Casein-catechin covalent complex, cosmetic composition, pharmaceutical composition, processes for the preparation thereof

Abstract

The present invention relates to a complex characterized by being formed of casein and catechins extracted from green tea, wherein the casein and catechins are combined by covalent bonds. The mass proportion of the casein to catechins in the formation of the complex ranges from 1:1 to 1:5. The complex is produced through a process involving dissolving casein in a first polar solvent, adjusting the pH to a range of 7.0 to 10.0, and similarly preparing a solution of catechins with a pH range of 7.5 to 9.0. The solutions are then reacted while maintaining a pH range of 7.5 to 8.5 to obtain the covalently bonded complex. This complex, obtainable by the described process, can be utilized in various applications, including the preparation of lyophilized compositions and as an active ingredient in cosmetic and pharmaceutical compositions for preventing or repairing cutaneous damages related to aging or treating dermatological ailments.

Description

Casein-catechin covalent complex, cosmetic composition, pharmaceutical composition, processes for the preparation thereof

[0001] The invention relates to the technical field of biochemistry and the development of novel complexes formed by the covalent bonding of casein, a milk-derived protein, and catechins, which are polyphenolic compounds extracted from green tea. The invention encompasses processes for preparing such complexes, which leverage the unique properties of both components, potentially for applications in the cosmetic and pharmaceutical industries. The complex is characterized by its formation through specific reaction conditions, including pH and temperature controls, and can be provided in various forms, including lyophilized compositions. The invention further extends to the use of the complex in cosmetic compositions for topical application aimed at preventing or repairing skin damage associated with aging, as well as in pharmaceutical compositions for treating dermatological conditions. The detailed process of creating the complex, as well as the resulting product's potential applications, places this invention at the intersection of biotechnology and therapeutic product development.

[0002] In the field of bioactive complexes, the interaction between proteins and polyphenols has been extensively explored due to their potential health benefits and applications in various industries, including pharmaceuticals and cosmetics. The formation of complexes can enhance the stability, solubility, and biological activity of polyphenolic compounds. Casein, a family of phosphoproteins commonly found in mammalian milk, has been recognized for its ability to bind with a variety of molecules, including polyphenols.

[0003] Catechins are a type of natural phenol and antioxidant present in green tea extracts. They have been widely studied for their multiple health-promoting properties, such as anti-inflammatory, antioxidant, and anticarcinogenic effects (Samaret al.; Alkufeidyet al.).Technical Problem

[0004] Catechins are known to have certain limitations in terms of stability and bioavailability when used independently. They are susceptible to degradation under physiological conditions and have limited absorption when ingested.

[0005] The technological challenge faced by researchers is to improve the delivery systems for these bioactive compounds to maximize their efficacy. A common approach has involved using different types of protein-polyphenol complexes that can potentially protect catechins from degradation and improve their bioavailability.

[0006] Addressing this challenge requires an advanced level of control over the interactions between these bioactive components. Techniques involving covalent bonding offer potential solutions but also pose difficulties related to ensuring that the reaction conditions do not denature the proteins or degrade the polyphenols involved.

[0007] Moreover, there is a continuing need for optimized ratios and concentrations within these complexes to achieve desired functional properties while maintaining biocompatibility. Ensuring precise control over pH levels during processing steps is crucial as it significantly affects both solubility and binding efficiency between proteins like casein and catechins.

[0008] Additionally, methods that allow controlled partial denaturation of casein could open up novel avenues by exposing more binding sites for catechins without compromising overall protein structure too much – leading possibly to enhanced complexity formation yielding better stabilization results.

[0009] Furthermore, selecting appropriate solvents for dissolving individual components prior to complexation remains critical since different solvents can significantly influence both dissolution rates as well as subsequent binding efficiencies between casein molecules and catechin constituents extracted from green tea.

[0010] The quest involves not only forming stable complexes but also devising scalable processes suitable for industrial applications—thereby bringing fortified health benefits into accessible consumer products ranging from dietary supplements through cosmetic formulations aimed at preventing or repairing cutaneous damages related to aging.Solution to Problem

[0011] The present invention provides a complex formed of casein and of catechins extracted from green tea. Casein, a high-quality protein derived from milk, is known for its nutritional profile rich in essential amino acids, fundamental for cell repair and regeneration. When combined with green tea catechins, recognized for their antioxidant and anti-inflammatory capabilities, the complex of the present invention offers an effective solution to combat the effects of ageing and protect the skin against environmental damage (Follieroet al.).

[0012] One of the main problems solved by the present invention is related to the efficient reaction of casein and green tea catechins, which involved optimizing the reaction conditions to form stable covalent bonds without compromising the bioactive properties of the components. The solution involved developing a set of reactional conditions that carefully balances pH and temperature to maximize reactivity and preserve the structural integrity of the molecules.

[0013] The present invention also encompasses a lyophilized composition comprising a complex formed of casein and of catechins extracted from green tea and process of preparation thereof. These embodiments ensure the long-term stability of the freeze-dried complex, minimize residual moisture and protect the product against degradation by environmental factors such as UV light and oxidation. In addition, the process of preparation of the lyophilized composition includes a step of purifying the complex using methods such as ultrafiltration and dialysis, which contributes to maintain the product’s purity without losing the product's efficacy.Advantageous Effects of Invention

[0014] The complex according to the present invention combines the rich bioactive properties of casein with the powerful antioxidant activity of green tea catechins. Developed in response to the growing demand for skin care products that offer multiple benefits, said complex stands out for its antioxidant, regenerative and healing properties. The complex of the present invention is designed to protect the skin against oxidative damage, promote cell regeneration and accelerate healing, offering a holistic approach to skin health and beauty. The complex of the present invention provides multiple benefits to the skin, including antioxidant protection, regenerative action and healing properties.

[0015] These and other advantages of the present invention will be clear and better understood from the following description and appended drawings and claims. Nevertheless, the examples and drawings are provided to illustrate the inventive concepts and are not intended to limit the scope of the invention.

[0016] In order to promote an understanding of the principles according to the modalities of the present invention, reference will be made to the modalities illustrated in the figures and the language used to describe them.

[0017] It should also be understood that there is no intention to limit the scope of the invention to the content of the figures and that modifications to the inventive features illustrated herein, as well as additional applications of the principles and embodiments illustrated, which would normally occur to a person skilled in the art having the possession of this description, are considered within the scope of the claimed invention.Fig.1

[0018] illustrates an in vitro test indicating a significant reduction (> 50%) of free radicals, proving the high antioxidant capacity of the complex formed of casein and of catechins extracted from green tea.Fig.2

[0019] illustrates cell viability tests of formulations comprising the complex according to the present invention.Fig.3

[0020] illustrates a Scratch Test for a healing action of a formulation comprising the complex according to the invention.Fig.4

[0021] illustrates results of the hemocompatibility test of the complex according to the invention.Fig.5

[0022] illustrates UV-Vis spectra of casein solutions.Fig.6

[0023] illustrates UV-Vis spectra of green tea solutions.Fig.7

[0024] illustrates UV-Vis spectra of solutions of casein, green tea and CAS / GT complex according to the invention ( 75ug / mL).

[0025] The present invention refers in a first aspect to a complex formed of casein and of catechins extracted from green tea, wherein the casein and catechins are combined by covalent bonds.

[0026] Casein is a family of related phosphoproteins commonly found in mammalian milk, making up about 80% of the proteins in cow's milk and between 20% to 45% of the proteins in human milk. In a broad sense, casein can be used in various applications such as food additives, adhesives, and in the production of certain plastics. In the context of this invention, casein is specifically used as a component in forming a complex with catechins extracted from green tea. The casein is dissolved in a polar solvent and its pH is adjusted to facilitate the formation of covalent bonds with catechins. In a narrower sense, the casein used in this invention is partially denatured under specific conditions to enhance its reactivity with catechins.

[0027] Casein is a phosphorylated protein made up of several subunits, mainly α, β and κ caseins, rich in amino acids such as proline, serine, glutamine and tyrosine. Some structural characteristics of casein are particularly important for its interaction with catechins:

[0028] - the Hydroxyl (-OH) and Amino (-NH2) groups: Present in amino acids such as tyrosine and serine, these groups are capable of forming hydrogen bonds;

[0029] - Carboxyl groups (-COOH): Present at the end of amino acid chains and in the side chains of some amino acids, they can take part in ionic interactions and covalent bonds;

[0030] - Phosphorylation: Phosphorylated serine residues help stabilize casein and increase solubility in neutral to slightly alkaline pH solutions.

[0031]

[0032] Catechins are a type of natural phenol and antioxidant, a part of the chemical family of flavonoids, predominantly found in green tea. Broadly, catechins are known for their health benefits, including anti-inflammatory, anti-carcinogenic, and cardiovascular protective effects (Al-Rawafet al.). In this invention, catechins are extracted from green tea and used to form a complex with casein. The catechins are dissolved in a polar solvent and their pH is adjusted to enable the formation of covalent bonds with casein. In a narrower sense, the catechins are used in specific concentrations and under controlled pH conditions to ensure optimal complex formation with casein.

[0033] Catechins are phenolic compounds (flavonoids) with a basic structure made up of three rings, two of them aromatic (A and B) and one heterocyclic (C ring). The main catechins in green tea include epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC) and epigallocatechin gallate (EGCG). Catechins are characterized by:

[0034] - Hydroxyl groups (-OH): In the A and B rings, allowing the formation of hydrogen bonds and van der Waals interactions;

[0035] Gallate acid (in ECG and EGCG): An additional phenolic acid group that can increase the ability to interact with proteins through hydrogen bonds and hydrophobic interactions.

[0036]

[0037] The term complex in this context refers to a molecular entity formed by the combination of casein and catechins through covalent bonds. Broadly, a complex can be any compound consisting of multiple molecules bonded together. In this invention, the complex specifically refers to the product formed by reacting casein and catechins under controlled conditions. This complex is characterized by its unique properties, which are leveraged in various applications such as cosmetics and pharmaceuticals. In a narrower sense, the complex is formed under specific pH and temperature conditions to ensure the stability and efficacy of the final product.

[0038] Casein-catechin complex formation can occur through various intermolecular interactions, as facilitated by the pH and temperature conditions of the conjugation process.

[0039] The hydroxyl groups present in catechins (particularly in the 3' and 4' positions of the B ring and in the phenolic groups of gallate acid) can form hydrogen bonds with the amino and carboxyl groups of the amino acid residues present in casein. This type of bond is particularly favored in slightly alkaline pH conditions (between 7.5 and 8.5), where the carboxyl groups of the casein may be partially deprotonated, making them more likely to form hydrogen bonds with the hydroxyl groups of the catechins.

[0040] Catechins have hydrophobic regions in their aromatic rings, which can interact with the non-polar regions of casein. Casein has a structure rich in hydrophobic amino acids, such as proline and valine, which can facilitate hydrophobic interactions with the aromatic rings of catechins. This type of interaction contributes to the stabilization of the complex, especially at moderate temperatures (20°C to 25°C), such as those used in the conjugation process.

[0041] Under certain conditions, such as in the presence of oxygen or oxidizing agents, catechins can be oxidized to form quinones, which react covalently with the nucleophilic groups of casein (such as the amino group of lysine residues). This covalent bond between the quinone of the catechins and the amino groups of the casein strengthens the complex and increases its stability. This mechanism can be facilitated by the addition of binding agents, such as citric acid or genipin, which help catalyze these reactions.

[0042] Under slightly alkaline conditions, the phosphorylated residues of the casein are negatively charged, while the phenolic groups of the catechins can form hydrogen bridges and ionic interactions with these charged groups. These electrostatic interactions can increase the affinity between the molecules and contribute to the stability of the complex.

[0043] The complex formed is stable and has synergistic antioxidant, anti-inflammatory, and healing properties. Casein acts as a biocompatible and controlled vehicle for the prolonged release of catechins, protecting them from degradation and ensuring that their activity is sustained for longer periods. The catechins, in turn, amplify the regenerative properties of casein, creating an effective therapeutic product for use in cosmetics and medicinal applications. Furthermore, Casein provides essential amino acids that accelerate cell repair and regeneration, improving healing (Kolahreezet al.).

[0044] The complex according to the present invention maintains its stability without degradation over time, verified by analytical methods such as DSC and spectroscopy. As a further technical advantage, the complex is stable at pH in the range of 5.5 to 7.5, preventing precipitation and color changes, ideal for cosmetic applications.

[0045] Preferably, in the complex of the present invention, the mass proportion of the casein and of catechins extracted from green tea in the formation of the complex is in the range from 1:1 to 1:5 or in the range from 1:1 to 5:1 (mass / mass).

[0046] Even more preferably, the ratio between casein and catechins ranges from 1:1 to 5:1 (mass / mass). This preferred range balances the structural stability provided by casein with the antioxidant and anti-inflammatory properties of the catechins. Based on experimental data and pharmaceutical formulation standards, the optimal ratio for the casein-catechin complex ranges from 1:1 to 5:1 (casein, mass / mass). When the Casein to Catechin Ratio is in a range of about 1:1, the balanced interaction where both components contribute equally to the structural integrity and bioactivity of the final formulation is ensured. A Casein to Catechin Ratio the range of 2:1 to 5:1 ratio increases the presence of casein, which is advantageous in formulations where protein stability and bioadhesive properties are crucial, such as in wound healing applications or slow-release formulations.

[0047] The present invention refers in a second aspect to a process for the preparation of the complex according to the first aspect of the invention, comprising the steps:

[0048] a1) Dissolving the casein in a first polar solvent, and adjusting the pH in the range of 7.0 to 10.0, obtaining a solution comprising casein; and

[0049] b1) Dissolving the green tea extract in a second polar solvent, and adjusting the pH in the range of 7.5 to 9.0, obtaining a solution comprising catechins; and

[0050] c1) Reacting the solution comprising casein and the solution comprising catechins, keeping the pH in the range of 7.5 to 8.5, obtaining a complex formed of casein and of catechins extracted from green tea.

[0051]

[0052] The green tea extract used in the process typically contains from 30% to 70% catechins (w / w), depending on the source and method of extraction. The ratio of casein to catechins is optimized based on the final concentration of catechins in the extract to ensure the bioactivity and stability of the casein-catechin complex

[0053] Preferably, it is advisable to use an extract standardized to contain from 40% to 60%, for example about 50% catechins (w / w), as this preferred range provides an optimal balance between antioxidant activity and stability of the extract. Preferably, epigallocatechin gallate (EGCG) should represent at least 50% (w / w) of the total catechin content to ensure the desired bioactive properties, such as free radical scavenging, anti-inflammatory effects, and antimicrobial activity.

[0054] For extracts containing lower concentrations of catechins in the range from 30% to 40% (w / w) (e.g., 30%), the ratio of casein to catechins may be adjusted to 1.5:1 or 2:1 (w / w).

[0055] For higher concentrations of catechins in the range from 60% to 70% (w / w) (e.g., 70%), the ratio of casein to catechins may be reduced to 0.8:1 or 1:1 (w / w). These adjustments ensure that the final product maintains the desired antioxidant properties and structural integrity.

[0056] The process begins in its step a1) with the preparation of casein, which is dissolved under conditions that maximize its solubility and functionality, followed by the preparation of green tea extract in step b1), which is rich in catechins, to ensure stability and antioxidant efficacy.

[0057] In the preferred embodiments, the first polar solvent, used to dissolve the casein, is selected from the group consisting of a tris(hydroxymethyl)aminomethane hydrochloride buffer, a borate buffer, a bicarbonate buffer, an amino acid buffer, and mixtures thereof.

[0058] The tris(hydroxymethyl)aminomethane hydrochloride buffer has a pH in the range of 7.5 to 9.0, and provides a proper compatibility with the skin and a stable buffering capacity. The borate buffer has a pH in the range of 8.0 to 10.0, and is a preferred selection when greater pH resistance is required. The bicarbonate buffer has a pH in the range of 8.0 to 8.5, and provides an easy and safe handling, but provides a lower buffering capacity. The amino acid buffer can be selected from the group including the glycine buffer or the histidine buffer. The glycine buffer is preferably adjusted with a strong base, for example sodium hydroxide to a pH in the range of 8.0 to 9.0. The histidine buffer is used in a pH range from 7.0 to 9.0 providing a good stability at a neutral or alkaline pH.

[0059] In the preferred embodiments of the process for the preparation of the complex, in step a1), the solution comprising casein comprises casein in a concentration of 3% to 5% (weight / volume), more preferably, in a concentration of 2,5% to 3,5% (weight / volume). Regarding the control of the parameter pH in step a1), preferably, the pH is adjusted in the range of 7.5 to 9.0, more preferably in the range of 7.6 to 8.0. A basic pH is necessary to keep casein in solution, facilitating interaction with green tea catechins during execution of step c1), besides maintaining the antioxidant properties of the casein. The concentration of casein can be adjusted by a vacuum distillation step in order to avoid thermal degradation of the casein.

[0060] In other preferred embodiments of the process for the preparation of the complex, a step a2) of partially denaturing the casein is executed after the step a1), wherein the partial denaturing is carried out in a temperature range of 40ºC to 65ºC for 1 min to 1 hour. The optional step a2) provides a controlled denaturation of the casein, wherein reactive functional groups of the casein are exposed, contributing for an effective binding with catechins, without compromising the overall structure of the protein. Preferably, a continuous and gentle stirring of the solution comprising casein is carried out in order to allow a proper heating transfer and avoiding the formation of aggregates. More preferably, the partial denaturing is carried out in a temperature range of 50ºC to 60ºC for 5 min to 30 min. The partially denatured solution comprising casein is clear and has a viscosity suitable for the next step, remaining stable under temporary storage conditions.

[0061] Casein, unlike globular proteins, has a relatively open structure and can withstand moderate temperatures without fully denaturing. The selected range of 40ºC to 65ºC is in line with the denaturation temperature of casein, which typically begins to partially denature above 40ºC.

[0062] Partial denaturation is achieved at lower temperatures (around 40ºC to 50ºC) where only limited unfolding occurs, exposing functional groups without leading to full denaturation. Higher temperatures (closer to 65ºC) cause more extensive denaturation, potentially leading to protein aggregation.

[0063] Preferably, at higher temperatures in the range from 55ºC to 65ºC, shorter durations (around 1 to 15 minutes) are sufficient to partially denature casein without leading to full denaturation. This allows for enough unfolding to expose reactive sites without causing excessive aggregation. Alternatively, at lower temperatures in the range from 40ºC to 50ºC, longer durations (up to 1 hour) may be required to achieve the desired degree of partial denaturation. The slower rate of denaturation at lower temperatures allows for more controlled unfolding, minimizing the risk of damaging the protein structure.

[0064]

[0065] In the preferred embodiments, the second polar solvent, which is used to dissolve the green tea extract, is selected from the group consisting of water, an aqueous solution of glycerin, an aqueous solution of propylene glycol, an aqueous solution of ethyl alcohol, and mixtures thereof.

[0066] In the most preferred embodiments, the second polar solvent is a solution of glycerin in water, e.g. distilled water, or purified water, wherein the concentration of glycerin is in the range of 3 to 10% (weight / volume), more preferably in the range of 4 to 6% (weight / volume). The solution of glycerin in water provides a proper solubilization and stabilization of the catechins, contributing to the enhancement of their antioxidant properties. Moreover, the solution of glycerin in water provides a proper compatibility and moisturizing effect.

[0067] In other preferred embodiments, the second polar solvent is a solution of propylene glycol in water, wherein the concentration of propylene glycol is in the range of 1 to 10% (weight / volume), wherein this solvent is choses when a greater skin penetration is desired.

[0068] In other preferred embodiments, the second polar solvent is a solution of ethyl alcohol in water, wherein the concentration of ethyl alcohol is in the range of 5 to 10% (weight / volume) to avoid skin irritation.

[0069]

[0070] In the preferred embodiments of the process for the preparation of the complex, in step b1 concentration of catechins in the solution comprising catechins is in the range of 3% to 5% weight / volume, more preferably, in a concentration of 2,5% to 3,5% (weight / volume). Regarding the control of the parameter pH in step a2), the pH is adjusted in the range of 7.5 to 9.0. A basic pH is necessary to keep casein in solution, facilitating interaction with green tea catechins during execution of step c1).

[0071] Preferably, the solution comprising catechins is submitted to a filtering step to remove insoluble particulates that might be present in said solution. The filtering step is executed by means of a filtering media selected from the group including a paper filter, a cellulose acetate membrane, a polypropylene filter, a polytetrafluoroethylene filter, a glass fiber filter, a nylon membrane filter, a stainless steel mesh or a woven wire cloth, a cellulose-based depth filter, a polyester felt filter or a polyethylene felt filter. A suitable range for the pores sizes is the range of 0.45 micrometers to 1 micrometer. As it will be understood by a person skilled in the art, the filtration unit operation can be a filtration driven by a positive pressure gradient or by a vacuum system. The filtered solution comprising casein is clear and stable for the chemical reaction between casein and catechins.

[0072] Preferably, in step c1) of the process for the preparation of the complex, the mass proportion of the casein and of catechins extracted from green tea in the formation of the complex is in the range from 1:1 to 1:5 or in the range from 1:1 to 5:1. As examples of suitable proportions, the mass proportion of the casein and of catechins extracted from green tea in the formation of the complex is 1:1, 2:1 or 5:1 (casein:catechins) or 1:2 or 1:5 (casein:catechins). These proportions are suitable to drive the formation of covalent bonds, guaranteeing the functionality of the complex with different intensities.

[0073] The proportions between casein and catechins abovementioned are defined in a dry mass proportion.

[0074] In the step c1) of the process for the preparation of the complex, the solution comprising casein and the solution comprising catechins is kept under stirring with the pH in the range of 7.5 to 8.5, which can be adjusted with a mineral acid, e.g. HCl, or a strong base, e.g. NaOH. This pH range contributes for the solubility and stability of the reactants, promoting an efficient reaction among them.

[0075] The pH range of 7.5 to 8.5 for the reaction step has been deliberately selected based on the optimal interaction conditions between casein and catechins. This range is critical for maintaining both the solubility and stability of the reactants, as well as promoting efficient bond formation during the conjugation process. Casein’s solubility is highly dependent on pH. At a slightly alkaline pH of 7.5 to 8.5, casein maintains an optimal structural state where it is sufficiently soluble to interact with catechins without undergoing excessive denaturation. This ensures that the protein remains bioavailable and that its functional groups (such as hydroxyl and amino groups) are accessible for interactions with catechins. Catechins, especially epigallocatechin gallate (EGCG), are sensitive to both acidic and highly alkaline environments. The pH range of 7.5 to 8.5 is optimal for maintaining the antioxidant properties of catechins, preventing their degradation into oxidative by-products (such as quinones) that would compromise their efficacy. The slightly alkaline environment (7.5 to 8.5) promotes the formation of hydrogen bonds, hydrophobic interactions, and covalent bonds between casein and catechins. This pH allows for optimal deprotonation of the functional groups necessary for these interactions.

[0076] Preferably, to ensure an optimal pH control, in-line pH sensors will be employed during the reaction to continuously monitor and maintain pH within the desired range. A buffering system, such as Tris-HCl, is utilized to stabilize pH and prevent unwanted fluctuations. The buffer concentration will be optimized to maintain a pH of about 7.8, allowing for minimal variation within the 7.5 to 8.5 range without compromising the product’s stability or bioactivity.

[0077] The casein-catechin solution exhibits a viscosity range of 10 to 50 mPa·s, ensuring fluidity during processing and stability for long-term storage, wherein the viscosity is measured at room temperature (from 22ºC to 25°C) and at an agitation speed of 350 rpm using a standard rotational viscometer.

[0078] At the beginning of the conjugation process, the viscosity is expected to be on the lower end of the range (closer to 10 mPa·s), as the molecular interactions between casein and catechins will not yet be fully established. As the reaction progresses and stable covalent and non-covalent bonds form between casein and catechins, the viscosity will gradually increase, potentially reaching 50 mPa·s as the solution becomes more structured due to these interactions. Viscosity adjustments can be made by altering the concentration of reactants or by adding buffered water to ensure that the solution remains within the desired range.

[0079] When the viscosity is in the range of 10 to 20 mPa·s, casein and catechins are present in equal or similar amounts. The interaction between casein and catechins will lead to the formation of a stable complex, but the viscosity will remain on the lower end of the range, as the network structure formed is not as dense compared to higher casein concentrations. This proportion is optimal for applications requiring moderate viscosity, such as lightweight topical creams or solutions.

[0080] When the viscosity is in the range of 20 to 35 mPa·s, and with a higher proportion of casein, there will be more protein molecules available to interact with the catechins, resulting in a denser network of bonds. This increases the viscosity of the final solution, as more covalent and non-covalent interactions form. This proportion would be suitable for thicker formulations, such as gels or ointments.

[0081] When the viscosity is in the range of 35 to 50 mPa·s, the solution will form a much more structured and viscous network due to the abundant protein available for binding with the catechins. The higher casein content leads to a more gel-like consistency, which would be ideal for applications that require thicker, highly viscous formulations such as wound care dressings or controlled-release pharmaceutical products.

[0082] The end of the reaction can be defined using the combination of viscosity measurements and other complementary analytical techniques such and FTIR to ensure that the reaction has progressed to the point of full conjugation between casein and catechins.

[0083] The reaction is considered complete when the viscosity reaches a steady-state plateau around 20-50 mPa·s (depending on the formulation proportion), with no further significant changes in viscosity over time. This stabilization reflects the completion of bond formation between casein and catechins and the cessation of any further structural development in the reaction medium.

[0084] The formation of new covalent bonds between the hydroxyl groups of catechins and the amide groups of casein can be tracked using FTIR. The reaction is complete when the FTIR spectra no longer show significant changes in key absorption bands, such as those related to amide bonds (1600 - 1700 1 / cm) and hydroxyl groups (3200 - 3500 1 / cm).

[0085]

[0086] Preferably, the reaction of the step c1) is carried out maintaining the stirring of the reactional media while maintaining a temperature in the range of 10ºC to 35ºC for 1 hour to 24 hours, more preferably in the range of 15ºC to 30ºC for 2 hours to 16 hours, even more preferably in the range of 20ºC to 25ºC for 4 hours to 8 hours, to avoid degradation of the catechins and excessive denaturation of the casein, and to ensure uniform distribution of the reagents and maximize the formation of covalent bonds.

[0087] Preferably, the reaction of the step c1) is carried out at a temperature in the range of 10ºC to 20ºC. In this condition, the reaction proceeds more slowly at lower temperatures, preserving the stability of catechins, which are prone to oxidation at higher temperatures. This range favors non-covalent interactions and is suitable for formulations sensitive to heat, allowing for gradual conjugation without degrading bioactivity. At lower temperatures (10ºC to 20ºC), longer times (up to 24 hours) are required for complete conjugation. This is beneficial for preserving bioactivity, especially with sensitive ingredients.

[0088] Alternatively, the reaction of the step c1) is carried out at a temperature in the range of 25ºC to 35ºC. In this condition, the reaction rate increases, promoting covalent bond formation between casein and catechins. This range allows quicker conjugation while still protecting the bioactivity of catechins, though exceeding 35ºC could lead to degradation. In this condition, the reaction completes more rapidly (1 to 6 hours), which is ideal for industrial efficiency. Monitoring viscosity and other indicators ensure the reaction's completion.

[0089] In other embodiments of the process for the preparation of the complex, a binding agent is added to reactional mixture of the step c1), wherein the binding agent is selected from the group consisting of citric acid, genipin, glutaraldehyde, carboiimides, terephthalaldehyde, and mixtures thereof. The role of the binding agent is to facilitate the formation of covalent bonds between the casein and the catechins while acting as a catalyst during the reaction.

[0090] The binding agent is preferably an aqueous solution of citric acid, an aqueous solution of genipin, or mixtures thereof, which are highly suitable to facilitate the formation of covalent bonds between the casein and the catechins, besides their proper safety and compatibility in cosmetic applications. The concentration of citric acid in the aqueous solution is in the range of 0.5% to 2.0% (mass / volume). The concentration of genipin in the aqueous solution is in the range of 0.05% to 0.2% (mass / volume).

[0091] Alternatively, the binding agent is an aqueous solution of glutaraldehyde, an aqueous solution of carbodiimide, an aqueous solution of terephthalaldehyde, or mixtures thereof. The concentration of glutaraldehyde in the aqueous solution is in the range of 0.01% to 0.1% (mass / volume). The concentration of carbodiimides in the aqueous solution is in the range of 0.1% to 0.5% (mass / volume). The concentration of terephthalaldehyde in the aqueous solution is in the range of 0.01% to 0.05% (mass / volume). Examples of carbodiimides include the 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or the N-hydroxysuccinimide (NHS).

[0092] The stability of the casein-catechin solution is primarily governed by the pH, light exposure, temperature, and the presence of stabilizing agents like glycerin. Based on these factors, the solution is expected to maintain its properties over a considerable period, as long as it is stored under optimal conditions.

[0093] The casein-catechin solution is stable for 6 to 12 months when stored at room temperature (from 22ºC to 25°C) in sealed containers that protect it from light and oxidation. The pH of the solution, which is maintained between 7.5 and 8.5, plays a critical role in this stability, as it prevents precipitation of casein and degradation of catechins.

[0094] Preferably, the presence of 1 to 10% of a stabilizing agent (e.g. 5% (w / v) of glycerin) in the green tea extract solution further enhances the stability by preventing oxidative degradation and ensuring that the solution remains homogenous over time.

[0095] The complex formed of casein and of catechins extracted from green tea formed in the step c1) comprises a concentration in the range of 0.5 % to 5 % (mass / volume), more preferably in the range of 1.0 % to 3 % (mass / volume), even more preferably in the range of 1.2 % to 2.5 % (mass / volume). These preferred concentration ranges are proper to an effective protection against free radicals.

[0096] The present invention refers in a third aspect to a complex formed of casein and of catechins extracted from green tea, which is obtained by the process according to the second aspect of the invention.

[0097]

[0098] The present invention refers in a fourth aspect to a process for the preparation of a lyophilized composition comprising a complex formed of casein and of catechins extracted from green tea, wherein said process comprises the steps:

[0099] i) obtaining a solution of the complex formed of casein and of catechins extracted from green tea by the process, as defined in the second aspect of the invention; and

[0100] ii) purifying the solution of the complex formed of casein and of catechins extracted from green tea by a unit operation selected from the group consisting of a filtration or a dialysis, obtaining a purified solution of the complex formed of casein and of catechins extracted from green tea; and

[0101] iii) freeze-drying the solution obtained in the previous step, obtaining the lyophilized composition comprising the complex formed of casein and of catechins extracted from green tea.

[0102]

[0103] The present invention refers in a fifty aspect to a lyophilized composition comprising the complex formed of casein and of catechins extracted from green tea, which is obtained by the process defined in the fourth aspect of the invention.

[0104]

[0105] For the purpose of the present invention, the term “lyophilized composition” refers to a product that has been freeze-dried to remove water content, resulting in a stable, dry powder. Broadly, lyophilization is a dehydration process typically used to preserve perishable materials or make them more convenient for transport. In this invention, the lyophilized composition comprises the complex formed of casein and catechins extracted from green tea. The process involves obtaining a solution of the complex, purifying it through filtration or dialysis, and then freeze-drying it. In a narrower sense, the lyophilized composition is specifically designed to maintain the stability and efficacy of the casein-catechin complex for use in cosmetic and pharmaceutical applications.

[0106] In one preferred embodiment, the purification of the solution of the complex formed of casein and of catechins extracted from green is executed by ultrafiltration. Even more preferably, the ultrafiltration system used in the step ii) of the process for preparation lyophilized composition is carried out by means of a pressure and flow control. The preferred ultrafiltration membranes are Polysulfone (PSF) or Polyethersulfone (PES), chosen for their chemical and thermal resistance. Preferably, the Molecular Weight Cut-off is adjusted in the range of 10 kDa to 30 kDa to retain the complex and remove minor impurities.

[0107] A pre-filtration can be executed prior to the ultrafiltration step in order to remove large particles to prevent membrane clogging. The preferred operational parameters of the pre-filtration are: pressure from 1 to 3 bar (100 kPa to 300 kPa) and a flow rate between 0.1 to 1 L / min to ensure efficient filtration.

[0108] Preferably, during the pre-filtration and the ultrafiltration step, the temperature is maintained in the range of 20°C to 25°C and the pH of 7.5 to 8.5.

[0109] Ultrafiltration is a fast and efficient method for removing low molecular weight impurities while retaining macromolecules such as the casein complex and catechins. This process is highly time-efficient, allowing purification in just a few hours. The ultrafiltration is ideal for large-scale production where time efficiency is a priority and it is desired to maintain a high concentration of the active product.

[0110] In another preferred embodiment, the purification of the solution of the complex formed of casein and of catechins extracted from green is executed by dyalisis. Even more preferably, the dialysis tubes have a cut-off of 3.5 kDa to 10 kDa, and the dialysis caps are configured to operate with a buffer in a pH in the range of 7.2 to 9.0. When the buffer is a solution of Phosphate (PBS), the pH is in the range of 7.2 to 7.4. When the buffer is a solution of Tris-HCl, the pH is in the range of 7.5 to 8.5, when the buffer is a solution of Borate, the pH is in the range of 8.0 to 9.0. When the buffer is a solution of Bicarbonate, the pH is in the range of 7.5 to 8.0.

[0111] The dialysis step is carried out under gentle agitation, preferably changing the buffer every 4-6 hours for 24-48 hours.

[0112] The dialysis step is particularly effective at removing ions, small molecules and other contaminants through a more gradual process. This method is highly effective in achieving extreme purity, ideal for sensitive products that require milder conditions. The dialysis is preferable for smaller batches or when the integrity of the product is crucial, such as in formulations that require very high purity levels. Dialysis better preserves the bioactive properties of the complex by avoiding exposure to high pressures, making it suitable for more delicate products.

[0113]

[0114] . The freeze-drying step of the process for the preparation of a lyophilized composition comprising a complex formed of casein and of catechins extracted from green tea is preferably carried out by executing a first stage of quick-freezing and a second stage of freeze-drying.

[0115] The first stage referred to the quick-freezing is preferably executed in a deep-freezer in a range of -40ºC to -80°C to prepare for freeze-drying. This stage ensures the formation of small ice crystals, preserving the structural integrity of the complex during freeze-drying. Preferably, the quick-freezing step during the freeze-drying process is ideally carried out in a range from -70ºC to -80°C (e.g. at -80ºC), ensuring that ice crystal formation is minimized, and the structural integrity of the casein-catechin complex is preserved. This temperature produces small, uniform ice crystals, preventing structural damage to the complex.

[0116] The second stage referred to the freeze-drying comprises a primary sublimation stage and a secondary desorption stage. The primary sublimation stage is preferably carried out in a temperature in the range -40 °C to -20 °C and in a pressure in the range of 100 mTorr to 200 mTorr (13 Pa to 27 Pa). The duration of the primary sublimation stage is adjusted according to the volume and nature of the sample, preferably in the range of 24 to 48 hours. The secondary desorption stage is preferably carried out in an increasing temperature, gradually increased from the first range -40 °C to -20 °C to a second range 20 °C to 30 °C and in a pressure in the range of 50 mTorr to 200 mTorr (6 Pa to 26 Pa). The duration of the secondary desorption stage is preferably in the range of 6 to 12 hours. During the transition between the primary sublimation stage and the secondary desorption stage, it is preferably used a heating in the range of 1°C to 2°C per hour to prevent the material from collapsing and preserve its structure. Moreover, preferably, the thickness of the frozen solution layer is maintained in the range of 1 to 2 cm to ensure uniform sublimation.

[0117] The obtained lyophilized composition comprising the complex formed of casein and of catechins extracted from green tea in the form of a powder is stored in hermetically sealed containers protected from light and humidity to maintain stability and prolong its shelf life.

[0118] The obtained lyophilized composition has an increased storage Stability with a minimum shelf life of 12 months, validated by accelerated stability tests, maintaining its antioxidant and cosmetic properties. The lyophilized composition is resistant to environmental factors, considering tests carried out to assess for resistance to UV light, heat and oxidation, showing a minimizing degradation of the complex formed of casein and of catechins extracted from green tea.

[0119] Residual Moisture: After freeze-drying, the powder has a moisture content of less than 5%, guaranteeing the prevention of microbial growth.

[0120]

[0121] The present invention refers in a sixty aspect to a cosmetic composition for topical application intended for preventing or repairing cutaneous damages related to aging, characterized by comprising as an active ingredient the complex according to the first aspect of the invention, in combination with any excipient which is physiologically compatible with skin.

[0122] Irritation and sensitization tests carried out for the complex formed of casein and of catechins extracted from green tea confirm that the complex is safe and non-irritating for all skin types. Moreover, clinical tests demonstrate efficacy and safety, confirming the absence of significant adverse effects.

[0123] Preferably, the cosmetic composition comprises a lyophilized composition including a complex formed of casein and of catechins extracted from green tea according to the present invention.

[0124]

[0125] The present invention refers in a seventy aspect to a pharmaceutical composition for use in the treatment of dermatological ailments characterized by comprising a complex as defined in the first aspect of the invention, in combination with any pharmaceutically acceptable carrier, or excipient.

[0126] The pharmaceutical composition of the present invention includes topical pharmaceutical compositions, e.g. an ointment, a gel, a lotion, a spray, a foam, a transdermal patch, or a cream for topical use. Oral pharmaceutical compositions comprise tablets, capsules, solutions, suspensions, syrups, powders or granules.

[0127] For topical pharmaceutical compositions, the concentration of the casein-catechin complex can range from 0.5% to 5% (w / v). This range provides sufficient active complex to deliver antioxidant, anti-inflammatory, and wound healing properties without compromising the formulation’s viscosity, spreadability, or patient comfort.

[0128] In oral pharmaceutical compositions, the concentration of the casein-catechin complex can range from 1% to 10% (w / v). This broader range ensures sufficient bioavailability and efficacy, particularly in controlled-release formulations, where the casein matrix aids in the slow and sustained release of catechins.

[0129] The defined ratios and concentration ranges ensure that the formulation delivers the therapeutic effects of both casein and catechins. Casein serves as a carrier for the catechins, enhancing their stability, bioavailability, and controlled release at the site of application. The proposed concentrations for both the complex and catechins are within ranges that promote stability under typical pharmaceutical storage conditions (e.g., room temperature, protection from light). The inclusion of casein, with its stabilizing and film-forming properties, further protects the catechins from oxidation and degradation, particularly during long-term storage.

[0130] The preferred ranges allow flexibility in adjusting the formulation to suit different pharmaceutical applications, from topical creams and gels to oral capsules or tablets, while ensuring that the active ingredients remain within the effective concentration range.

[0131] The integration of casein and green tea catechins creates a complex with antioxidant, anti-inflammatory and healing properties. These characteristics are fundamental for therapeutic formulations aimed at treating skin conditions such as eczema, psoriasis, and skin lesions, where inflammation and oxidative stress play critical roles in worsening symptoms. In addition, the complex formed of casein and of catechins extracted from green tea promote cell regeneration and is beneficial in skin recovery treatments, facilitating natural healing and repair processes.

[0132] Examples

[0133] Antioxidant action

[0134] illustrates the antioxidant action of the complex formed of casein and of catechins extracted from green tea determined by the DPPH method, which is an in vitro test, indicating a significant reduction (> 50%) of free radicals, proving the high antioxidant capacity of the complex according to the invention. In, concentration is displayed in micrograms / mL and ROS stands for Reactive Oxygen Species.

[0135] : A solution of (2,2-Diphenyl-1-picrylhydrazyl) DPPH (0.1 mM) was prepared in absolute ethanol. The samples evaluated include prepared solutions of the main active ingredients (green tea, casein) and a cream formulation in different concentrations. The samples were prepared by mixing 100 µL of each sample in 2.9 mL of the DPPH solution. The mixtures were incubated in the dark for 30 minutes at room temperature. The absorbance was measured at 517 nm using a spectrophotometer. The concentration required to reduce DPPH activity by 50% (IC50) was determined, using the ISO 14502-1 as a normative of reference, wherein the results are plotted in.

[0136] : In vitro tests showed that complex according to the invention significantly (> 50%) reduces free radicals, demonstrating a high antioxidant capacity. This result confirms that the product can effectively protect the skin against oxidative damage, in line with the protection and anti-aging claims.

[0137] Green tea contains catechins, which are powerful antioxidants that neutralize free radicals. Casein, as well as being an effective carrier, can act as a stabilizing matrix for catechins, increasing their bioavailability and prolonging their antioxidant activity in the skin.

[0138]

[0139] Cytotoxicity

[0140] Cells of the cell line NCTC clone 929 (ATCC CCL 1) were grown in Eagle's minimal medium (EMM) supplemented with 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acids and 1 mM sodium pyruvate. The cells were incubated at 37 °C in a humid atmosphere with 5% CO₂ until a cell monolayer was formed.

[0141] The sample preparation included serial dilutions of the face cream, which were prepared in PBS culture medium. Negative (PBS) and positive (0.02% phenol solution) controls were used.

[0142] During the test procedure, 200 microliters of a suspension of 2.5 x 105cells / mL was added to the wells of a 96-well microplate and incubated for 24 hours for adhesion. The culture medium was replaced by dilutions of the Complex and the controls, in triplicate. The plates were incubated for 24, 48 and 72 hours at 37oC.

[0143] After the incubation period, the neutral red incorporation method was used to assess cell viability. Live cells incorporated the dye, while dead cells did not. 0.2 mL of MEM containing 50 mg / mL of neutral red was added, incubated for 3 hours, washed with PBS and fixed with a solution of CaCl₂ in formaldehyde. The dye was solubilized in acetic acid in ethanol and the optical density was read at 540 nm.

[0144] illustrates the percentage of cell viability, which was calculated and compared to the negative control for the formulations comprising Casein 5% (mass / volume), Complex 0.3% (mass / volume), Complex 0.6% (mass / volume), and Complex 9% (mass / volume).

[0145] The cytotoxicity assay was conducted using 10% DMSO (v / v) as the positive control, a concentration known to induce cytotoxic effects through cell membrane disruption and apoptosis. This concentration has been validated in the NCTC clone 929 cell line and is commonly used in cytotoxicity assays to benchmark the effects of test compounds.

[0146] Phosphate-buffered saline (PBS) was used as the negative control to represent normal, non-toxic cell conditions. The results of the assay were compared against these controls to assess the cytotoxic potential of the casein-catechin formulations.

[0147] The complex showed a low percentage of hemolysis (<5%), indicating that the product is hemocompatible and safe for topical application, minimizing risks of adverse reactions.

[0148] Healing action

[0149] Cells of the cell line NCTC clone 929 (ATCC CCL 1) were cultured in 24-well plates until they reached confluence.

[0150] A scratch test was carried out, wherein a pipette created a straight line in the middle of the confluent cells. The cells were washed with PBS to remove cell debris.

[0151] Solutions of the complex in different concentrations were added to the wells. Culture medium was used as a negative control.

[0152] The plates were incubated at 37°C with 5% CO₂. Photographs were taken immediately after scratching and after 24, 48 and 72 hours to monitor cell migration.

[0153] In order to quantify cell migration, the area of the scratch was measured using image analysis software. The rate of scratch closure was compared between the different treatments and the control.

[0154] illustrates the Scratch Test for a healing action of a formulation comprising the complex according to the invention 0.6% (mass / volume).

[0155] The healing trials indicated that the complex significantly accelerates wound closure by promoting cell migration. Therefore, the complex according to the invention aids cell regeneration and improves healing, reinforcing its efficacy in post-treatment applications.

[0156] Casein is rich in essential amino acids that support cell regeneration, while the catechins in green tea reduce inflammation and accelerate tissue recovery. When combined, these components act synergistically to accelerate healing and regeneration, especially under conditions of oxidative stress.

[0157] Hemocompatibility

[0158] A sample of the complex according to the invention was dissolved in PBS at different concentrations.

[0159] The blood collection was carried out by obtaining Fresh human blood from healthy volunteer donors (Origin), and by preparing a solution of 3.8% sodium citrate was used to prevent coagulation (Anticoagulant).

[0160] The Procedure of the hemolysis test was: 0.2 mL of anticoagulated blood was added to the saline solution containing the samples. Positive (distilled water) and negative (saline solution) control tubes were prepared simultaneously.

[0161] The tubes were incubated at 37°C for 1 hour under gentle agitation.

[0162] After incubation, the samples were centrifuged at 2500 rpm for 15 minutes. The absorbance of the supernatant was measured at 540 nm to quantify the hemoglobin released. The percentage of hemolysis was calculated in comparison to the positive and negative controls.

[0163] Results with hemolysis of less than 5% are considered hemocompatible. The hemolysis between the samples containing the complex according to the invention and the controls was compared to assess the compatibility of the material with blood, as illustrated in, which illustrates results of the hemocompatibility test for a Base sample (5% Casein), a first sample of complex 0.3% (mass / volume), and a second sample of Cas-GT 0.6% (mass / volume).

[0164] Irritability test (HET-CAM Test (Hen Egg Allantois Membrane Test))

[0165] The objective of this teste is to evaluate the irritation potential of hydrogels according to the ICCVAM protocol.

[0166] Materials used: Eggs: Eggs of the White Essex strain (White Leghorn hybrids; Euribrid N.V., Aarschot, Belgium) weighing 60.0 ± 5.0 g.

[0167] Equipment: Rotary tray incubator (Petersime, Zulte-Olsene, Belgium), laser source lamp (Schott KL I500 electronic, Zaventem, Belgium), dentist's rotary saw (Dentimex, Zeist, Netherlands), Kuhne tweezers (Wironit 5862, Vel, Leuven, Belgium), vacuum pump system (Inotech, Brussels, Belgium), curved tip tweezers (Laborimpex, Brussels, Belgium).

[0168] The selected eggs were incubated for 10 days with the larger end facing upwards in an incubator at 37.0 ± 0.5°C and 62.5 ± 1.5% relative humidity.

[0169] On day 9, the eggs were placed at the bottom of the incubator and incubation continued without rotation.

[0170] On the 10th day, the eggs were candled with a laser lamp to ensure viability. Unfertilized or non-viable eggs were discarded.

[0171] For the egg preparation, the area delimited by the inner membrane at the larger end of the egg was marked and the shell was removed using a rotary saw and Kuhne tweezers.

[0172] : The inner membrane, in direct contact with the CAM, was moistened with 1.5-2.0 mL of 0.9% saline solution and the egg was returned to a temperature of 37°C for a maximum of 20 minutes.

[0173] After incubation, the saline solution was gently removed with a vacuum pump and the inner membrane was carefully removed with curved-tip forceps, without damaging the underlying blood vessels.

[0174] 100 UL of the formulations containing the complex according to the invention were placed in contact with the inner membrane for 5 minutes.

[0175] During the microscopic examination, the CAM was exposed and examined microscopically to confirm its integrity. The presence of vascular reactions such as hemorrhage, coagulation and vasodilation was observed to assess the irritation potential of the hydrogels tested.

[0176] : Tests have shown that the complex does not cause significant irritant reactions, ensuring that it is suitable for use on all skin types, including the most sensitive. This corroborates the product's hypoallergenicity features.

[0177] The Table 1 summarizes the conditions that were tested during development of the complex according to the invention.

[0178] StageConditions studied to determine optimum parametersParameterTested ConditionPreferred rangeObservationsPreparation of Casein (CAS)CAS concentration0,50% - 5%1% - 2%<0.50%: insufficient interaction; >2%: excessive viscositypH of the solution07-097,6-8,5<7.5: precipitation; >9: excessive denaturationDenaturation Temperature40°C - 75°C50°C - 60°C<50°C: insufficient denaturation; >60°C: impaired functionalityDenaturation Time5 min - 30 min20 min - 30 min<20 min: incomplete denaturation; >30 min: risk of aggregationPreparation of Green Tea Extract (GT)Glycerin concentration in the solvent0% - 3%1% - 3%0%: instability of catechins; <1%: insufficient solubilityCatechin concentration0% - 10%0,30% - 5%<0.30%: reduced efficacy; >5%: precipitation, toxicity problemsComplexCasein Conjugation (CAS) and Green Tea Extract (GT)Mixture Proportion (CAS / GT)1:1 - 5:12:1 - 5:1 e 3:21:1 - 1:5: high proportions of extract dilute the effectiveness of caseinpH of the mixture07-0908_09<7.5: precipitation of components; >9: excessive denaturationReaction temperature4°C - 60°C20°C - 25°C<20°C: slow reaction; >25°C: degradation of catechinsReaction time30 min - 12h2h - 12h<2h: incomplete formation of the complex; >12h: instability of the complexStirring speed<200 rpm - >400 rpm200 rpm - 400 rpm<200 rpm: inadequate mixing; >400 rpm: foaming and instability

[0179]

[0180] The subject matter described above is provided as an illustration of the present invention and, therefore, should not be construed to limit it. The terminology employed for the purpose of describing preferred embodiments of the present invention should not be restricted to them.

[0181] As used in the description, defined and indefinite articles, in their singular form, are intended for interpretation to also include plural forms, unless the context of the description explicitly indicates otherwise.

[0182] Undefined articles "one" should generally be interpreted as "one or more", unless the meaning of a singular modality is clearly defined in a specific situation.

[0183] It will be understood that the terms "understand" and "include", when used in this description, specify the presence of characteristics, elements, components, steps and related operations, but do not exclude the possibility of other characteristics, elements, components, steps and operations as well contemplated.

[0184] As used throughout this patent application, the term "or" is used in an inclusive sense rather than an exclusive sense, unless the exclusive meaning is clearly defined in a specific situation. In this context, a phrase of the type "X uses A or B" should be interpreted as including all relevant inclusive combinations, for example "X uses A", "X uses B" and "X uses A and B".

[0185] In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

[0186] All changes, provided they do not modify the essential characteristics of the following claims, must be considered within the scope of the protection of the present invention.

Claims

A complex characterized by being formed of casein and of catechins extracted from green tea, wherein the casein and catechins are combined by covalent bonds.The complex, according to claim 1, wherein the mass proportion of the casein and of catechins extracted from green tea in the formation of the complex is in the range from 1:1 to 1:5 or in the range from 1:1 to 5:1 (dry mass / dry mass).A process for the preparation of the complex defined in any one of claims 1 to 2, characterized by comprising the steps:a1) Dissolving the casein in a first polar solvent, and adjusting the pH in the range of 7.0 to 10.0, obtaining a solution comprising casein; andb1) Dissolving the green tea extract in a second polar solvent, and adjusting the pH in the range of 7.5 to 9.0, obtaining a solution comprising catechins; andc1) Reacting the solution comprising casein and the solution comprising catechins, keeping the pH in the range of 7.5 to 8.5, obtaining a complex formed of casein and of catechins extracted from green tea.The process, according to claim 3, wherein the first polar solvent is selected from the group consisting of a tris(hydroxymethyl)aminomethane hydrochloride buffer, a borate buffer, a bicarbonate buffer, an amino acid buffer, and mixtures thereof.The process, according to any one of claims 3 to 4, wherein a step a2) of partially denaturing the casein is executed after the step a1), wherein the partial denaturing is carried out in a temperature range of 40ºC to 65ºC for 1 min to 1 hour.The process, according to any one of claims 3 to 5, wherein the second polar solvent is selected from the group consisting of water, an aqueous solution of glycerin, an aqueous solution of propylene glycol, an aqueous solution of ethyl alcohol, and mixtures thereof.The process, according to any one of claims 3 to 6, wherein the solution comprising casein comprises casein in a concentration of 3% to 5% (weight / volume).The process, according to any one of claims 3 to 7, wherein the concentration of catechins in the solution comprising catechins is in the range of 3% to 5% weight / volume.The process, according to any one of claims 3 to 8, wherein the reaction of the step c1) is carried out in a temperature in the range of 10ºC to 35ºC for 1 hour to 24 hours.The process, according to any one of claims 3 to 9, wherein a binding agent is added to reactional mixture of the step c1), wherein the binding agent is selected from the group consisting of citric acid, genipin, glutaraldehyde, carboiimides, terephthalaldehyde, and mixtures thereof.A complex formed of casein and of catechins extracted from green tea, characterized by being obtained by the process defined in any one of the claims 3 to 10.A process for the preparation of a lyophilized composition comprising a complex formed of casein and of catechins extracted from green tea, characterized by comprising the steps:obtaining a solution of the complex formed of casein and of catechins extracted from green tea by the process, as defined in any one of the claims 3 to 10; andpurifying the solution of the complex formed of casein and of catechins extracted from green tea by a unit operation selected from the group consisting of a filtration or a dialysis, obtaining a purified solution of the complex formed of casein and of catechins extracted from green tea; andfreeze-drying the solution obtained in the previous step, obtaining the lyophilized composition comprising the complex formed of casein and of catechins extracted from green tea.A lyophilized composition comprising the complex formed of casein and of catechins extracted from green tea, characterized by being obtained by the process defined in claim 12.A cosmetic composition for topical application intended for preventing or repairing cutaneous damages related to aging, characterized by comprising as an active ingredient the complex as defined in any one of claims 1 to 2, in combination with any excipient which is physiologically compatible with skin.The cosmetic composition, according to claim 14, wherein the complex formed of casein and of catechins extracted from green tea is comprised in a lyophilized composition, according to claim 13.A pharmaceutical composition for use in the treatment of dermatological ailments characterized by comprising a complex as defined in any one of claims 1 to 2, in combination with any pharmaceutically acceptable carrier, or excipient.

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