CN118240014A - Polypeptide with anti-wrinkle effect, composition and application thereof - Google Patents

Abstract

The invention discloses a polypeptide with an anti-wrinkle effect, a composition and application thereof, belonging to the field of biology. The polypeptide of some examples of the invention can remove DPPH free radical, repair aging cells, promote the growth of human skin fibroblast HSF, promote the expression of collagen, has good anti-skin aging effect, and can play an important role in medical and aesthetic products and anti-aging treatment.

Description

Polypeptide with anti-wrinkle effect, composition and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a polypeptide with an anti-wrinkle effect, a composition and application thereof.

Background

The human skin gradually ages with age, and simultaneously, due to external factors, mainly ultraviolet irradiation damages DNA, protein and lipid of the skin, so that the level of antioxidants in the skin is reduced, and the skin aging speed is increased, the appearance of aging is mainly represented by darkening complexion, growing spots, rough skin, loosening, wrinkling and the like, and pathological characteristics are represented by thinning epidermis, damaging basal lamina, collagen degradation of dermis, elastosis and telangiectasia (the damage, repair and research progress of mechanism of photoaged basal lamina of the skin, journal of beauty and plastic surgery of China, 2020,31 (08)).

With the development of economic level, there is a growing demand for skin care products, which are expected to effectively solve various problems of skin aging, and thus, related researches are increasingly conducted in order to obtain or find products capable of effectively combating the problems of skin aging. For example, chinese invention CN114805601a discloses a polypeptide analogue which is a fusion peptide of MYMD with an argireline, palmitoyl pentapeptide or SYN-AKE, wherein the argireline residue, palmitoyl pentapeptide residue or SYN-AKE residue is linked to MYMD via an amide bond, and the invention combines MYMD-1 with anti-wrinkle and skin cosmetic peptide SYN-AKE, pentapeptide or ARGIRELINE, and the obtained polypeptide analogue has a bifunctional anti-aging effect.

In addition, many skin care products achieve a combination of effects by adding multiple active ingredients simultaneously in order to achieve the desired effect. For example, chinese invention CN115364017a discloses a collagen active peptide skin care composition, the main ingredients of which include: 5-10% of active peptide, 5-12% of plant extract, 1-3% of nano titanium dioxide, 1-6% of vitamin E, 15-20% of humectant, 8-10% of glycerin and the balance of deionized water. Wherein the active peptide is oligopeptide-1 and tripeptide-1 copper; the plant extract is radix Arnebiae extract and Hamamelis mollis extract. The composition of the invention utilizes active peptide to promote the synthesis of collagen and elastin, utilizes plant extract to improve the repairing effect of skin care products on skin, and simultaneously obtains sun-proof effect and delays photoaging through nano titanium dioxide and vitamin E. Chinese invention CN202211205397.9 also discloses a skin care product for relieving and resisting aging, which can block the connection of nerves to muscles by utilizing acetyl hexapeptide, prevent skin from forming wrinkles, promote muscle growth by utilizing whey protein in milk protein extract, remove free radicals harmful to skin by utilizing the antioxidant activity of caffeine, and increase the relieving property of the skin care product by utilizing the antibacterial property of Cordyceps extract and rosmarinic acid and utilizing amino acid and ursolic acid, thereby ensuring that the skin care product has the effects of resisting aging, inhibiting bacteria and relieving simultaneously. However, due to the diversity of the nature of each substance, interactions between the different components may occur, which may reduce or lose the original activity, resulting in a product with a practical effect that does not meet the expected requirements.

The challenge for those skilled in the art is to develop an anti-wrinkle product that is both effective in penetrating the skin barrier and safe and efficient. To this end, the present invention provides a novel anti-wrinkle solution based on polypeptides, aimed at overcoming the limitations of the prior art. Through accurate biological function assessment, the polypeptides of the invention exhibit significant biological functions in experiments.

Disclosure of Invention

The invention aims to solve the limitations in the prior anti-wrinkle technology, and provides a polypeptide-based composition with remarkable anti-wrinkle effect and application thereof, which are used for preparing skin care products, medical products or anti-wrinkle effect products for preventing and/or treating skin aging, and can be used singly or in combination with other skin care components to enhance the anti-wrinkle and anti-aging effects. The invention fully utilizes the advanced application of the polypeptide in the fields of skin care and anti-aging, and provides a scientific basis for anti-wrinkle skin care products through an accurate synthesis method and biological function evaluation.

The main principles of skin aging to produce wrinkles include reduction of collagen and elastin, loss of skin moisture, and decreased skin cell viability. With age, naturally occurring collagen and elastin of the skin gradually degrade, which results in the skin losing elasticity and firmness, forming fine lines and wrinkles. In addition, external environmental factors (especially ultraviolet radiation) accelerate this process, increasing oxidative stress, further compromising the structure of skin cells and collagen. The development of an anti-aging product with high efficiency and deep effect, which delays skin aging and reduces wrinkle formation, is a problem to be solved by those skilled in the art. Polypeptides are considered to be key components effective against skin aging, mainly because they mimic the natural biological signals of skin cells, promoting the synthesis of collagen and elastin, which are critical to maintaining the structural integrity and elasticity of the skin. In addition, the polypeptide has strong antioxidation property, can neutralize free radical and relieve oxidative damage caused by environmental pressure (such as ultraviolet irradiation).

The present invention addresses this problem by employing Fmoc solid-phase polypeptide synthesis (example 1), precisely synthesizing polypeptides with optimized sequences, ensuring their purity and bioactivity. The polypeptides provided by the invention show remarkable biological functions in experimental study, such as higher clearance rate of DPPH free radical (DPPH. Cndot.) and show that the polypeptides can effectively resist oxidative stress (example 2); the polypeptides provided by the present invention proved to promote the growth of human skin fibroblast HSF, contributing to the repair and renewal of skin cells (example 3); zebra fish model experiments further demonstrate the effect of the polypeptides of the invention in promoting collagen expression, thus combating skin sagging and wrinkle formation (example 4).

In one embodiment, the polypeptide exhibits high antioxidant activity. Free radicals are one of the major factors in skin aging and can destroy DNA, proteins and lipids of skin cells, leading to cell dysfunction and cell death. The antioxidant properties of polypeptides are derived from their chemical structure, allowing them to directly capture and neutralize free radicals, or to enhance the antioxidant defenses of the cell itself, such as increasing the activity of antioxidant enzymes. Thus, these polypeptides are capable of protecting the skin from oxidative damage caused by environmental stresses, such as ultraviolet radiation and contamination. In the long term, this helps to slow down the skin aging process, improve the skin's color and texture, and reduce the formation of stains and wrinkles. The polypeptide is effective in protecting skin from damage by environmental factors such as ultraviolet radiation by neutralizing oxidative stress factors responsible for skin aging. This powerful antioxidant capacity is critical to the resistance to the skin aging process associated with oxidative stress, helping to slow the skin aging rate and improve the overall health and appearance of the skin.

In one embodiment, the polypeptide is capable of substantially promoting the growth of human skin fibroblast HSF. The polypeptide acts as a signaling molecule capable of stimulating cell growth and division by binding to specific receptors on the surface of skin cells, activating intracellular signaling pathways. Such a mechanism may involve increasing the expression of intracellular growth factors or activating cell cycle related proteins. By promoting the natural regeneration and repair process of skin cells, these polypeptides help to maintain the healthy state of the skin and to combat skin damage due to aging or external stress such as environmental pollution and ultraviolet radiation. In addition, the ability to support skin cell turnover means that polypeptides may have a positive effect on reducing fine lines and wrinkles, thereby preserving the youthful and shiny appearance of the skin.

In one embodiment, the polypeptide is effective to increase the relative expression of the col1a1a gene. Collagen is a key component of skin elasticity and firmness, responsible for maintaining skin firmness and smoothness. In the zebra fish model, the polypeptide can increase the expression level of the col1a1a gene, which encodes the alpha 1 (I) chain in collagen. This suggests that the polypeptide may stimulate collagen synthesis and accumulation, potentially helping to reduce skin sagging and wrinkle formation, and restore skin firmness. This feature is particularly valuable in the skin care and medical fields, and provides a non-invasive method to improve skin quality and appearance, particularly in anti-aging applications. Thus, these polypeptides have significant potential in reducing wrinkles and improving the appearance of skin.

In conclusion, the polypeptide of the present invention not only resists skin aging on a cellular and molecular level, but also comprehensively improves the appearance and health of skin by increasing the expression of collagen and enhancing the antioxidant defense mechanism. The comprehensive effect of the polypeptides makes the polypeptides ideal choices for improving and preventing wrinkles, and opens up a new way for developing high-efficiency and deep-acting anti-aging skin care products.

The polypeptide with the anti-wrinkle effect is characterized by comprising an amino acid sequence EEMQRRAD shown in SEQ ID NO. 7, and the amino acid sequence of the polypeptide comprises 15-20 amino acids.

In a preferred embodiment, the amino acid sequence of the polypeptide comprises 16-19 amino acids.

In a preferred embodiment, the amino acid sequence of the polypeptide is increased by 1-20 amino acids at the N-terminus of the EEMQRRAD sequence.

In a preferred embodiment, the amino acid sequence of the polypeptide is increased by 8-11 amino acids at the N-terminus of the EEMQRRAD sequence.

In a preferred embodiment, the amino acid sequence of the polypeptide comprises an amino acid sequence selected from the group consisting of:

RADSRADCEEMQRRAD，SEQ ID NO:8、

YGRKKRRQRRREEMQRRAD，SEQ ID NO:9、

ACSSSPSKHCGEEMQRRAD，SEQ ID NO:10、

ACTGSTQHQCGEEMQRRAD, SEQ ID NO 11, or

TRCFRRCCEEMQRRAD，SEQ ID NO:12。

In a preferred embodiment, the amino acid sequence of the polypeptide comprises an amino acid sequence selected from the group consisting of:

RADSRADCEEMQRRAD，SEQ ID NO:8、

ACSSSPSKHCGEEMQRRAD, SEQ ID NO 10, or

ACTGSTQHQCGEEMQRRAD，SEQ ID NO:11。

In a preferred embodiment, the polypeptide is modified at the N-or C-terminus with auxiliary groups that are carefully selected to enhance the overall performance and function of the polypeptide. Preferably, these auxiliary groups may include acetyl, amino, alkyl, aryl, fatty acid, sugar, phosphate, sulfate, polyethylene glycol (PEG) groups, or peptide linking groups. These modifying groups help to improve the bioavailability, stability, solubility or targeting properties of the polypeptide, thereby enhancing its effectiveness in anti-wrinkle applications.

In a preferred embodiment, the modifying group is an alkyl group to enhance lipid solubility of the polypeptide and promote skin absorption. The introduction of PEG group can raise the water solubility and biocompatibility of polypeptide, and this makes the polypeptide easy to be applied in various water-base cosmetics and skin care products.

In a preferred embodiment, the modifying group is a phosphate or sulfate group to enhance the hydrophilicity and stability of the polypeptide. Such modifications help to preserve the structural integrity of the polypeptide in an aqueous environment, thereby preserving its biological activity.

In a more preferred embodiment, the modifying group is an N-terminal acetyl group (Ac-). The introduction of acetyl can not only effectively protect the C end of the polypeptide from hydrolysis and enzymolysis, but also improve the stability and biological activity of the polypeptide. Acetyl modified polypeptides are more suitable for long-term storage and use, and are critical for commercial production and applications.

In a more preferred embodiment, the C-terminal end of the polypeptide is blocked by amidation, so that the stability of the molecule is further improved, and the molecule is more resistant to enzymolysis in organisms, so that the half-life of the polypeptide in vivo is prolonged, which is important for ensuring that the polypeptide reaches target cells deeper in the skin.

The N-terminal acetyl-modified and C-terminal amidated blocked polypeptides are more suitable for long-term storage and use, which is of critical importance for commercial production and application, especially in anti-wrinkle and skin care products.

In a preferred embodiment, the polypeptide amino acid sequence is selected from any one of SEQ ID NO. 1-SEQ ID NO. 5 or a variant thereof. Wherein, the sequence information of SEQ ID NO:1-SEQ ID NO:6 is as follows in Table 1.

Table 1 amino acid sequence listing and LC-MS identification results of polypeptides 1-6

Note that: the term-NH ₂ in the table indicates that the C-terminus of the polypeptide is amidated closed.

In one embodiment, the polypeptide anti-wrinkle products of the present invention include, but are not limited to, pharmaceutical compositions, food compositions, health care compositions, dietary supplements, and the like. In particular, the polypeptides may be formulated into various pharmaceutical compositions, such as tablets, capsules, powders, granules, solutions, lozenges, jellies, cream formulations, spirits, suspensions, tinctures, cataplasms, liniments, lotions, aerosols, and the like. These pharmaceutical compositions may be prepared using generally known preparation techniques and appropriate pharmaceutical excipients, such as stabilizers, antioxidants and preservatives, may be added to maintain product stability and effectiveness.

In one embodiment, the polypeptide anti-wrinkle product of the present invention is in the form of cream and emulsion, which is suitable for direct application to the skin surface, providing immediate moisturizing and anti-wrinkle effects to the user.

In one embodiment, the polypeptide anti-wrinkle product is in the form of aqueous solution and gel, is lighter and is suitable for oily or mixed skin.

In one embodiment, the polypeptide anti-wrinkle product is in the form of oil and powder, is suitable for dry skin, and provides deep nourishing and long-acting moisture preservation.

In one embodiment, the polypeptide anti-wrinkle product of the present invention is a mud and aerosol type dosage form, providing more options for the user, and being suitable for specific skin care needs and situations.

In one embodiment, the polypeptide anti-wrinkle product of the present invention is in the form of patches and freeze-dried forms, and provides an innovative way of using the polypeptide anti-wrinkle product of the present invention, so that the active ingredient can act on the target area more directly and effectively.

In one embodiment, the polypeptide anti-wrinkle product of the invention is a capsule or tablet type dosage form, provides an oral route, exerts anti-aging and anti-wrinkle effects by oral administration, and improves skin conditions from the body.

In one embodiment, the polypeptide composition of the invention may further comprise a pharmaceutically, food, nutraceutical or dietary acceptable carrier. For example, a variety of compatible solid or liquid fillers, gel materials or solvents may be employed, and these carriers should be suitable for human use, of sufficient purity and low toxicity. Common carriers include cellulose and its derivatives, polyols such as glycerol, and other pharmaceutical excipients such as talc, magnesium stearate, and the like.

In one embodiment, the mode of administration of the polypeptide composition of the present invention is not particularly limited and may include, but is not limited to, oral, parenteral (e.g., intravenous, intramuscular), topical, and the like. In oral formulations, the polypeptide may be admixed with conventional inert excipients or carriers, such as starch, lactose, sucrose, or other fillers; and may be added with binder, humectant, disintegrating agent, slow solvent, wetting agent, adsorbent, lubricant, etc. Solid dosage forms such as tablets, capsules, and the like, may also be prepared with coatings and shells to improve their stability and bioavailability.

In one embodiment, the polypeptide compositions of the present invention are useful in liquid dosage forms for oral administration or administration, including but not limited to emulsions, solutions, suspensions, syrups or tinctures, and the like, wherein various inert diluents, solubilizers, emulsifiers, sweeteners, flavoring agents and flavorants may be included in addition to the active compound. Particularly in suspension, suspending agents may be added to improve product stability and ease of use.

In one embodiment, the polypeptides of the invention may be used in combination with a humectant, such as hyaluronic acid, glycerol, allantoin, natural Moisturizing Factor (NMF), or polyglutamic acid. These ingredients help to increase the moisture content of the skin, and increase the softness and elasticity of the skin, thereby enhancing the anti-wrinkle effect of the polypeptide.

In one embodiment, the polypeptides of the invention may be combined with an antioxidant, such as vitamin E, vitamin C, green tea extract or coenzyme Q10. These antioxidants help neutralize free radicals in the skin, reduce oxidative damage caused by environmental factors, and further enhance the anti-aging efficacy of the polypeptide.

In one embodiment, the polypeptides of the invention may comprise stabilizers, such as vitamin B3 (nicotinamide), vitamin a derivatives or lipoic acid, etc., to maintain stability and activity of the polypeptide and to extend its useful life in the product.

In one embodiment, the polypeptides of the invention may be combined with an emulsifying agent, such as lecithin, polysorbate or stearyl stearate, to stabilize the emulsifying system of the product, ensuring uniform distribution of the ingredients.

In one embodiment, the polypeptides of the present invention may be used in combination with conditioning agents and plant extracts, such as aloe vera extract, green tea extract or chamomile extract, to provide skin relief and other additional skin care benefits to the user.

In one embodiment, the polypeptides of the invention may comprise an ultraviolet absorber or sunscreen agent, such as benzophenone, zinc oxide, or titanium dioxide, to provide sun protection and reduce ultraviolet damage to the skin.

In one embodiment, the polypeptides of the present invention may incorporate permeation enhancers, such as liposomes, nanoparticles, or dimethylsulfoxide, to enhance the skin permeability of the polypeptide, ensuring that the active ingredients act deeper into the skin.

In one embodiment, the polypeptides of the invention may be combined with excipients and fillers, such as silicic acid, microcrystalline cellulose, or starch, etc., to improve the texture and appearance of the product.

In one embodiment, the polypeptides of the present invention may comprise fragrances and flavoring agents to improve the scent of the product and enhance the user's use experience.

The polypeptide anti-wrinkle product provided by the invention provides a multifunctional, efficient and safe anti-wrinkle solution through the combination of carefully designed polypeptide active ingredients and proper auxiliary materials and carriers, is suitable for various dosage forms and application modes, and meets the requirements of different crowds.

Drawings

FIG. 1 shows experimental results of measurement of DPPH clearance of polypeptides.

Fig. 2 shows the results of the effect of the polypeptide on Human Skin Fibroblasts (HSF), compared to a blank, wherein p <0.05, p <0.01, p <0.001, and p <0.0001.

Fig. 3 shows the effect of the polypeptide on the relative expression of the zebra fish col1a1a gene, wherein p <0.05, p <0.01, p <0.001, and p <0.0001 are shown in comparison with the normal control group.

Detailed Description

Definition and description

In order that the application may be more readily understood, certain technical and scientific terms are defined below. Unless defined otherwise herein, all other 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. It is to be understood that this application is not limited to particular methods, reagents, compounds, compositions or biological systems, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "amino acid" refers to a molecule comprising both amino and carboxyl functionalities, the amino and carboxyl groups of an alpha-amino acid being attached to the same carbon atom (alpha carbon). The alpha carbon may additionally have 1-2 organic substituents. Amino acids comprise the L and D isomers and racemic mixtures. Unless otherwise indicated, all amino acid residues in polypeptide sequences in this disclosure are L isomers, i.e., L-amino acids, D-amino acids are indicated by the lower case letter "D" preceding the amino acid name or abbreviation, e.g., dK.

The amino acid composition of the polypeptides of the present disclosure may be altered without substantially affecting the biological activity thereof. For example, a polypeptide sequence may comprise one or more conservative amino acid substitutions. Conservative amino acid substitutions are substitutions of one amino acid residue with another amino acid residue having a similar side chain. Amino acid residues are classified in the literature according to the nature of the side chain of the amino acid residue. Amino acid residues containing basic side chains include lysine, arginine, histidine; amino acid residues comprising an acidic side chain and amide side chains thereof include aspartic acid, glutamic acid, asparagine, glutamine; small aliphatic, nonpolar or weakly polar side chain amino acid residues include glycine, alanine, threonine, serine, proline; the large aliphatic nonpolar side chain amino acid residues comprise leucine, isoleucine and valine; aromatic amino acid residues include phenylalanine, tryptophan, tyrosine; sulfur-containing side chain amino acid residues include cysteine, methionine.

The term "ancillary group" refers to a moiety that alters the characteristics of a multivalent presenter and/or a multivalent presenter component (e.g., a framework moiety, a functional group moiety, a spacer group, etc.). Properties that may be altered include, for example, solubility (in water, fat, lipids, biological fluids, etc.), hydrophobicity, hydrophilicity, flexibility of the framework, antigenicity, molecular size, molecular weight, in vivo half-life, in vivo distribution, biocompatibility, immunogenicity, stability, strength of binding to multivalent targets, and the like.

Those skilled in the art will appreciate that many, if not all, of these properties are substantially overlapping with each other and that the ancillary groups will affect the change in these properties. For example, it is expected that the introduction of one or more poly (ethylene glycol) (PEG) groups onto the framework of a multivalent presenter will increase hydrophilicity and water solubility, increase molecular weight and molecular size, and, depending on the nature of the non-pegylated (unPEGylated) framework, increase in vivo retention time. In addition, PEG is expected to reduce antigenicity and increase the overall rigidity of the polymeric presenter through hydrogen binding to solvent molecules (e.g., water). Similar overlap regions between the nature of the framework and the ancillary groups that can affect these characteristics will be apparent to those skilled in the art.

Auxiliary groups capable of enhancing the water solubility and/or hydrophilicity of the polyvalent presenters are useful in the practice of the invention. Thus, it is within the scope of the invention to use auxiliary groups to increase the water solubility and/or hydrophilicity of the multivalent presenters, including, for example, poly (ethylene glycol), alcohols, polyols (e.g., glycerol, propoxylated glycerol, sugars, including monosaccharides, oligosaccharides, and polysaccharides, etc.), carboxylates, polycarboxylates (e.g., polyglutamic acid, polyacrylic acid, etc.), amines, polyamines (e.g., polyglycine, poly (aziridine), etc.

Also within the scope of the invention is the use of auxiliary groups that enable the incorporation of the multi-valent presenters into vesicles, such as liposomes or micelles. The term "lipid" refers to all fatty acid derivatives capable of forming a bilayer, which have a hydrophobic portion facing the bilayer while a hydrophilic portion of the lipid material faces the aqueous phase. The hydrophilicity is derived from the presence of phosphate, carboxylate, sulfate, amino, mercapto, nitro, and the like groups. The presenters after inclusion of long chain saturated or unsaturated aliphatic hydrocarbon groups, for example, but not limited to, acquire hydrophobicity, and these groups may be substituted with one or more aromatic, cycloaliphatic or heterocyclic groups. Preferred lipids are phosphoglycerides and (neuro) sphingolipids, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearoyl phosphatidylcholine or dioleoyl phosphatidylcholine. Other phosphorus-free compounds, such as sphingolipids and glycosphingolipids, also belong to the lipid group. Alternatively, the amphipathic lipids may be mixed with other lipids, including triglycerides and sterols, for use.

It is also within the scope of the invention to alter the antigenicity of the polyvalent presenter by judicious selection of the auxiliary groups. In some applications it may be desirable to reduce the antigenicity of the multivalent presenter. As mentioned above, masking groups, such as poly (ethylene glycol), are groups known in the art that are capable of reducing the antigenicity of monovalent and multivalent molecules. In other applications (people) it may be desirable to increase the antigenicity of the polyvalent presenter, thus eliciting an immune response. Auxiliary groups in these applications may include groups known in the art to enhance hapten immunogenicity. Groups suitable for enhancing immunogenicity of the multivalent presenters include, but are not limited to, proteins such as Keyhole Limpet Hemocyanin (KLH) and Bovine Serum Albumin (BSA). Other groups capable of increasing the antigenicity of the polyvalent presenter will be known to those skilled in the art.

The term "composition" or "formulation" means a mixture comprising one or more polypeptides of the application with other components, such as physiologically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to organisms, facilitate the absorption of active ingredients and thus exert biological activity.

The term "aqueous formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "lyophilized powder for injection" also refers to sterile powder for injection prepared by lyophilization.

As used herein, "about" when referring to a measurable value (e.g., amount, duration, etc.) is intended to encompass variations of + -20% or + -10% relative to the particular value, including + -5%, + -1% and + -0.1%, as these variations are suitable for carrying out the disclosed methods.

The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or salts thereof (e.g., arginine hydrochloride), glycine, alanine (α -alanine, β -alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ -aminobutyric acid (GABA), opioids (opines), alanines, octopine, glycine (strombine)) and the N-oxides of Trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α -casein, globulin, α -lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, and the like, may also act to control osmotic pressure. The stabilizer used in the present invention is one or more selected from the group consisting of polyhydric alcohols, amino acids, salts and saccharides. The preferred salts are sodium chloride, the preferred sugars are sucrose and trehalose, and the preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or salts thereof (e.g., arginine hydrochloride), glycine, proline. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.

The term "surfactant" generally includes agents that protect proteins such as antibodies from air/solution interface induced stress, solution/surface induced stress to reduce aggregation of the antibodies or minimize the formation of particulates in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycols, polyoxyethylene-stearates, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamer), sodium Dodecyl Sulfate (SDS).

The preparations to be used on the human or animal body must be sterile. This can be easily achieved by sterile filtration through sterile filtration membranes.

Examples

Materials and methods

HaCaT cells to which the present invention relates are provided by the company Wuhanprinus (product number: CL 0090) and HSF cells are provided by the company Guangzhou Ji Ni European Biotechnology Co., ltd (product number: JNO-H0599). The media used included DMEM medium supplied by Gibco, fetal bovine serum and trypsin to ensure optimal conditions for cell culture. CCK8 reagent is provided by biosharp (product number: BS 350D) for use in cell proliferation and cytotoxicity assays. N-acetyl-cysteine was supplied by Sigma (product number: A9165) for cytoprotection and experimental control. 2, 2-biphenyl-1-picrylhydrazyl was provided by Tao Shu organisms for antioxidant experiments.

In terms of chemical and biological experiments, unless otherwise specified, all reagents used in the present invention were analytically pure and solvents were supplied by Shanghai Taitan. To achieve efficient purification of the polypeptide, a reverse phase C18 preparation column (46 mm. Times.250 mm) with a size of 5.0cm was used. High Performance Liquid Chromatography (HPLC) analysis was performed using the instrument of the samer company, while mass spectrometry analysis was performed using a Waters mass spectrometer.

Laboratory equipment aspects, including ultra clean Bench (BAKER), carbon dioxide incubator (HF 240), centrifuge (eppendorf), inverted microscope (Nikon), microplate reader (TECAN). The choice of these equipment and materials is intended to ensure the accuracy and reliability of the experiments, providing a solid experimental support for the present invention.

Example 1: synthesis of polypeptide 1

In this example, fmoc solid-phase polypeptide synthesis was used to synthesize the specific sequence of the polypeptide "Arg-Ala-Asp-Ser-Arg-Ala-Asp-Cys-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH ₂" (abbreviated RADSRADCEEMQRRAD-NH ₂). Fmoc-Asp (OtBu) -OH is taken as an initial group, and is synthesized from a carboxyl end to an amino end, and the amino acid sequences are sequentially connected.

Step 1: synthesis of Main peptide resin

First, 1.47 g WANG RESIN (loading 0.47 mmol/g) was added to a reaction column containing N, N-Dimethylformamide (DMF) to swell for 30 minutes. Then, starting from the C-terminus, fmoc-Asp (OtBu) -OH was used as the starting group, t-butoxycarbonyl (OtBu) to prevent the side chain from participating in the coupling reaction. DIC was added to the reaction mixture for 2 hours by using a coupling agent and an activator (HOBT, DMAP) to give Fmoc-Asp (OtBu) -WANG RESIN resin. Subsequently, fmoc-protected forms of the remaining amino acids of polypeptide 1, including Fmoc-Arg (Pbf) -OH, fmoc-Ala-OH, fmoc-Ser (tBu) -OH, etc., are added sequentially. Before each new amino acid addition, 20% piperidine/DMF solution was used to remove Fmoc protecting group from the previous amino acid and the coupling effect was monitored by ninhydrin to ensure successful reaction.

The amidation and closure of the C end can be realized through the selection of the amino acid of the terminal raw material, so that the stability of the C end is ensured, and the nonspecific hydrolysis of the C end is prevented; the acetyl group can be introduced to modify the N end by reacting with an acetylating reagent (such as acetic anhydride), so as to prevent nonspecific hydrolysis or oxidation and improve the stability of the polypeptide; alkyl can be introduced through the reaction of alkyl halide and amino at the N end of amino acid, so that the fat solubility of the polypeptide is increased, and the absorption of skin is facilitated; polyethylene glycol (PEG), phosphate group, sulfate group and other groups are introduced during or after synthesis, so that the water solubility of the polypeptide and the solubility in blood are improved.

Step 2: full protection against polypeptide removal

After the synthesis of the main peptide chain was completed, the polypeptide resin was treated with a lysate (TFA, DODT, m-cresol, H ₂ O in a volume ratio of 92.5:2.5:2.5:2.5) and frozen in a refrigerator for 2H to remove all protecting groups. The lysate was added to the dried polypeptide resin at 10mL lysate/g polypeptide resin, and the cleavage reaction was performed at room temperature for 3 hours. After completion of the cleavage, the filtrate was slowly poured into ice methyl tert-butyl ether to form a precipitate. The supernatant is removed, centrifuged and washed to obtain the crude compound.

Step 3: refining and purifying crude compounds

The crude compound after cleavage was dissolved in a mixed solution of acetonitrile and water and then purified using a reverse phase C18 preparative HPLC column. The column was eluted at a flow rate of 10mL/min for 60 minutes using 45% acetonitrile in water containing trifluoroacetic acid as eluent at a rate of 0.33%/min to obtain a sample with an HPLC purity of greater than 90%. Repeating HPLC purification once, using 31% acetonitrile/20 mM sodium dihydrogen phosphate aqueous solution to adjust pH to 6.5 with 1M sodium hydroxide solution as the start, gradually increasing acetonitrile proportion at 0.33%/min, eluting the column at 10mL/min for 60 min, collecting polypeptide-containing component, and freeze-drying to obtain high-purity polypeptide product.

Step 4: product validation

And finally, adopting a liquid chromatography-mass spectrometry technology to confirm the structure and purity of the polypeptide. Elution analysis was performed using 5% ACN/H2O containing 0.1% formic acid as the starting mobile phase, increasing the ACN ratio at a rate of 6%/min and a flow rate of 0.4 mL/min. This procedure enables the theoretical molecular weight of the polypeptide and the MS discrimination value (m+2h) ⁴⁺ to be determined to verify its structure and purity. Polypeptide 1 has a theoretical molecular weight of 1906.85 and an MS discrimination value (m+2h) ⁴⁺ of 477.7, confirming it as the target compound.

The polypeptides 2-6 of Table 1 can be synthesized from the corresponding starting materials by the synthesis method of reference example 1. The synthesis method ensures that the amino acid sequence of the polypeptide is accurately synthesized according to a predetermined sequence, and polypeptide products with high purity and correct structure can be obtained through subsequent steps of cleavage, purification and analysis. This method is suitable for laboratory scale polypeptide synthesis and can be extended to larger synthesis scales as desired.

EXAMPLE 2DPPH clearance measurement

In this example, the DPPH clearance assay used is a classical method widely used to evaluate antioxidant activity. DPPH (2, 2-diphenyl-1-picrylhydrazyl) is a stable free radical compound, the most remarkable feature of which is a strong absorption peak at 517nm, which is attenuated after capturing free radicals. Therefore, by measuring the change in absorbance at a specific wavelength, the DPPH-scavenging ability of an antioxidant can be quantitatively evaluated to reflect its antioxidant activity.

The grouping method comprises the following steps:

Sample group: PBS solutions (10 mg/mL) of polypeptides 1-6 were mixed with equal volumes of 1mM DPPH absolute ethanol solution, respectively.

Control group: the PBS solution was mixed with 1mM DPPH absolute ethanol solution in equal volume.

Blank group: equal amounts of polypeptide and PBS were mixed with equal volumes of absolute ethanol solution, respectively.

The experimental method comprises the following steps:

the polypeptides 1-6 were dissolved in PBS to prepare 10mg/mL solutions, respectively. Next, a DPPH absolute ethanol solution of 1mM concentration was prepared. 50. Mu.L of polypeptide PBS solution and 50. Mu.L of DPPH absolute ethanol solution are mixed in a 96-well plate, and then the mixture is shaken and allowed to stand at room temperature for reaction for 30 minutes. The absorbance value A of the sample group was measured at a wavelength of 517 nm. The blank group replaced DPPH solution with equal volume of absolute ethanol solution, and the control group replaced polypeptide solution with equal volume of PBS.

Data processing and result analysis:

And (3) data processing: the clearance of DPPH from the panel polypeptides was calculated using the following formula.

DPPH clearance = [1- (Ai-Aj)/Ao ]. Times.100%

Wherein Ao is absorbance of a control group; ai is the absorbance of the sample group; aj is the absorbance of the blank group.

The DPPH clearance for the sample and blank groups was plotted using GRAPHPAD PRISM software (fig. 1). In the figure, the DPPH clearance of the blank control was = [1- (Ao-Aj)/Ao ] ×100%.

Analysis of results: according to the data of FIG. 1, the results show that DPPH clearance of polypeptide 1 and polypeptide 4 exceeds 80% at a concentration of 5mg/mL, indicating that they have a strong antioxidant capacity. Antioxidants can prevent or slow down oxidative stress caused by free radicals by neutralizing the free radicals, thereby protecting skin tissues from damage and delaying the aging process. Thus, polypeptides 1 and 4 may have a significant effect in preventing skin aging caused by ultraviolet light, environmental pollutants or other oxidative stress. In contrast, the clearance of polypeptides 2, 5 and 6, although slightly lower, was still more than 60%, showing good antioxidant effect. These polypeptides may still function as antioxidants in skin care products, although they may not be as potent as polypeptides 1 and 4. However, the DPPH clearance of polypeptide 3 is relatively low, probably because its structural features do not react as efficiently with DPPH as other polypeptides.

The embodiment not only proves the antioxidant capacity of the polypeptide, but also provides scientific basis for the application of the polypeptide in the anti-aging field. These polypeptides, while protecting skin cells from oxidative stress against free radical damage, also help maintain the physiological function and appearance of skin, and in particular show potential advantages in delaying skin aging and reducing wrinkle formation. Thus, these polypeptides are powerful candidates for the development of novel anti-aging skin care products.

EXAMPLE 3 growth promoting effect of Polypeptides 1-6 on Human Skin Fibroblasts (HSF)

Fibroblasts play a central role in the structure and function of the skin, they are not only the main producers of collagen and other matrix proteins, but also play a key role in wound healing and skin regeneration processes. By promoting proliferation of fibroblasts, the repair ability of the skin can be effectively enhanced, thereby counteracting the aging process of the skin. This example is directed to the study of the growth promoting effect of polypeptides 1-6 on human skin fibroblast HSF at a concentration of 0.2 mM. The effect of polypeptides on HSF cell proliferation was assessed by comparing cell viability in the different polypeptide treated groups and the control group using CCK8 assay.

The grouping method comprises the following steps:

sample group: HSF cells were seeded and contained 0.2mg/mL of polypeptide 1-6 in DMEM.

Culture medium group: only DMEM medium was contained, no cells were inoculated, and no polypeptide was added.

Blank control group: no polypeptide was added, other conditions were the same as in the sample group.

The experimental method comprises the following steps:

Inoculating HSF cells into a 96-well plate at a density of 10000 cells/well, incubating 100 mu L of each well in a 5% CO ₂ constant-temperature incubator at 37 ℃ for 24 hours, removing the solution in the well, adding 0.2mg/mL polypeptide solution prepared by using DMEM as a solvent, and taking 100 mu L of each well as a sample group; a medium group containing only the medium and a blank group containing no polypeptide and only the cells were simultaneously set, and 5 parallel wells were set in each group. After 48h incubation in the cell incubator, the original medium was discarded, washed 2 times with PBS, and incubated for 4h in the dark with 10% CCK8 in DMEM medium. The absorbance was measured at 450nm in a microplate reader, and the cell viability value (cell viability) was calculated.

Data processing and result analysis:

And (3) data processing: sample group HSF cell viability was calculated using the following formula.

Cell viability (%) = (a ₁-A)/(A₀ -a) ×100%,

Wherein A ₁ is the absorbance of the sample group; a is the absorbance of the culture medium group; a ₀ is the absorbance of the blank.

Cell viability histograms of the sample and control groups were plotted using GRAPHPAD PRISM software (fig. 2).

Analysis of results: from the data in fig. 2, analysis of the results showed that polypeptide 1 significantly increased the viability of HSF cells at a concentration of 0.2mg/mL, which had a very significant effect on promoting cell viability and was statistically significant (p < 0.01). This may be due to the ability of polypeptide 1 to activate intracellular growth signaling pathways, or to be directly involved in the regulation of the cell cycle, thereby promoting cell proliferation. Polypeptides 2, 3 and 4 also showed an effect of enhancing HSF cell viability, which was significantly improved (p < 0.05) over the blank, although less effective than polypeptide 1. This suggests that these polypeptides may also promote the growth of HSF cells by similar or other mechanisms, such as increasing the uptake of trophic factors by cells, increasing antioxidant defenses, and the like. However, the results for polypeptide 6 showed no significant differences, meaning that their effect in promoting cell viability was not significant. This may be due to differences in structural features or amino acid sequences from other polypeptides, thereby affecting their interaction with specific targets within the cell.

These experimental results show that polypeptides 1,2,3 and 4 have the potential ability to promote proliferation of skin fibroblasts and maintain skin health, significantly promote growth of HSF cells, with cell viability exceeding 100% of the blank, while the effects of polypeptides 5, 6 are not significant. In particular, polypeptide 1 showed the strongest effect. This provides strong evidence for the development of new anti-aging skin care ingredients and may play an important role in medical and cosmetic products and anti-aging treatments.

Example 4 evaluation of Effect of Polypeptides 1-6 on the expression of the zebra fish col1a1a Gene

Skin growth, repair, nutrition, elasticity, and tension are all related to collagen, and its loss can reduce skin smoothness and generate wrinkles. In quadruped, type I collagen is a trimer, consisting mainly of two α1 chains and one α2 chain, encoded by the col1a1a and col1a2 genes, respectively, performing collagen-related biological functions in connective tissue and bone. Three type I collagen genes exist in zebra fish, col1a1a, col1a1b and col1a2, encoding the α1 (I), α2 (I) and α3 (I) chains, respectively. Therefore, by detecting the relative expression amounts of the col1a1a or (and) col1a1b or (and) col1a2 genes, it can be shown whether the sample has anti-wrinkle efficacy. The present study evaluates the anti-wrinkle efficacy of samples by detecting the relative expression level of the col1a1a gene.

Test system:

the animal model was a wild type AB strain zebra fish, 4 days after fertilization (4 dpf).

The grouping method comprises the following steps:

Sample group: administering 0.3mM polypeptide treatment, and culturing for 24 hr;

normal control group: no polypeptide treatment was given, other culture conditions were the same as for the sample group;

Each group had 30 zebra fish (three biological replicates, n=3).

The experimental method comprises the following steps:

Zebra fish of 4dpf were randomly selected in 6-well plates with 30 tails per well. The final concentration of the water-soluble administered samples was 0.3mM, while the normal control group was set to a capacity of 3mL per well (three biological replicates, n=3). Incubate at 28℃for 24h in the absence of light. Extracting total RNA of zebra fish of each experimental group, synthesizing cDNA, and detecting gene expression of beta-actin and target genes by q-PCR. The relative expression quantity of RNA of the target gene is calculated by using beta-actin as an internal reference of gene expression.

Data processing and result analysis:

and (3) data processing: the relative expression amount of RNA of the target gene is calculated by adopting the following formula:

RNA relative expression = 2 ΔΔc (t)

ΔΔC(t)＝ΔC(t) _{Normal control group} -ΔC(t) _{Sample group}

ΔC(t)＝C(t) _{Target gene} -C(t)_β-actin

In the method, in the process of the invention, ΔΔΔC (t) is one the number of values to be normalized is one, reflecting the change in the relative expression level of a specific gene. Δc (t) represents the difference between the expression levels of the target gene and the reference gene, and is used to normalize the expression level of the target gene so as to reduce the error caused by experimental operation variables. Δc (t) _{Sample group} is the difference in C (t) values (threshold cycle) of the target gene and the reference gene for the experimental sample set samples; Δc (t) _{Normal control group} is the difference in C (t) values of the target gene and the reference gene of the control group sample; c (t) _{Target gene} is the number of cycles required for the target gene to reach the detection threshold in the experiment. C (t) _β-actin is the number of cycles required for the reference gene (β -actin in this example) to reach the detection threshold.

RNA relative expression levels of the target genes of the sample group and the normal control group were plotted using GRAPHPAD PRISM software (FIG. 3). Wherein ΔΔc (t) =Δc (t) _{Normal control group} -ΔC(t) _{Normal control group} =0 and rna relative expression amount=1 in the normal control group.

Experimental results: the relative expression levels of the zebra fish col1a1a genes shown in FIG. 3. From the experimental results, it was found that the polypeptides 1, 4 significantly increased the expression level of the col1a1a gene (p < 0.0001) in zebra fish at a polypeptide test concentration of 0.3mM, which is directly related to skin elasticity and collagen synthesis to prevent wrinkle formation. Among these, polypeptides 1 and 4 exhibited particularly strong effects in these experiments, and this extremely remarkable statistical result not only suggests that polypeptides 1 and 4 have a strong promotion of col1a1a gene expression, but also suggests that they may provide a remarkable anti-wrinkle effect in clinical applications.

In addition, polypeptides 2 and 3 also showed the ability to increase the expression level of the col1a1a gene, and their effect was statistically significant, indicating that they also have a positive effect on wrinkle resistance, although their efficacy was not as strong as that of polypeptides 1 and 4. However, at the same test concentrations for polypeptides 5 and 6, they did not reach statistical significance in increasing col1a1a gene expression, which may mean that their anti-wrinkle effect is not sufficiently pronounced under the current experimental conditions, or that higher concentrations or longer times are required to exhibit their potential effects.

In conclusion, the experimental results reveal that the polypeptides 1 and 4 have strong potential in improving the expression level of collagen, and have important significance in developing novel anti-wrinkle products. These polypeptides are expected to become potent anti-aging components of the skin by promoting the expression of the col1a1a gene, providing a new strategy for keeping the skin young.

The above description of the present application is further illustrated in detail and should not be taken as limiting the practice of the present application. All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. It should be understood that those skilled in the art to which the present application pertains can easily derive or replace the present application without departing from the spirit of the present application.

Claims (12)

1. A polypeptide with anti-wrinkle effect is characterized in that the polypeptide comprises an amino acid sequence EEMQRRAD shown in SEQ ID NO. 7, and the amino acid sequence of the polypeptide comprises 12-25 amino acids, preferably 16-19 amino acids.

2. Polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide is increased by 1-20 amino acids, preferably by 8-11 amino acids, at the N-terminal end of the EEMQRRAD sequence;

Preferably, the polypeptide is selected from the following amino acid sequences or variants thereof:

RADSRADCEEMQRRAD，SEQ ID NO:8、

YGRKKRRQRRREEMQRRAD，SEQ ID NO:9、

ACSSSPSKHCGEEMQRRAD，SEQ ID NO:10、

ACTGSTQHQCGEEMQRRAD, SEQ ID NO 11, or

TRCFRRCCEEMQRRAD，SEQ ID NO:12；

Preferably, the amino acid sequence of the polypeptide comprises an amino acid sequence selected from the group consisting of: RADSRADCEEMQRRAD, SEQ ID NO 8,

ACSSSPSKHCGEEMQRRAD, SEQ ID NO 10, or

ACTGSTQHQCGEEMQRRAD，SEQ ID NO:11。

3. The polypeptide of claim 1 or 2, wherein the N-terminus or C-terminus of the amino acid sequence of the polypeptide comprises a modification;

Preferably, the modification is capable of increasing the lipid solubility of the polypeptide, promoting absorption of the polypeptide by the skin, increasing the water solubility of the polypeptide, increasing the biocompatibility of the polypeptide, increasing the hydrophilicity of the polypeptide, increasing the stability of the polypeptide, increasing the bioactivity of the polypeptide, and/or protecting the polypeptide from hydrolysis and/or enzymolysis;

Preferably, the modification is a modification with an auxiliary group selected from one or more of acetyl, amino, alkyl, aryl, fatty acid, sugar, phosphate, sulfate, polyethylene glycol (PEG) groups, or peptide linking groups;

Preferably, the modification is selected from: c-terminal amidation blocking and/or N-terminal modification of acetyl groups.

4. A polypeptide according to any one of claims 1 to 3, wherein the polypeptide comprises any one of the amino acid sequences selected from SEQ ID NOs 1 to 5 or variants thereof.

5. The polypeptide of any one of claims 1-4, wherein the polypeptide is effective to combat and/or repair skin loss and/or aging;

Preferably, the effects of the polypeptide include, but are not limited to: neutralizing oxidative stress factors responsible for skin aging, promoting the growth of human skin fibroblasts, or promoting the gene expression of collagen;

preferably, the oxidative stress factors that neutralize and cause skin aging are neutralizing free radicals;

Preferably, the radicals are DPPH radicals;

preferably, the human skin fibroblasts are Human Skin Fibroblasts (HSF);

Preferably, the collagen gene is a type I collagen gene;

Preferably, the type I collagen gene is one or more of col1a1a, col1a1b, col1a 2;

preferably, the type I collagen gene is col1a1a.

6. A nucleic acid molecule encoding the polypeptide according to any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A cell comprising the vector of claim 7.

9. A composition comprising an adjuvant and an active ingredient, wherein the active ingredient comprises a polypeptide according to any one of claims 1 to 5, or a nucleic acid molecule according to claim 6, or a vector according to claim 7, or a cell according to claim 8;

Preferably, the auxiliary materials include diluents, fillers, binders, wetting agents, absorption enhancers, surfactants, lubricants and stabilizers.

10. The composition of claim 9, wherein the composition is in a formulation suitable for administration orally, by painting or subcutaneously.

11. The composition of claim 9 or 10, wherein the formulation of the composition comprises one or more of a cream, emulsion, aqueous, gel, oil, powder, mud, aerosol, patch, freeze-dried, capsule, or tablet.

12. Use of the polypeptide of any one of claims 1-5, or the nucleic acid molecule of claim 6, or the vector of claim 7, or the cell of claim 8, or the composition of claim 9 or 10, for the preparation of a product having anti-wrinkle efficacy; preferably, the product with anti-wrinkle effect is one or more of a pharmaceutical composition, a personal care product, a cosmetic product, and a medical product.