Tripeptides or derivatives thereof and uses thereof, moisturizing compositions, moisturizing preparations, cosmetics or pharmaceuticals

Abstract

This application relates to the field of oligopeptide technology, and particularly to tripeptides or their derivatives and their applications, moisturizing compositions, moisturizing formulations, cosmetics, or pharmaceuticals. The tripeptide has an amino acid sequence of Phe-Pro-Gly from the N-terminus to the C-terminus, which can bind to aquaporin-3 and filaggrin, promoting the expression of aquaporin-3 and filaggrin, maintaining the moisture content of the stratum corneum, and exhibiting good moisturizing effects. It can be used in the preparation of moisturizing formulations, cosmetics, and / or pharmaceuticals.

Description

Technical Field

[0001] This application relates to the field of oligopeptide technology, and in particular to tripeptides or their derivatives and their applications, moisturizing compositions, moisturizing preparations, cosmetics or pharmaceuticals. Background Technology

[0002] Moisturizing is a fundamental and core function of cosmetics. The moisture content of the stratum corneum directly affects its suppleness, barrier function, and appearance. When the moisture content of the stratum corneum decreases, the skin becomes dry, flaky, rough, and may even crack. Long-term dehydration can also accelerate skin aging, leading to fine lines and decreased elasticity. The moisturizing mechanism of cosmetics mainly includes three aspects: first, forming a occlusive oil film on the skin surface to reduce transepidermal water loss; second, absorbing and locking in moisture from the external environment or deeper layers of the skin through hygroscopic ingredients; and third, repairing and strengthening the skin barrier function to enhance the skin's own moisturizing ability.

[0003] Currently, the commonly used moisturizing ingredients in cosmetics are mainly divided into the following categories: (1) Occlusive agents, such as petrolatum, lanolin, squalane and other oily ingredients, which prevent water evaporation by forming a hydrophobic barrier on the skin surface, but generally have defects such as heavy skin feel, greasiness, and easy acne; (2) Humectants, such as glycerin, propylene glycol, butylene glycol, hyaluronic acid and other polyhydroxy compounds, which rely on hydrogen bonds to bind water, but high concentrations often produce a sticky feeling, and may absorb water from the skin in the opposite direction in low humidity environment; (3) Natural moisturizing factors and their analogues, such as sodium pyrrolidone carboxylic acid (PCA), sodium lactate, urea, etc., although they have good biocompatibility, their moisturizing effect is limited when used alone; (4) Biomacromolecules, such as collagen, elastin, hyaluronic acid, etc., among which collagen is widely used because of its good biocompatibility and film-forming properties. It can reduce water loss by forming a moisturizing film on the skin surface and has certain repair effects.

[0004] However, synthetic moisturizing ingredients pose potential risks of irritation or sensitization, and moisturizing methods that rely solely on hygroscopic or occlusive mechanisms cannot fundamentally improve the skin's own water-retention capacity. Collagen, as the main structural protein of the skin's natural extracellular matrix, possesses excellent biocompatibility, low immunogenicity, and safety, and has a long history of application in the cosmetics industry. Its molecular chain is rich in hydrophilic amino acid residues such as glycine, proline, and hydroxyproline, which can bind a large number of water molecules through hydrogen bonds, forming a breathable moisturizing film on the skin surface, effectively reducing transepidermal water loss. At the same time, collagen degradation products can also provide nutritional support for skin cells, promote fibroblast proliferation and matrix synthesis, and have both moisturizing and repairing effects. With the development of biohydrolysis technology, collagen can be prepared into hydrolyzed collagen (collagen peptides) through enzymatic hydrolysis. These hydrolysates not only retain the amino acid composition and hydrophilic characteristics of collagen, but also significantly reduce the molecular weight. Their water solubility and permeability are significantly improved compared to the original collagen, making them easier for the skin to absorb and utilize. They can also stimulate the synthesis of endogenous hyaluronic acid and collagen in the skin, enhancing the skin's water retention capacity from deep within. Therefore, they are widely used in high-end moisturizing cosmetics.

[0005] However, using collagen as a moisturizing ingredient still has shortcomings. Traditional large-molecule collagen, due to its large molecular size, is unable to penetrate the skin's stratum corneum barrier and can only remain on the skin surface to exert a physical moisturizing effect, unable to penetrate into the dermis to exert its biological effects, thus limiting its full moisturizing efficacy. Although lower molecular weight collagen peptides can be obtained through hydrolysis technology, existing hydrolysates are mostly mixtures with a wide molecular weight distribution (usually containing peptides ranging from hundreds to tens of thousands of Daltons), with unclear active ingredients and difficulty in ensuring efficacy stability. Furthermore, some hydrolysates still retain a large molecular size, and the transdermal absorption efficiency needs to be improved.

[0006] Therefore, there is an urgent need to develop a novel moisturizing ingredient with a well-defined molecular structure, good transdermal absorption, safety and gentleness, and biological activity, especially specific small molecule peptides that can penetrate deep into the skin to exert moisturizing effects, in order to overcome the shortcomings of existing technologies. Summary of the Invention

[0007] Therefore, it is necessary to provide a tripeptide or its derivatives and their applications, moisturizing compositions, moisturizing preparations, cosmetics or pharmaceuticals.

[0008] In a first aspect, a tripeptide or a derivative thereof is provided, wherein the amino acid sequence of the tripeptide from the N-terminus to the C-terminus is Phe-Pro-Gly.

[0009] Secondly, the application of the tripeptide or its derivatives in the first aspect for non-diagnostic therapeutic purposes is provided in any of (I) to (V):

[0010] (I) Upregulate the expression of at least one of filaggrin and aquaporin-3;

[0011] (II) Preparation of a formulation for upregulating the expression of at least one of filaggrin and aquaporin-3;

[0012] (III) Preparation of formulations for skin moisturizing;

[0013] (IV) Preparation of cosmetics;

[0014] (V) Preparation of drugs for use on the skin.

[0015] In an optional embodiment, the subjects of the formulation for upregulating the expression of at least one of filaggrin and aquaporin-3 include subjects with UV damage.

[0016] In an optional embodiment, the upregulation of the expression of at least one of filaggrin and aquaporin-3 includes the in vitro upregulation of the expression of at least one of filaggrin and aquaporin-3 in cells.

[0017] Thirdly, a moisturizing composition is provided, the moisturizing composition comprising the tripeptide or its derivative described in the first aspect and excipients.

[0018] Fourthly, a moisturizing agent, cosmetic, or pharmaceutical is provided, which contains the tripeptide or a derivative thereof described in the first aspect; or, contains the moisturizing composition of the third aspect.

[0019] In an optional embodiment, the concentration of the tripeptide or its derivative in the cosmetic product is 0.1 wt% to 1 wt%.

[0020] In optional embodiments, the cosmetic includes creams, lotions, and liquids.

[0021] In an optional embodiment, the cosmetic is a cream containing the tripeptide or its derivatives at 0.1 wt% to 1 wt%, butylene glycol at 3 wt% to 10 wt%, propylene glycol at 1 wt% to 10 wt%, polyglycerol-10 at 1 wt% to 3 wt%, glycerin at 1 wt% to 10 wt%, β-glucan at 0.1 wt% to 1 wt%, carbomer at 0.1 wt% to 2 wt%, xanthan collagen at 0.1 wt% to 1 wt%, allantoin at 0.01 wt% to 5 wt%, sodium hyaluronate at 0.01 wt% to 5 wt%, glucose at 0.1 wt% to 5 wt%, trehalose at 0.1 wt% to 5 wt%, triethanolamine at 0.1 wt% to 5 wt%, sunflower seed oil at 5 to 10 wt%, and water at 28 wt% to 88.4 wt%.

[0022] In an optional embodiment, the drug is a topical preparation for use on the skin.

[0023] The amino acid sequence of the tripeptide (FPG peptide) or its derivative provided in this application is derived from the enzymatic hydrolysis product of tilapia skin identified by mass spectrometry. The FPG peptide can bind to aquaporin-3 and filaggrin, thereby regulating the water permeability of aquaporin-3 and promoting the gradual degradation of filaggrin during keratinocyte differentiation according to physiological rhythms, continuously generating amino acid-based NMFs (natural moisturizing factors). Simultaneously, the FPG peptide promotes the expression of aquaporin-3 and filaggrin in the subjects. Experimental verification shows that the FPG peptide has good moisturizing effects, maintaining the moisture content of the stratum corneum, and has no adverse reactions such as erythema, edema, or papules when applied to the skin. Cosmetics containing FPG peptide can maintain the moisture of the stratum corneum, playing a moisturizing role for the skin, and have no adverse reactions such as erythema, edema, or papules. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0025] Figure 1 This is a secondary mass spectrum of the enzymatic hydrolysis products of tilapia skin;

[0026] Figure 2 The results show the liquid phase purity verification of the chemically synthesized FPG peptide in Example 1;

[0027] Figure 3 The mass spectrum of the chemically synthesized FPG peptide in Example 1;

[0028] Figure 4 The molecular formula and three-dimensional structure of FPG peptide;

[0029] Figure 5 Molecular docking diagram of FPG peptide with Aquaporin-3 (A) and Filaggrin (B);

[0030] Figure 6 The results of immunoblotting detection of Aquaporin-3 and Filaggrin protein expression in HaCaT cells of each experimental group in Example 3 after treatment;

[0031] Figure 7The expression levels of Aquaporin-3 and Filaggrin proteins in HaCaT cells of each experimental group in Example 3 after treatment;

[0032] Figure 8 The moisturizing rate-time curve in Example 4;

[0033] Figure 9 The curve showing the change in skin stratum corneum moisture content over time in Example 5 is shown. Detailed Implementation

[0034] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0036] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0037] The terms “and / or,” “or / and,” and “and / or” as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. “Any and all combinations” includes any two related listed items, any more related listed items, or a combination of all related listed items. For example, “A and / or B” includes three parallel options: A, B, and “a combination of A and B.”

[0038] In this application, the terms "multiple", "various", "multiple times", "several", "several", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more or more.

[0039] In this application, "optionally", "optional", and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without".

[0040] In this application, "separately independent", "each...independently selected" and "...separately independently selected" and "...independently selected" are interchangeable and should be interpreted broadly. They refer to the range or options that each member of a set of variables or components can choose independently, that is, the choice of each variable or component is independent and is not affected by the choice of other variables or components.

[0041] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0042] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0043] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0044] In one aspect, some embodiments provide a tripeptide (FPG peptide) or a derivative thereof, wherein the amino acid sequence of the FPG peptide from the N-terminus to the C-terminus is Phe-Pro-Gly, i.e., phenylalanine-proline-glycine. This application obtained the amino acid sequence of the tripeptide by mass spectrometry identification of the enzymatic hydrolysis product of tilapia skin and then synthesized the tripeptide using a chemical method for subsequent functional verification. Molecular docking verification showed that the FPG peptide can stably bind to the functional pocket region near the aquaporin-3 channel, regulating the water permeability of aquaporin-3; the FPG peptide can bind to filaggrin, promoting the gradual degradation of filaggrin according to physiological rhythms during keratinocyte differentiation, thereby continuously generating amino acid-based NMFs (natural moisturizing factors). Simultaneously, the FPG peptide promotes the expression of aquaporin-3 and filaggrin in the subjects. Experimental verification showed that the FPG peptide has good moisturizing effects, maintaining the moisture content of the stratum corneum, and no adverse reactions such as erythema, edema, or papules were observed when applied to the skin.

[0045] In this application, the derivative of the tripeptide refers to a compound formed by chemical modification or structural alteration of the aforementioned FPG peptide, which retains the core amino acid sequence and has the same or enhanced biological activity. The derivative of the tripeptide includes, but is not limited to, the following modification types: salt form, such as a pharmaceutically acceptable salt formed with an inorganic acid (hydrochloric acid, sulfuric acid, phosphoric acid, etc.), an organic acid (acetic acid, citric acid, tartaric acid, etc.), or an inorganic base (sodium hydroxide, potassium hydroxide, calcium hydroxide, etc.); and acylated form, esterified form, terminal modified form, polyethylene glycolation, glycosylation, and glycolipidation; and prodrug form, such as being linked to a protecting group through a cleavable linker (ester bond, amide bond, carbonate bond, or enzyme-sensitive peptide bond), releasing the FPG peptide in vivo via enzymatic or chemical cleavage.

[0046] In an optional implementation, the FPG peptide is an isolated tripeptide, where "isolated" means that the peptide is not in its native medium or in its native form. Therefore, the term "isolated" includes peptides removed from their original environment, such as those naturally occurring if they are present. For example, isolated peptides typically do not contain at least certain proteins or other cellular components that are normally bound to or mixed with or in solution. Isolated peptides include naturally produced peptides contained in cell lysates, peptides in purified or partially purified forms, recombinant peptides, peptides expressed or secreted by cells, and peptides in heterologous cells or cultures.

[0047] In an optional implementation, the FPG peptide is a peptide obtained through chemical synthesis.

[0048] Based on the functions of the FPG peptides described above, in a second aspect, some embodiments also provide the application of the tripeptide or its derivatives of the first aspect in any one of (I) to (V), wherein the application of any one of (I) to (V) may be for diagnostic and therapeutic purposes or for non-diagnostic and therapeutic purposes:

[0049] (I) Upregulate the expression of at least one of filaggrin and aquaporin-3.

[0050] In optional implementations, examples of non-diagnostic and therapeutic applications of upregulating at least one of filaggrin and aquaporin-3 include, but are not limited to, using it to construct cell models, such as cell models with high expression of at least one of filaggrin and aquaporin-3; constructing in vitro reconstructed skin models, using FPG peptides to regulate the expression levels of filaggrin and aquaporin-3 in the model to make it closer to the physiological state of natural skin, etc.

[0051] (II) Preparation of formulations for upregulating the expression of at least one of filaggrin and aquaporin-3. Examples of formulations for upregulating the expression of at least one of filaggrin and aquaporin-3 include, but are not limited to, reagents for constructing cell models or in vitro reconstructed skin models as described above; reagents for studying the molecular mechanisms of filaggrin and aquaporin-3 related signals; or standards or references for screening substances that regulate filaggrin and aquaporin-3.

[0052] In an optional implementation, the upregulation of at least one of filaggrin and aquaporin-3 in the application of the first aspect (I) includes upregulating the expression of at least one of filaggrin and aquaporin-3 in subjects with UV damage.

[0053] In an optional implementation, the subject of the formulation for upregulating the expression of at least one of filaggrin and aquaporin-3 in the application of the first aspect (II) includes a subject with UV damage.

[0054] In an optional embodiment, the upregulation of at least one of filaggrin and aquaporin-3 in the first aspect of application (I) and (II) includes the upregulation of at least one of filaggrin and aquaporin-3 in vitro.

[0055] In an optional implementation, the cells include keratinocytes.

[0056] (III) Preparation of formulations for skin moisturizing.

[0057] In optional embodiments, examples of preparations for skin moisturizing include, but are not limited to, preparations for the treatment of skin diseases and preparations for the repair of skin wounds.

[0058] In an optional embodiment, the skin moisturizing agent is used to maintain the moisture content of the stratum corneum of the skin.

[0059] (IV) Preparation of cosmetics: FPG peptides or their derivatives can be combined with cosmetic-acceptable carriers and excipients to prepare cosmetics with skin moisturizing effects.

[0060] (V) Preparation of drugs for use on the skin.

[0061] FPG peptides or their derivatives can be combined with pharmaceutically acceptable carriers and excipients to prepare drugs with skin moisturizing effects, such as drugs for the treatment of skin diseases, drugs for wound repair, drugs for postoperative care, medical moisturizing excipients, and skin care preparations during treatment.

[0062] Thirdly, some embodiments also provide a moisturizing composition containing the tripeptide or its derivative from the first aspect and excipients. This moisturizing composition, containing the FPG peptide or its derivative from the first aspect, can maintain the moisture of the stratum corneum and thus provide a moisturizing effect.

[0063] In optional embodiments, exemplary excipients include, but are not limited to, at least one of solvents, diluents, fillers, pH adjusters, stabilizers, thickeners, emulsifiers, penetrants, carriers, and matrices.

[0064] Fourthly, some embodiments also provide a moisturizing agent, cosmetic, or pharmaceutical. The moisturizing agent, cosmetic, or pharmaceutical independently contains the tripeptide or its derivative as described in the first aspect, or contains a moisturizing composition as described in the third aspect, and can provide good moisturizing effects on the skin.

[0065] In an optional embodiment, the concentration of the tripeptide or its derivative in the cosmetic is 0.1 wt% to 1 wt%, preferably 0.5 wt%.

[0066] In an optional embodiment, the cosmetic contains excipients acceptable for use in the cosmetic field, including but not limited to at least one of the following: matrix, carrier, emulsifier, thickener, stabilizer, preservative, antioxidant, colorant, fragrance, pH adjuster, humectant, binder and disintegrant.

[0067] In optional embodiments, cosmetics include, but are not limited to, toners, creams, lotions, serums, masks, and eye creams.

[0068] In optional embodiments, the cosmetic formulation may include, but is not limited to, creams, lotions, and aqueous solutions.

[0069] In an optional embodiment, the cosmetic is a cream or ointment, and also contains at least one of a base, a moisturizer, a thickener, a pH adjuster, a skin emollient, and other functional ingredients.

[0070] In an optional embodiment, the cosmetic is a cream containing 0.1 wt% to 1 wt% of tripeptide or its derivative, 3 wt% to 10 wt% of butylene glycol, 1 wt% to 10 wt% of propylene glycol, 1 wt% to 3 wt% of polyglycerol-10, 1 wt% to 10 wt% of glycerin, 0.1 wt% to 1 wt% of β-glucan, 0.1 wt% to 2 wt% of carbomer, 0.1 wt% to 1 wt% of xanthan collagen, 0.01 wt% to 5 wt% of allantoin, 0.01 wt% to 5 wt% of sodium hyaluronate, 0.1 wt% to 5 wt% of glucose, 0.1 wt% to 5 wt% of trehalose, 0.1 wt% to 5 wt% of triethanolamine, 5 to 10 wt% of sunflower seed oil, and 39 wt% to 88.4 wt% of water.

[0071] In an optional embodiment, the drug in the fourth aspect includes a topical preparation for the skin. Since the drug for the skin contains FPG peptides or their derivatives, it has a moisturizing effect and can assist other active ingredients of the drug to exert their effects in a better skin environment, or use its moisturizing function to assist in the repair of skin wounds.

[0072] In an optional embodiment, the topical formulation for skin further includes pharmaceutical excipients, including but not limited to at least one of solvents, bases, emulsifiers, stabilizers, thickeners, transdermal absorption enhancers, antibacterial agents, preservatives, pH adjusters, and buffering components.

[0073] The following are some examples.

[0074] The embodiments of this application will be described in detail below with reference to some examples. It should be understood that these embodiments are only for illustrating this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0075] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0076] Example 1

[0077] Preparation and characterization of tripeptides

[0078] 1. Enzymatic hydrolysis of tilapia skin

[0079] Take an appropriate amount of tilapia skin, soak it in 5-10% NaOH solution to remove impurities, then rinse it repeatedly with water until the pH of the washing solution is neutral; then add a complex protease for enzymatic hydrolysis for 5 hours, then inactivate the enzyme activity by boiling water bath, filter the hydrolysate through filter paper to remove impurities, and finally freeze dry to obtain the tilapia skin enzymatic hydrolysate.

[0080] 2. Mass spectrometry identification

[0081] (1) Experimental Methods: The enzymatic hydrolysis products of tilapia skin were subjected to mass spectrometry. Electrospray ionization (ESI) was used to convert sample molecules into gaseous ions, which then entered the mass analyzer of the mass spectrometer. In the mass analyzer, ions were separated according to their mass-to-charge ratios, and the signal intensity of each ion was recorded by the detector to obtain a secondary mass spectrum. By analyzing the secondary mass spectrum, peptide sequences with higher reliability and peak area were selected for further study.

[0082] (2) Experimental results: such as Figure 1 The image shows the secondary mass spectrum of the screened peptide sequence FPG, a tripeptide consisting of glycine-proline-histidine (Phe-Pro-Gly) from the N-terminus to the C-terminus. The characteristic peaks of the FPG peptide sequence are clearly visible in the secondary mass spectrum, and their positions and intensities are highly consistent with theoretical predictions. In-depth analysis of the mass spectrometry data not only confirmed the existence of the FPG peptide sequence but also revealed its possible modification sites and structural features, providing crucial data support for subsequent functional studies and application development.

[0083] 3. Chemical Synthesis

[0084] (1) Experimental Method: FPG was chemically synthesized. First, the required amino acid raw materials were accurately weighed. According to the specific sequence of the FPG (phenylalanine-proline-glycine) peptide sequence, the amino acids were activated sequentially to make them reactive. Then, they were added to the peptide synthesis reactor, and the reactor was placed in a shaker at 30°C for 2 hours. A small amount of resin was tested using the ninhydrin method. If the resin was colored, it indicated that the condensation was incomplete, and the reaction was continued. If the resin was colorless, it indicated that the reaction was complete. After the reaction was complete, the resin was washed four times with DMF and then dried. The FPG peptide chain was gradually constructed. After the reaction was completed, the synthesized product was separated and purified to remove unreacted raw materials, by-products and other impurities, and finally high-purity FPG peptides were obtained. The purity was verified by liquid chromatography, and the results were as follows. Figure 2 As shown, the mass spectrum of chemically synthesized FPG was analyzed by mass spectrometry. Figure 3 As shown.

[0085] (2) Experimental results: Figure 4The molecular and three-dimensional structures of FPG peptides are shown. The molecular structure of FPG peptides exhibits a specific arrangement and combination, and its three-dimensional structure also shows a unique spatial conformation. This specific structure determines its possible biological activity and functional properties.

[0086] Example 2

[0087] Molecular docking:

[0088] (1) Experimental Methods: The three-dimensional crystal structures of aquaporin-3 and filaggrin were obtained from the Protein Data Bank (PDB). After downloading the structures, the proteins were preprocessed using AutoDock Tools. The two-dimensional structure of the ligand peptide FPG was drawn using ChemDraw and converted into a three-dimensional structure using PyMOL. Molecular docking calculations were performed using AutoDock Vina software. The docking grid was set with the potential binding cavities around Aquaporin-3 and filaggrin as the center, and the grid size covered the entire possible binding region to ensure that the ligand could fully search for target sites. During docking, the receptor protein remained rigid, and the ligand peptide FPG was set to be completely flexible. Multiple possible conformations were generated for each docking, and the results were sorted according to the binding free energy. The complex with the lowest binding energy and reasonable conformation was selected for subsequent analysis.

[0089] (2) Experimental results: Molecular docking results show that ( Figure 5 The peptide FPG can stably bind to the functional pocket region near the Aquaporin-3 channel. This binding site is close to a key region of Aquaporin-3 involved in the transport of water and glycerol molecules, suggesting that the binding of the peptide FPG may regulate the channel's permeability. From a structural perspective, after the peptide FPG is embedded around the channel, it may affect the transport efficiency of Aquaporin-3 for water molecules through spatial occupancy effects or by inducing local conformational changes in the protein. Moderately regulating the water permeability of Aquaporin-3 can help reduce excessive epidermal water loss, thereby improving the skin's water retention capacity to some extent.

[0090] Filaggrin is a key protein for maintaining the structural integrity of the stratum corneum and the generation of natural moisturizing factors (NMFs), and its metabolic processes directly affect the stratum corneum's water content and barrier function. Molecular docking results show that the peptide FPG can stably bind to specific binding pockets on the surface of filaggrin, and enhance the stability of the complex through multiple hydrogen bonds and hydrophobic interactions. The binding of peptide FPG to filaggrin may help maintain its structural stability and promote the gradual degradation of filaggrin according to physiological rhythms during keratinocyte differentiation, thereby continuously generating amino acid-based NMFs. This mechanism of action helps enhance the stratum corneum's ability to absorb and retain moisture, constituting a second regulatory mechanism for skin hydration.

[0091] The FPG peptides prepared in Example 1 were used in Examples 3-6 below.

[0092] Example 3

[0093] Effects of FPG peptides on the expression of Aquaporin-3 and Filaggrin proteins in HaCaT cells (Human adult skin keratinocytes transformed, human immortalized keratinocytes).

[0094] 1. Experimental Methods: Human immortalized keratinocyte cell line HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO2. Cells were subjected to experimental treatment after reaching the logarithmic growth phase. The experimental groups were as follows:

[0095] 1) Blank control group (Control);

[0096] 2) UV treatment group (UV);

[0097] 3) UV + GPH treatment group (UV+GPH), where GPH is a tripeptide, consisting of glycine-proline-histidine from the N-terminus to the C-terminus;

[0098] 4) UV + FPG treatment group (UV+FPG).

[0099] After UV treatment, cells were cultured for a certain period of time with either 2 mg / mL GPH or FPG. After treatment, cells were lysed using RIPA lysis buffer, incubated on ice, and centrifuged. The supernatant was collected as the total protein sample. An equal volume of protein was separated by SDS-PAGE gel electrophoresis, and the proteins were then transferred to a PVDF membrane. After transfer, the membrane was blocked with 5% skim milk powder at room temperature for 1 h. After incubation overnight at 4 ℃, the membrane was washed with TBST and incubated with the corresponding HRP-labeled secondary antibody. Finally, chemiluminescence staining was used for color development, and band images were acquired using a gel imaging system.

[0100] 2. Experimental results: See Figure 6 and Figure 7 Western blot results showed that, compared with the blank control group, UV treatment significantly reduced the expression level of Filaggrin protein in HaCaT cells, suggesting that UV damage inhibits the expression of key structural proteins in the stratum corneum, thereby weakening the skin barrier and moisturizing ability.

[0101] Adding FPG to UV treatment significantly upregulated Filaggrin protein expression, with levels significantly higher than the UV group, approaching or even returning to normal control levels, and superior to the UV+GPH treatment group. This result indicates that FPG can effectively alleviate the inhibitory effect of UV on Filaggrin expression and help maintain or promote normal Filaggrin expression.

[0102] Aquaporin-3 is an important water and glycerol channel protein in keratinocytes, crucial for skin water transport and hydration. Western blotting results showed that UV treatment significantly decreased Aquaporin-3 protein expression, suggesting that UV damage disrupts the skin's water metabolism balance. Compared with the UV group, the UV+FPG treatment group showed a significantly increased Aquaporin-3 protein expression level, with a better recovery than the UV+GPH group, indicating that FPG can effectively promote or maintain Aquaporin-3 expression.

[0103] Example 4

[0104] In vitro hygroscopicity test

[0105] Accurately weigh 1.000 g of the test sample (0.5 wt% FPG aqueous solution) and the same amount of positive control sample (5 wt% glycerol aqueous solution, ready to use) into an open weighing bottle. Then, quickly transfer the weighing bottle to a desiccator using silica gel as a desiccant. Under experimental conditions of 20–26 °C and 40%–50% RH (relative humidity), remove the weighing bottle and weigh it every hour, immediately returning it to the container after each weighing. Continuously measure, record the data, calculate the moisture retention rate, and finally plot the moisture retention rate-time curve.

[0106] Based on the results of in vitro hygroscopicity tests, the moisturizing efficacy of the sample can be determined by comparing the moisturizing rate-time curves of the FPG aqueous solution with those of the sample and the positive control (5% glycerin). Figure 8 As shown, the moisturizing rate-time curve of the test sample with a concentration of 0.5 wt% FPG aqueous solution was above the positive control curve throughout the entire process, indicating that under the experimental conditions, the test sample showed significantly better hygroscopic and moisturizing ability than the glycerol standard control, confirming its significant moisturizing effect.

[0107] Example 5

[0108] stratum corneum moisture measurement

[0109] 1. Subject and Site Selection

[0110] Ten qualified participants were recruited based on the pre-set screening criteria. The test area was selected on the inner side of each participant's left and right arms, 5 cm from the base of the palm, with a uniform test area of ​​3 cm × 3 cm on each side.

[0111] 2. Samples and Grouping

[0112] Apply equal amounts of the following two samples to the test areas on both arms:

[0113] Sample group: Test face cream containing 0.5wt% FPG active ingredient.

[0114] Matrix control group: A matrix cream without 0.5wt% FPG active ingredient. A self-controlled left-right design was used to eliminate the influence of individual skin differences on the results.

[0115] 3. Testing Process and Environment Control

[0116] The test was conducted in a constant temperature and humidity environment: temperature (21±1)℃, relative humidity (50±5)%. After cleaning their arms, the subjects sat quietly in this environment for 30 minutes to achieve skin stabilization.

[0117] Immediately after the sitting period, the initial moisture content of the stratum corneum of the skin at each test site was measured before application. After evenly applying the corresponding sample, the immediate moisture content of the skin at each site was measured at 1, 2, 3, 4, and 5 hours. Each site and each time point was measured three times, and the arithmetic mean was taken as the final test value. The results were uniformly expressed as stratum corneum moisture content.

[0118] Figure 9 The experimental results show that the face cream containing 0.5wt% FPG active ingredient has significantly better moisturizing effects than the base control group. Within 1 to 5 hours after application, the moisture content of the stratum corneum in the experimental group remained at a higher level, demonstrating both rapid moisturizing and long-lasting moisturizing properties. This effective moisturizing duration of over 5 hours can well meet the daily skincare needs in dry environments, confirming that the 0.5wt% FPG active ingredient is the key functional factor that gives this face cream its long-lasting moisturizing ability.

[0119] Example 6

[0120] Cream preparation and human skin patch experiment

[0121] 1. Prepare samples according to Table 1:

[0122] Table 1

[0123]

[0124] (1) According to the above formula, first dissolve the carbomer, then add xanthan gum until it is completely dissolved;

[0125] (2) Redissolve butylene glycol, propylene glycol, polyglycerol-10, glycerol, β-glucan, allantoin, sodium hyaluronate, glucose and trehalose until all raw materials are completely dissolved, and then add sunflower seed oil; homogenize until all raw materials are completely dissolved.

[0126] (3) Formula 1 includes FPG; Formula 2 does not.

[0127] (4) Triethanolamine was added to each sample, and water was added to each sample to 100g to prepare cream and base cream containing FPG.

[0128] 2. In accordance with the requirements of the "Cosmetic Safety Technical Specifications" (2015 edition), this study used a standard human skin occlusive patch test to assess the skin irritation of face cream containing 0.5wt% FPG.

[0129] (1) Subject information

[0130] Ten healthy volunteers were recruited, five men and five women, aged 19 to 30. None of the selected volunteers had a known history of skin allergies or active skin diseases.

[0131] 2. Test Methods

[0132] A sample of face cream containing 0.5 wt% FPG was evenly filled into a standard patch applicator at a dosage of approximately 0.02 g to 0.025 g and applied to healthy skin on the inner forearm of each volunteer. The sample was kept sealed on the skin surface.

[0133] 3. Observation and Evaluation

[0134] After 0.5 hours, 24 hours, and 48 hours of application, the patch applicator was removed, and a professional observed the skin reaction at the test site 30 minutes after removal. The skin reaction level was strictly determined and recorded according to the scoring criteria in the appendix of the guidelines (e.g., 0 points for no reaction, 1 point for mild erythema, etc.).

[0135] Table 2. Grading Criteria for Skin Reaction in Closed Patch Tests

[0136]

[0137] According to the results of the human skin occlusive patch test conducted in accordance with the "Cosmetic Safety Technical Specifications" (2015 edition) (Table 2), among all 10 healthy subjects (5 males and 5 females), no positive reactions such as skin erythema, edema, or papules were observed after 0.5 hours, 24 hours, and 48 hours of application. The skin reaction scores of all subjects were 0.

[0138] The results clearly demonstrate that, under the conditions of this experiment, the face cream containing 0.5 wt% FPG did not exhibit skin irritation or sensitization, indicating that its formulation is gentle and safe for the skin at the tested concentration, meeting the basic safety requirements for cosmetics on human skin. This provides important safety data support for the further development and use of the product.

[0139] Table 3 Results of human patch experiment

[0140]

[0141] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0142] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A tripeptide or a derivative thereof, characterized in that, The amino acid sequence of the tripeptide from the N-terminus to the C-terminus is Phe-Pro-Gly.

2. The use of the tripeptide or its derivative as described in claim 1 for non-diagnostic therapeutic purposes in any one of (I) to (V): (I) Upregulate the expression of at least one of filaggrin and aquaporin-3; (II) Preparation of a formulation for upregulating the expression of at least one of filaggrin and aquaporin-3; (III) Preparation of formulations for skin moisturizing; (IV) Preparation of cosmetics; (V) Preparation of drugs for use on the skin.

3. The application according to claim 2, characterized in that, Subjects for the formulation used to upregulate the expression of at least one of filaggrin and aquaporin-3 include subjects with UV damage.

4. The application according to claim 2, characterized in that, The upregulation of the expression of at least one of filaggrin and aquaporin-3 includes the in vitro upregulation of the expression of at least one of filaggrin and aquaporin-3 in cells.

5. A moisturizing composition, characterized in that, It contains the tripeptide or its derivative as described in claim 1 and excipients.

6. A moisturizing preparation, cosmetic, or pharmaceutical, characterized in that, It contains the tripeptide or its derivative as described in claim 1; or, it contains the moisturizing composition as described in claim 5.

7. The moisturizing preparation, cosmetic, or pharmaceutical according to claim 6, characterized in that, The concentration of the tripeptide or its derivative in the cosmetic product is 0.1 wt% to 1 wt%.

8. The moisturizing preparation, cosmetic, or pharmaceutical according to claim 6 or 7, characterized in that, The cosmetics include creams, lotions, and liquids.

9. The moisturizing preparation, cosmetic, or pharmaceutical according to claim 8, characterized in that, The cosmetic product is a cream containing the tripeptide or its derivatives at 0.1 wt% to 1 wt%, butylene glycol at 3 wt% to 10 wt%, propylene glycol at 1 wt% to 10 wt%, polyglycerol-10 at 1 wt% to 3 wt%, glycerin at 1 wt% to 10 wt%, β-glucan at 0.1 wt% to 1 wt%, carbomer at 0.1 wt% to 2 wt%, xanthan collagen at 0.1 wt% to 1 wt%, allantoin at 0.01 wt% to 5 wt%, sodium hyaluronate at 0.01 wt% to 5 wt%, glucose at 0.1 wt% to 5 wt%, trehalose at 0.1 wt% to 5 wt%, triethanolamine at 0.1 wt% to 5 wt%, sunflower seed oil at 5 to 10 wt%, and water at 28 wt% to 88.4 wt%.

10. The moisturizing preparation, cosmetic, or pharmaceutical according to claim 6, characterized in that, The drug is a topical preparation for use on the skin.

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