quantity of visible protons present in the sample (AU / g); T2: average proton mobility (ms); Ampl / P: quantity of free protons (AU / g); T2(l): mobility of bound protons (ms); T2(2): mobility of very free protons (ms); A(l) / P: quantity of bound protons (AU / g); A(2) / P: quantity of very free protons (AU / g).

[0068] These different parameters can be measured and combined in order to determine the repairing action of an ingredient depending on whether it is applied to the skin or a skin expansive or to the hair.

[0069] Method for analyzing the repairing action of an ingredient on a hair sample

[0070] The compartmentalization of water in hair varies depending on its environment. The interactions between the macromolecules present in the hair (keratin and melanin) and the surrounding water can be measured by low-field NMR. To better understand the significance of the measurements performed by low-field NMR, [Fig. 1] represents the distribution of water in a water-binding model such as hair.

[0071] Highly bound water undergoes strong interactions with macromolecules (melanin and keratin). This water population can be considered the "structural" water of the hair. Another population, called bound water, is indirectly influenced by macromolecules. This fraction of water is more readily exchanged with the surrounding atmospheric water. The bound water within the hair is more or less mobile. This mobility is measured and is representative of the water's binding forces. The lower the mobility, the stronger the binding forces, and vice versa.

[0072] In hair, two water populations are characterized: highly bound water and bound water. The Applicant has demonstrated that the following parameters, when applied to hair, are excellent markers of an ingredient's repairing action: - the total quantity of protons (corresponding to the total water content), possibly combined with the distribution of protons through the respective quantities of highly bound protons and bound protons (corresponding to the compartmentalization of water) - the mobility of protons (corresponding to the mobility of water).

[0073] These different parameters will make it possible to measure the moisturizing and restructuring effectiveness of the ingredient or the finished product.

[0074] The terms "moisturizing efficacy" or "moisturizing capacity" may be used interchangeably in this application. "Moisturizing efficacy" or "moisturizing capacity," when applied to hair, refers to the ability of the ingredient or finished product to maintain or increase the water content in the hair, thereby preserving the hair's suppleness, elasticity, and / or shine.

[0075] The total quantity of protons (i.e. the total water content) is representative of the hydration state of the hair since the penetration of water leads to an increase in the total quantity of protons visible inside the hair.

[0076] Thus, according to one embodiment, the total quantity of water is correlated with the moisturizing effectiveness of the ingredient.

[0077] An increase in the total quantity of protons in the hair compared to a placebo or a control under the same conditions, reflects a greater penetration of water.

[0078] The control may be a control, a "water" control or a placebo.

[0079] The term "control" means a hair sample to which no product or active ingredient has been applied. The term "control" may also be used.

[0080] The term “water” control means a hair sample to which water is applied.

[0081] A "placebo" is defined as a hair sample to which a product without any cosmetic or dermatological ingredients is applied. The composition of the product is comparable to that of the product being tested, but it does not include the ingredient being evaluated.

[0082] Thus, according to one embodiment, the method of analyzing the repairing action according to the invention is characterized in that the total quantity of water is measured, the total quantity of water being correlated with the moisturizing effectiveness of the ingredient.

[0083] Additional parameters may be evaluated to determine the moisturizing efficacy of an ingredient or product. The distribution of protons through the respective quantities of tightly bound and bound protons is a good indicator of moisturizing activity. Thus, the content of tightly bound and bound water can be evaluated.

[0084] Indeed, a decrease in the fraction of highly bound protons (and therefore of highly bound water) and / or an increase in the fraction of bound protons (and therefore of bound water), compared to a placebo or a control, is significant of hydrating efficacy.

[0085] Thus, according to one embodiment, the method of analyzing the repairing action according to the invention is characterized in that the fraction of bound water and highly bound water are In addition, the fractions of bound water and highly bound water, associated with the total amount of water, were measured and correlated with the moisturizing efficacy of the ingredient.

[0086] The terms "restructuring effect" and "restructuring efficacy" may be used interchangeably. "Restructuring effect" and "restructuring efficacy" refer to a property of a hair product that helps repair damaged hair and restore its original structure. For example, hair products with a restructuring effect can help reduce split ends, frizz, and breakage, while improving hair softness and shine.

[0087] According to one embodiment, proton mobility will be measured to determine the restructuring effect. Proton mobility is representative of the bonding forces between water, the constituent molecules of the ingredients or finished products, and the hair. The lower the proton mobility, the stronger the bonding forces, and vice versa. This parameter is therefore representative of the restructuring effect of the ingredient.

[0088] A decrease in proton mobility compared to a placebo or a control is therefore significant of restructuring efficacy.

[0089] Thus, according to one embodiment, the method of analyzing the repairing action according to the invention is characterized in that the water mobility is measured, the water mobility being correlated with the restructuring effectiveness of the ingredient.

[0090] The term "proton mobility" refers to the mobility of bound protons. Indeed, under the conditions of this application, the measured mobility is representative of bound protons. The mobility of tightly bound protons cannot be measured due to the excessively rapid return of their spins to their equilibrium position.

[0091] Thus, according to one embodiment, the mobility of the bound protons is determined.

[0092] Skin or skin expanses

[0093] The distribution of water in an ex vivo model such as skin expiants varies according to its environment. The interactions between the macromolecule(s) constituting the skin and the surrounding water are measurable by low-field NMR. To better understand the significance of the measurements performed by low-field NMR, [Fig. 7] represents the distribution of water in a biological tissue or any system capable of binding water.

[0094] According to [Fig. 7], structural water, indicated as "1" in the figure, contributes to the cohesion of the macromolecular structure. Hydration water, indicated as "2" in [Fig. 7], is called highly bound water because it undergoes strong interactions with macromolecules. Another population, called free water, is divided into two groups: "relatively bound" water, indicated as "3" in [Fig. 7], which is indirectly influenced by macromolecules, and "very free" water, indicated as "4" in [Fig. 7], which is not subject to any interaction. Free water within biological tissue is mobile. This mobility is measured and is representative of the binding forces of water in the sample. It is also possible to determine the mobility of "very free" water and "relatively bound" water.

[0095] The Applicant has demonstrated that the following parameters, when applied to the skin, are excellent markers of the restorative action of an ingredient: - the total quantity of protons (corresponding to the total water content), possibly combined with the mobility of protons (corresponding to the mobility of water), - the quantity of free protons and / or bound protons (corresponding to the free water and bound water content), possibly combined with the mobility of protons (corresponding to the mobility of water).

[0096] These different parameters will make it possible to measure the moisturizing and structuring effectiveness of the ingredient or the finished product.

[0097] According to one embodiment, the quantity of highly bound protons (corresponding to the quantity of highly bound water) may also be determined in order to measure the hydrating and / or structuring effectiveness of the ingredient or the finished product.

[0098] The terms "moisturizing efficacy" or "moisturizing capacity" may be used interchangeably in this application. "Moisturizing efficacy" or "moisturizing capacity," when applied to the skin, refers to the ability of an active ingredient to maintain or increase the water content in the superficial layers of the epidermis, thereby preserving the skin's suppleness, elasticity, and comfort.

[0099] Moisturizing efficacy can be assessed by the increase in the total quantity of protons (i.e., the total quantity of water), possibly combined with the change in proton mobility (i.e., the increase in water mobility). The Applicant has indeed advantageously demonstrated that the total quantity of protons and water mobility are correlated with the moisturizing efficacy of an ingredient.

[0100] Thus, according to one embodiment, the method of analyzing the repairing action of an ingredient according to the invention is characterized in that the total quantity of water is measured, the total quantity of water being correlated to the moisturizing effectiveness of the ingredient.

[0101] According to one embodiment, the method for analyzing the repairing action of an ingredient according to the invention is characterized in that the water mobility is further measured, the water mobility, associated with the total quantity of water, being correlated with the moisturizing effectiveness of the ingredient.

[0102] An increase in the total amount of water and proton mobility or a lesser decrease in the total amount of water and proton mobility, compared to the control under the same conditions, will be significant of a "moisturizing efficacy" or a "hydrating capacity" on the skin of the ingredient.

[0103] The term "smaller decrease" means a decrease in the quantity of water, but less pronounced than that observed in the control condition, under the same conditions.

[0104] The control may be a control, a "water" control or a placebo.

[0105] The term "control" means a skin sample on which no product or active ingredient is applied. is not applied. The term "control" may also be used.

[0106] The term “water” control means a skin sample to which water is applied.

[0107] A "placebo" is defined as a skin sample onto which a product without a cosmetic or dermatological ingredient is applied. The composition of the product is comparable to that of the product being tested, but it does not include the ingredient being evaluated.

[0108] Structuring effectiveness can be assessed by measuring the fractions of free water and / or bound water (i.e., the quantities of free and / or bound protons), possibly combined with measuring water mobility (i.e., proton mobility). The Applicant has advantageously demonstrated that the quantity of free and / or bound water, as well as water mobility, are correlated with the structuring effectiveness of an ingredient.

[0109] The "structuring efficacy" of an ingredient is based on its ability to act on the Key components of the epidermis, such as collagen and hyaluronic acid, and of the extracellular matrix, and / or its ability to induce stronger water-binding forces in the tissue, either through intrinsic skin molecules like hyaluronic acid or collagen, or through the active ingredient itself. By promoting the synthesis of collagen, hyaluronic acid, and other extracellular matrix components, and / or by increasing the fraction of bound water in the skin, and / or by inducing stronger water-binding forces in the tissue, a structuring active ingredient contributes to healthier, firmer, and better-hydrated skin.

[0110] The change in the fractions of free protons and / or bound protons compared to the control under the same conditions will be indicative of the ingredient's structuring efficacy on the skin. A decrease in the fraction of free protons, possibly combined with an increase in the fraction of bound protons compared to the control, will be indicative of the ingredient's structuring efficacy.

[0111] Thus, according to one embodiment, the present invention relates to a method for analyzing the repairing action of an ingredient, characterized in that the fractions of free water and / or bound water are measured, the fractions of bound water and / or free water being correlated with the structuring effectiveness of the ingredient.

[0112] Water mobility (i.e., proton mobility) can also be measured to determine the structuring activity of an ingredient. Indeed, a decrease Greater proton mobility compared to a control under the same conditions will be significant of the ingredient's restorative action.

[0113] Thus, according to one embodiment, the present invention relates to a method for analyzing the repairing action of an ingredient, characterized in that the water mobility is further measured, the water mobility, associated with the bound water and free water fractions, being correlated with the structuring effectiveness of the ingredient.

[0114] According to one embodiment, the present invention relates to a method for analyzing the repairing action of an ingredient, characterized in that the total quantity of water, the water mobility or the water compartmentalization measured in the sample of keratinous materials is compared to a control.

[0115] The control may be a control, a "water" control or a placebo.

[0116] The term "control" means a skin sample on which no product or active ingredient is applied. is not applied. The term "control" may also be used.

[0117] The term “water control” means a skin sample to which water is applied.

[0118] A "placebo" is defined as a skin sample onto which a product without a cosmetic or dermatological ingredient is applied. The composition of the product is comparable to that of the product being tested, but it does not contain the ingredient being evaluated.

[0119] Kinetic monitoring of the total quantity and mobility of protons under appropriate expiant survival conditions will allow monitoring of the moisturizing efficacy of the ingredient over time.

[0120] Kinetic monitoring of the evolution of the fractions of free protons and / or bound protons, and possibly of the mobility of protons under appropriate conditions of expiant survival will make it possible to monitor the structuring efficiency over time.

[0121] Thus, according to one embodiment, the present invention also relates to the kinetic monitoring of the repairing action of an ingredient on a sample of keratinous materials, preferably skin, by measuring the repairing action of the ingredient by the method according to the invention at given time intervals.

[0122] According to one embodiment, the repair action as previously defined will be measured on a skin expiant according to the desired kinetics.

[0123] According to one embodiment, the remedial action will be measured: - immediately after application of the ingredient to the keratin sample (T0) and then at one of the following times, preferably all of them: - 2 hours after T0, - 4 hours after T0, - 6 hours after T0, - 8 hours after T0, - 10 hours after T0, - 12 hours after TO, - 14 hours after TO, - 16 hours after TO, - 18 hours after TO, - 20 hours after TO, - 22 hours after TO, - 24 hours after TO, - 36 hours after TO, - 48 hours after TO, - 60 hours after TO, - 72 hours after TO.

[0124] The kinetic evaluation of the restorative efficacy of an ingredient on hair or skin excipient over a prolonged period, ideally from 30 minutes to 72 hours, offers significant advantages. By observing the effects over an extended period, it is possible to detect subtle changes that would not be immediately visible. This allows for a more comprehensive evaluation of the ingredient's efficacy and helps identify when it begins to act, reaches its maximum efficacy, and eventually diminishes. This dynamic approach helps to understand the mechanisms of action and to adapt treatment protocols to maximize benefits. Examples

[0125] Example 1: Measurement of the moisturizing efficacy and restructuring effect of an active ingredient on the hair fiber

[0126] Materials and Methods

[0127] 1. Setting up the acquisition conditions by NMR (20MHz) to measure the moisturizing and restructuring efficacy of an active ingredient or a finished product formula

[0128] Low-field NMR analyses were performed on a Bruker Minispec mq20 operating at 20 MHz (18 mm absolute probe). In order to obtain usable and interpretable information for this purpose, different NMR sequences were optimized and used: - FID sequence allowing the retrieval of information on the total quantity of visible protons in the sample (FID), - CPMG sequence with monoexponential signal processing allowing recovery of the average value of the relaxation time T2 and the amplitude of the signal at the origin (Ampl).

[0129]

[0130] 2. Implementation of a protocol adapted to the preparation of hair strands for performing NMR measurements

[0131] Before treating the hair strands with the active ingredient or formula to be tested, they undergo a preparation step as described below: - Moisten the strands under cold water, - Wash the strands with neutral shampoo, massaging for 1 minute. - Rinse under cold water, then pat dry gently. - Allow to dry at room temperature for 1 hour. - Gently comb through and dry with a hairdryer on the cool setting for 2 minutes.

[0132] The protocol for processing the wicks and preparing the samples for NMR analyses is as follows: - Dampen the wicks under cold water, - Apply the tested shampoo - Massage for 1 minute, - Rinse under cold water and pat dry gently - Allow to dry at room temperature for 1 hour - Gently comb through and blow-dry on the cool setting for 2 minutes. - Cut 2 cm from the tips (discard them) - Cut 6 cm (3x2 cm) pieces and insert them into the NMR tubes. - Allow the samples to re-equilibrate overnight at +4°C - Analyze by low-field NMR.

[0133] Results

[0134] Two shampoo-type formulas were tested on natural hair and damaged hair: - a shampoo not containing the active ingredient to be tested (= placebo formula), - a shampoo containing the active ingredient to be tested.

[0135] Fig. 2 summarizes the treatment carried out on the different strands.

[0136] Figures 3a and 3b show respectively the total quantity of protons visible in the six normal and damaged hair strands, as well as the percentage change in the total quantity of protons compared to the controls.

[0137] The total quantity of protons is representative of the hydration state of the hair since the penetration of water leads to an increase in the total quantity of protons visible inside the hair.

[0138] Moisturizing efficacy of the active ingredient on normal hair

[0139] Compared to the control, the total quantity of protons increases after application of the placebo and the shampoo with active ingredient. This increase is greater with the latter, reflecting greater water penetration into the hair.

[0140] Moisturizing efficacy of the active ingredient on damaged hair

[0141] Compared to the control, the total quantity of protons increases similarly after application of the placebo and the shampoo with the active ingredient. On damaged hair, application of the shampoo containing the active ingredient has no greater impact on water penetration than the placebo shampoo.

[0142] Restructuring efficacy of the active ingredient on normal hair

[0143] Figures 4a and 4b show respectively the average mobility of the constituent protons of the six normal and damaged hair strands, as well as the percentage change in proton mobility compared to the controls.

[0144] The average proton mobility is representative of the bonding forces between water, the constituent molecules of shampoos, and hair. Indeed, the lower the proton mobility, the stronger the bonding forces, and vice versa. This parameter is representative of the restructuring effect of the ingredient.

[0145] Compared to the control, proton mobility decreases after application of the placebo and the shampoo with active ingredient. This decrease is greater in the presence of the latter, reflecting stronger water-binding forces and a restructuring effect on the hair.

[0146] Restructuring efficacy of the active ingredient on damaged hair

[0147] Compared to the control, proton mobility decreased after application of the placebo and the shampoo with the active ingredient. As with normal hair, this decrease in proton mobility was more pronounced with the shampoo containing the active ingredient. It should be noted that the shampoo with the active ingredient had a greater impact on damaged hair than on normal hair.

[0148] Fig. 5 shows the distribution of water and associated mobility in the different hair strands analyzed.

[0149] In hair, only two proton populations are differentiated: tightly bound protons and bound protons. The measured mobility is representative of bound protons under our experimental conditions. Indeed, the mobility of tightly bound protons cannot be measured due to a too-rapid return to spin equilibrium. The application of shampoos (with and without active ingredients) has a similar effect on normal and damaged hair. In fact, the fraction of bound water increases at the expense of the fraction of tightly bound water, with a parallel decrease in proton mobility. Whether on normal or damaged hair, the shampoo's effectiveness is greater in the presence of the active ingredient.

[0150] Fig. 6 shows the total quantity of protons as a function of the average mobility of protons in the different strands of hair analyzed.

[0151] On normal hair, the shampoo with active ingredient is more moisturizing than the placebo compared to untreated controls. On damaged hair, the shampoo with active ingredient is as moisturizing as the placebo compared to untreated controls. However, hair (normal or damaged) treated with the shampoo containing the active ingredient has stronger water-binding strength than hair treated with the placebo, demonstrating a structuring effect on the hair fiber. This effect is more pronounced on damaged hair.

[0152] Conclusions

[0153] The Applicant has advantageously developed a low-field NMR method for measuring the moisturizing and restructuring efficacy of a shampoo-type formula applied to normal or damaged hair. This application can be extended to measuring the efficacy of isolated active ingredients or other finished product formulas (conditioner, balm, serum, etc.).

[0154] This measurement of the hydrating and restructuring efficacy is possible by identifying the specific NMR parameters allowing us to assess: - The hydrating efficacy through the total quantity of protons, - The restructuring effect through the mobility of the protons, - The distribution of protons through the respective quantities of highly bound protons and / or bound protons.

[0155] Example 2: Comparison of the efficacy of active ingredients on skin expiants - Kinetic monitoring of hydration by low-field NMR (20 MHz)

[0156] Materials and Methods

[0157] 1. Setting up the acquisition conditions by NMR (20MHz) to measure hydration capacity

[0158] Low-field NMR analyses were performed on a Bruker Minispec mq20 operating at 20 MHz (18 mm absolute probe). In order to obtain usable and interpretable information for this problem, different NMR sequences were optimized and used: - FID sequence allowing the retrieval of information on the total quantity of visible protons in the sample (FID), - CPMG sequence with monoexponential signal processing allowing recovery of both the average relaxation time T2 and the original signal amplitude (Ampl), - CPMG sequence with bi-exponential signal processing allowing recovery of the relaxation time values ​​T2(l) and T2(2), as well as the amplitudes of the corresponding original signals (A(l) and A(2)).

[0159] 2. Development of protocols adapted to the evaluation of hydrating efficacy of active ingredients over relatively long periods

[0160] Upon receipt, the expiry samples are incubated in a 70% ethanol solution for 3 minutes. They are then rinsed in PBS 3 times for 3 minutes each time before being dispensed. on inserts and stabilized on a sterile compress, soaked in 8mL of DMEM medium for 24h at 37 °C 5%CO2.

[0161] The active ingredients are then deposited onto the surface of the skin excipients at a concentration of 7.5 mg / cm² and then spread. For kinetic monitoring, the excipients are then placed in the incubator at 37°C on a compress lightly impregnated with DMEM medium so as not to saturate the excipients with water but to promote their survival.

[0162] Results

[0163] Kinetic study of the repairing efficacy of skin excipients after application of different active ingredients

[0164] 1. Kinetic study of the hydration of skin expiants after application of various assets

[0165] In this study, the total quantity of protons as well as the proton mobility of dermatome expiants was measured at 30 minutes, 2 hours, 4 hours and 24 hours.

[0166] High molecular weight hyaluronic acid at 0.2% (HA-HMP 0.2%) and low molecular weight hyaluronic acid at 0.2% (HA-LMB 0.2%) were evaluated and compared to: -a control (no active ingredient or formulation was applied to the dermatomed expiant), - a "water" control, i.e., water was applied to the dermatometic expiratory area, - 2% glycerin and 2% betaine as references. Glycerin and betaine are indeed known for their moisturizing effect.

[0167] Fig. 8 shows the percentage change in the total quantity of protons measured in the expiants after application.

[0168] The results show that for the Control, Water, and HA-HPM conditions, the total quantity of protons tends to decrease during the first 4 hours. A slight increase is observed at T24h, likely related to the fact that the expiants are repositioned on the DMEM-impregnated blotting paper. Conversely, in the HA-BPM, Glycerin, and Betaine conditions, the total quantity of protons increases from 2 hours after application until 24 hours after application, demonstrating water penetration into the expiants.

[0169] Thus, due to the increase in the total quantity of protons, it is possible to conclude that HA-BPM, glycerin and betaine are better moisturizing agents.

[0170] The determination of the hydrating action in relation to the measurement of the total quantity of water can be refined by measuring the mobility of water (i.e. the mobility of protons).

[0171] Figure 9 shows the percentage change in proton mobility measured in the expiants after application. Under all conditions, proton mobility decreases as early as 30 minutes or 2 hours after application. This decrease reflects a structuring of the tissue over time. Depending on the ingredient tested, maximum structuring is not observed after the same duration. Similarly, after the application of water or an active ingredient, the decrease in proton mobility is less pronounced. This result is explained by the entry of water, which intrinsically possesses a much higher proton mobility (on the scale of 2000 ms) than that of the expiratory tissue. The mobility of water alone is on the order of 2000 ms under our analytical conditions. The mobility of the expiratory tissue is on the order of 40 ms. Therefore, the penetration of water into the expiratory tissue will induce an increase in the average mobility of the expiratory tissue and thus a smaller decrease in proton mobility compared to the control.

[0172] Advantageously, the measurement of the total quantity of protons, combined with the measurement of water mobility over 24 hours, compared to a control, makes it possible to analyze the repairing action of a cosmetic ingredient.

[0173] 2. Kinetic study of the structuring of skin expiants after application of various assets

[0174] In this study, the fractions of free protons, bound protons and highly bound protons, as well as the proton mobility of dermatome expiants, were measured at 30 minutes, 2 hours, 4 hours and 24 hours.

[0175] High molecular weight hyaluronic acid at 0.2% (HA-HMP 0.2%) and low molecular weight hyaluronic acid at 0.2% (HA-LMB 0.2%) were evaluated and compared to: -a control (no active ingredient or formulation was applied to the dermatomed expiant), - a "water" control, i.e., water was applied to the dermatometic expiratory area, - 2% glycerin and 2% betaine as references. Glycerin and betaine are indeed known for their restructuring effect.

[0176] Figure 10 shows the evolution of the bound water fraction in the excipients for each of the tested conditions and for each kinetic point. Compared to the untreated control excipients, the bound proton fraction increases much more significantly throughout the kinetic analysis under the conditions of 0.2% low molecular weight hyaluronic acid (HA-BPM 0.2%) and 2% glycerin. Conversely, despite an increase in the total quantity of protons from T2h after betaine application, a decrease in the bound proton fraction is observed under this condition. This result reflects a better structuring effect of low molecular weight hyaluronic acid and glycerin compared to betaine. Although all three active ingredients have hydrating efficacy, their ability to bind water within the skin is not the same.

[0177] Advantageously, the measurement of the fractions of free protons and bound protons will allow us to conclude that the ingredient has a structuring action.

Claims

Demands

1. Method for analyzing the repairing action of an ingredient on a sample of keratinous materials, comprising the steps of: - applying said ingredient to a sample of keratinous materials - measuring by low-field Nuclear Magnetic Resonance, in said sample of keratinous materials, at least one parameter chosen from the total amount of water, water mobility and water compartmentalization, the total amount of water, water mobility and water compartmentalization being correlated with the repairing action of the ingredient, the repairing action being chosen from a restructuring action, a structuring action and a hydrating action.

2. Method for analyzing the repairing action of an ingredient according to claim 1, characterized in that the sample of keratinous materials is a hair sample.

3. Method for analyzing the repairing action of an ingredient according to claim 2, characterized in that the water mobility is measured, the water mobility being correlated with the restructuring effectiveness of the ingredient.

4. Method for analyzing the repairing action of an ingredient according to claim 2, characterized in that the total amount of water is measured, the total amount of water being correlated with the moisturizing effectiveness of the ingredient.

5. Method for analyzing the repairing action of an ingredient according to claim 4, characterized in that the bound water and highly bound water fractions are further measured, the bound water and highly bound water fractions, associated with the total amount of water, being correlated with the moisturizing effectiveness of the ingredient.

6. Method for analyzing the repairing action of an ingredient according to claim 1, characterized in that the sample of keratinous materials is a skin sample.

7. Method for analyzing the repairing action of an ingredient according to claim 6, characterized in that the total amount of water is measured, the total amount of water being correlated with the moisturizing effectiveness of the ingredient.

8. Method for analyzing the repairing action of an ingredient according to claim 7, characterized in that the water mobility is further measured, the water mobility, associated with the total amount of water, being correlated with the moisturizing effectiveness of the ingredient.

9. Method for analyzing the repairing action of an ingredient according to claim 6, characterized in that the free water and / or bound water fractions are measured, the bound water and free water fractions being correlated with the structuring effectiveness of the ingredient.

10. Method for analyzing the repairing action of an ingredient according to claim 9, characterized in that the water mobility is further measured, the water mobility, associated with the bound water and / or free water fractions, being correlated with the structuring effectiveness of the ingredient.

11. Method for analyzing the repairing action of an ingredient according to any one of the preceding claims, characterized in that the total quantity of water, the water mobility or the water compartmentalization measured in the sample of keratinous materials is compared to a control.

12. Method for analyzing the repairing action of an ingredient according to any one of the preceding claims, characterized in that the ingredient is in the form of a cream, lotion, serum, oil, shampoo, conditioner, mask or balm.

13. Kinetic monitoring of the repairing action of an ingredient on a sample of keratinous materials, characterized in that the repairing action of the ingredient is measured by the method according to any one of the preceding claims at given time intervals.

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