CN117510617A

CN117510617A - Active peptide for improving skin activity and application thereof in medicines or cosmetics

Info

Publication number: CN117510617A
Application number: CN202311405067.9A
Authority: CN
Inventors: 陈磊; 王振
Original assignee: Beijing Tailiang Biotechnology Co ltd
Current assignee: Beijing Tailiang Biotechnology Co ltd
Priority date: 2023-10-27
Filing date: 2023-10-27
Publication date: 2024-02-06

Abstract

The invention relates to an active peptide for improving skin activity and application thereof in medicines or cosmetics. According to the invention, the specific polypeptide with antioxidant and anti-wrinkle properties is obtained from the specific pelteobagrus fulvidraco by means of enzymolysis, column separation, mass spectrometry and other methods, has strong antioxidant properties, can repair the influence of ultraviolet irradiation and oxidative stress on cell damage, can be prepared into medicines and cosmetics, and has excellent application prospects.

Description

Active peptide for improving skin activity and application thereof in medicines or cosmetics

Technical Field

The present application relates to the field of biology, and more particularly, to an active peptide for improving skin activity and its use in medicine or cosmetics.

Background

Skin is the tissue that constitutes the outermost layer of the human body and is composed of epidermis, dermis and subcutaneous tissue. The epidermis is the outermost layer of the skin, and is mainly composed of keratinocytes, melanocytes, langerhans cells, and mercer cells. Keratinocytes are the most abundant cells found in the epidermis and act as a barrier against the external environment [5]. The dermis, which includes three structures of elastin, collagen and glycosaminoglycan, is the extracellular matrix that maintains the skin structure (Extracellular matrix). Skin aging manifests itself as telangiectasia, pigmentation, sallowness, dryness, sagging and deep wrinkles. The intrinsic aging and the extrinsic aging can be classified according to the mechanism of action. Intrinsic aging is a latent degradation process of organisms over time, associated with genetic susceptibility and physiological components, and is a necessary consequence of physiological changes in humans. Extrinsic aging is the result of damage in exogenous environments, such as ultraviolet radiation (UV), environmental pollution, chemicals, and the like. Among several external stimuli, the most serious skin aging is ultraviolet radiation, which is classified into long-wave ultraviolet UVA (320 to 400 nm), medium-wave ultraviolet UVB (280 to 320 nm) and short-wave ultraviolet UVC (200 to 280 nm) according to the wavelength of photons. UVB can penetrate the epidermis, leading directly to DNA mutations. UVA can penetrate deep into the dermis, inducing the production of Matrix Metalloproteinases (MMPs) that break down collagen and elastin fibers, leading to skin aging and wrinkling. Exposure of the skin to uv radiation causes various physiological effects such as sunburn, immunosuppression, photoaging, skin pigmentation, mutations, DNA and protein denaturation, cancer, and the production of Reactive Oxygen Species (ROS).

Wrinkles are a typical symptom of skin aging, caused by loss of elasticity, which is associated with decreased collagen and elastin associated with the elasticity of dermal tissue of the skin. Collagen accounts for about 90% of the extracellular matrix (ECM) of human dermis, type I collagen is the most abundant structural protein in skin and connective tissue, accounting for 80% of total collagen, and its structural changes are believed to be the major cause of skin aging and wrinkle formation. Reactive Oxygen Species (ROS) and Matrix Metalloproteinases (MMPs) are upregulated and nuclear factor- κB (NF- κB) is also activated in skin exposed to acute ultraviolet radiation. Matrix metalloproteinases are zinc-dependent endopeptidases, the family of MMP enzymes responsible for the degradation of connective tissue and transcription of a variety of MMPs, mainly regulated by nuclear factor- κB (NF- κB) and activin 1 (AP-1), which specifically up-regulate MMP-1 (interstitial collagenase), MMP-3 (matrix degrading enzyme-1) and MMP-9 (gelatinase B), directly involved in collagen degradation. ROS can react with DNA, proteins, fatty acids, and carbohydrates, causing oxidative damage. ROS are essential participants in a variety of MAPK (mitogen activated protein kinase) pathways, including the three ERK, p38 and JNK pathways, responsible for activation of the activator protein (AP-1) and nuclear factor- κb (NF- κb), and thereby up-regulate MMP expression. JNK is mainly activated by ROS and is involved in the production of AP-1 transcription factors. In addition, MAPKs phosphorylate NF-. Kappa. B p65 (Ser 276) by phosphorylation of ERK and p38, ultimately inducing MMP production, skin aging and wrinkling. In addition, ultraviolet radiation also down-regulates transforming growth factor beta (TGF-beta), a multifunctional cytokine that induces the downstream protein Smad 2/3 to form heterodimeric complexes with Smad 4 and transfer into the nucleus, activating procollagen promoters, thereby inducing collagen and elastin production and blocking MMP-1 and MMP-3 production.

Functional active peptides are the research hot spot of functional foods in recent years, and particularly collagen has a great deal of application and research report in aspects of beauty, including oral administration and external use, and generally, the active peptides are required to have a specific function in the target organ by passing through the gastrointestinal tract, entering the blood circulation and then entering the target organ in the form of active factors. Skin, the largest organ of the human body, provides another route for some active functional factors to enter the body, and there are also some studies reported on the absorption of active peptides through the skin. For example, mary et al review that a number of active peptides can be absorbed through the skin and thus exert anti-skin aging effects. Through animal experiments, hairless mice are orally taken from fish scale collagen hydrolysate for 6 weeks, and the effect of the collagen hydrolysate on ultraviolet rays on skin is studied, so that the model group is relatively irradiated by ultraviolet rays, the thickness of the collagen hydrolysate and an ultraviolet irradiation sample group is close to that of a normal group, and the model group is remarkably higher than that of the sample group and the normal group. The skin collagen content was significantly higher in both the normal and sample groups than in the model group. The results show that the collagen hydrolysate can enhance the capability of skin to resist ultraviolet injury. Gelatin was extracted from Pacific cod and then enzymatically hydrolyzed to produce peptide samples of 2000-6000 u and <2000 molecular weight. The animal experiment shows that the collagen peptide can raise the activity of antioxidant enzyme system of male ICR mouse skin tissue, such as superoxide dismutase, glutathione peroxidase, catalase, etc. and raise the content of skin reduced glutathione and hydroxyproline and lower the content of skin malondialdehyde, so as to raise the antioxidant capacity of skin and reduce photodamage of skin. The experimental results also show that both molecular weight samples protect collagen fibers in the skin. In the current research, the common antioxidant peptide has the function of resisting wrinkle.

The natural product has the advantages of nature, mildness, no stimulation, high safety and the like, and along with the continuous development and optimization of the extraction technology and the biotechnology, the stability of the active ingredients of the natural plants is continuously improved, and the plant raw materials are used in the functional cosmetics for resisting wrinkles, whitening and the like. The population of China is numerous, the plant resources are rich, and the anti-aging cosmetic market and research and development potential are huge. The development of environment-friendly, healthy and safe natural component cosmetics is a remarkable trend, and the natural return is gradually a trend. Therefore, the preparation optimization of peptide, the separation and purification of peptide, the structure identification, the development of the mechanism combining skin aging, the screening of the efficient anti-skin aging active peptide functional factors and the utilization of the screened target peptide segment, and the further improvement of the effect of polypeptide by the related technical means are important research directions in the future.

Disclosure of Invention

The present invention is based on the drawbacks of the prior art and provides a polypeptide for improving skin activity.

The polypeptide has the activity polypeptide of resisting oxidation and wrinkle and increasing skin moisturizing property, and concretely, the polypeptide is Y2-2-T3, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

Furthermore, the polypeptides may be conservatively substituted or modified while still retaining the activity of the corresponding polypeptide.

In particular, "conservatively modified variants" refers to amino acid sequences that contain conservative substitutions. Exemplary conservatively modified variants include substitutions, deletions, or insertions of the polypeptide sequence which alter, add, or delete a single amino acid or a small percentage of amino acids in the polypeptide sequence or encoded polypeptide sequence, e.g., up to 1, 2, 3, 4, or 5 amino acids, or up to 0.5%, 1%, 1.5%, 2%, 2.5%, or 3.5% of the amino acids in the polypeptide sequence or encoded polypeptide sequence, optionally amino acid substitutions which may be or include chemically similar amino acids. Conservative substitutions that provide functionally similar amino acids are known to those of ordinary skill in the art. Such conservatively modified variants are added to, but do not exclude, polymorphic variants, interspecies homologs and alleles of the disclosed modified relaxin polypeptides.

Conservative substitutions that provide functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions between each other:

1) Alanine (a or Ala), glycine (G or Gly);

2) Aspartic acid (D or Asp), glutamic acid (E or Glu);

3) Asparagine (N or Asn), glutamine (Q or Gln);

4) Arginine (R or Arg), lysine (K or Lys), histidine (H or His);

5) Isoleucine (I or Ile), leucine (L or Leu), methionine (M or Met), valine (V or Val);

6) Phenylalanine (F or Phe), tyrosine (Y or Tyr), tryptophan (W or Trp);

7) Serine (S or Ser), threonine (T or Thr); and

8) Cysteine (C or Cys), methionine (M or Met)

The term "identical" or "percent identity" in the context of two or more polypeptide sequences refers to two or more identical sequences or subsequences. When comparing and aligning the maximum correspondence over a comparison window, or measuring a designated region as using one of the following sequence comparison algorithms (or other algorithms available to one of ordinary skill in the art) or by manual alignment and visual inspection, a sequence is "substantially identical" if it has amino acid residues or nucleotide percentages that are identical (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% identity over the designated region).

In some embodiments, the polypeptides of the invention may be substituted and modified with an unnatural amino acid sequence. Still further, the at least one non-naturally encoded amino acid may comprise a carbonyl group, an aminooxy group, a hydrazide group, a hydrazine group, a semicarbazide group, an azide group, or an alkyne group. The non-naturally encoded amino acid may comprise a phenylalanine derivative. The non-naturally encoded amino acid may be selected from para, ortho or meta substituted phenylalanine. The non-naturally encoded amino acid may be selected from para-, ortho-, or meta-substituted phenylalanine comprising carbonyl, aminooxy, hydrazide, hydrazino, semicarbazide, azide, or alkyne groups. The non-naturally encoded amino acid may comprise p-acetyl-L-phenylalanine. The non-naturally encoded amino acid may be linked to the pharmacokinetic enhancer. For example, the non-naturally encoded amino acid may be linked to the pharmacokinetic enhancer via an oxime linkage or a triazole linkage (e.g., oxime linkage).

A wide variety of non-naturally encoded amino acids are suitable for use in the present disclosure. Any number of non-naturally encoded amino acids may be introduced into the modified polypeptide. In general, the introduced unnatural encoded amino acid is essentially chemically inert for the 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). In some embodiments, the non-naturally encoded amino acid includes side chain functionalities (including, but not limited to, azido, keto, aldehyde, and aminoxy) that effectively and selectively react with functionalities found in 20 common amino acids to form stable conjugates. For example, a modified polypeptide comprising a non-naturally encoded amino acid containing an azido functional group may be reacted with a polymer or PK extender,

the invention also provides a pharmaceutical composition comprising a polypeptide of the invention and at least one pharmaceutically acceptable excipient or carrier; and the use of a composition of molecules of such polypeptides in the methods of the invention as further described herein. Certain embodiments of the invention are pharmaceutical compositions comprising any of the polypeptides of the invention; and at least one pharmaceutically acceptable excipient or carrier. When a therapeutically effective amount of a polypeptide of the invention is designed for administration by, for example, intravenous, cutaneous or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable protein solutions are within the skill in the art in view of appropriate pH, isotonicity, stability, and the like. In addition to the binding agent, preferred pharmaceutical compositions for intravenous, cutaneous, or subcutaneous injection will contain an isotonic vehicle such as sodium chloride injection, ringer's injection, dextrose and sodium chloride injection, lactated ringer's injection, or other vehicle known in the art. The pharmaceutical compositions of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those skilled in the art.

Specifically, the pharmaceutical composition provided by the invention contains the polypeptide provided by the invention, and is used for improving skin activity, and increasing anti-wrinkle activity and antioxidant property of skin.

The pharmaceutical composition of the invention further comprises a buffer, which may be selected from the group consisting of: potassium phosphate, acetic acid/sodium acetate, citric acid/sodium citrate, succinic acid/sodium succinate, tartaric acid/sodium tartrate, histidine/histidine HCl, glycine, tris, glutamic acid, acetate and mixtures thereof, and in particular selected from potassium phosphate, citric acid/sodium citrate, succinic acid, histidine, glutamic acid, acetate and combinations thereof. Suitable buffer concentrations encompass concentrations of about 200mM or less, for example about 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 80, 70, 60, 50, 40, 30, 20, 10, or 5mM. The skilled artisan will be able to readily adjust the buffer concentration to provide stability of the pharmaceutical compositions as described herein. The buffer concentration contemplated in the pharmaceutical compositions of the present invention specifically ranges from about 5 to about 200mM, preferably from about 5 to about 100mM, more preferably from about 10 to about 50mM.

Salts may be used according to the invention, for example, to adjust the ionic strength and/or isotonicity of a pharmaceutical formulation and/or to further improve the solubility and/or physical stability of an antibody construct or other component. It is well known that ions can stabilize the natural state of a protein by binding to charged residues on the surface of the protein and by shielding charged and polar groups in the protein and reducing the strength of their electrostatic interactions, attraction and repulsion interactions. The ions may also stabilize the denatured state of the protein by specifically binding to the denatured peptide bond (- -CONH) of the protein. In addition, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions and thereby prevent or reduce protein aggregation and insolubilization. The ion species have different effects on proteins. A variety of classification ratings have been developed for the ions and their effects on proteins that can be used to formulate pharmaceutical compositions according to the invention. One example is the Hofmeister series, which rates ionic and polar nonionic solutes by their effect on the conformational stability of proteins in solution. The stabilizing solute is referred to as "lyophile". The "labile solute" is referred to as "chaotropic". "high concentrations of a nucleophile (e.g., >1 mole of ammonium sulfate) are typically used to precipitate proteins from solution (" salting out "). Chaotropic agents are commonly used to denature and/or solubilize proteins ("saline"). The relative effectiveness of ion pairs "salting-in" and "salting-out" defines their positions in the Hofmeister series. The free amino acids can be used in pharmaceutical compositions as bulking agents, stabilizers and antioxidants as well as other standard uses. Lysine, proline, serine and alanine can be used to stabilize proteins in the formulation. Glycine can be used to freeze-dry to ensure proper cake structure and characteristics. Arginine can be used to inhibit protein aggregation in both liquid and lyophilized formulations. Methionine can be used as an antioxidant. Particularly useful excipients for formulating pharmaceutical compositions include sucrose, trehalose, mannitol, sorbitol, arginine, lysine, polysorbate 20, polysorbate 80, poloxamer 188, pluronic and combinations thereof. The excipients can be present in the pharmaceutical composition in varying concentrations, so long as the composition exhibits the desired properties as exemplified herein, and in particular promotes the stabilization of the bispecific single chain antibody construct contained. For example, sucrose may be present in the pharmaceutical composition at a concentration of 2% (w/v) to 12% (w/v), i.e., at a concentration of 12% (w/v), 11% (w/v), 10% (w/v), 9% (w/v), 8% (w/v), 7% (w/v), 6% (w/v), 5% (w/v), 4% (w/v), 3% (w/v), or 2% (w/v). The preferred sucrose concentration range is 4% (w/v) to 10% (w/v), and more preferably 6% (w/v) to 10% (w/v). Polysorbate 80 may be present in the pharmaceutical composition at a concentration of 0.001% (w/v) to 0.5% (w/v), i.e. at a concentration of 0.5% (w/v), 0.2% (w/v), 0.1% (w/v), 0.08% (w/v), 0.05% (w/v), 0.02% (w/v), 0.01% (w/v), 0.008% (w/v), 0.005% (w/v), 0.002% (w/v) or 0.001% (w/v). Preferred polysorbate 80 concentrations range from 0.002% (w/v) to 0.5% (w/v), and preferably from 0.005% (w/v) to 0.02% (w/v).

The pharmaceutical compositions of the present invention can be formulated in a variety of forms, for example, in solid, liquid, frozen, gaseous, or lyophilized form, and may be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, among others.

For administration of the pharmaceutical composition of the invention, the dosage range will typically be about 0.0001-100 milligrams per kilogram (mg/kg) of host body weight, and more typically 0.01-5mg/kg of host body weight. Exemplary dosages may be 0.25mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight or in the range of 1-10 mg/kg. An exemplary treatment regimen is once or twice daily, or once or twice weekly, once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months, or once every three to 6 months. The dosage may be selected and readjusted by the skilled healthcare professional as needed to maximize therapeutic benefit for a particular patient.

The pharmaceutical compositions of the present invention will typically be administered to the same patient in a variety of situations. The interval between single doses may be, for example, 2-5 days, weekly, monthly, every two or three months, every six months, or annually. The interval between administrations may also be irregular based on modulating blood levels or other markers in the subject or patient. The dosage regimen of the compounds of the invention comprises intravenous administration of 1mg/kg body weight or 3mg/kg body weight, wherein the compounds are administered once every two to four weeks for up to six doses, followed by administration at 3mg/kg body weight or 1mg/kg body weight once every three months.

The pharmaceutical compositions of the present invention may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As the skilled artisan will appreciate, the route and/or mode of administration will vary depending on the desired result. Routes of administration of the polypeptides and pharmaceutical compositions of the invention include, for example, skin surface, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. In other embodiments, the polypeptides or pharmaceutical compositions of the invention may be administered by a non-parenteral route, such as topical, epidermal or mucosal route of administration, e.g., intranasal, oral, skin surface application.

Further, the invention provides a cosmetic for improving skin characteristics, which contains the polypeptide of the invention, and can be used for improving skin activity, and increasing anti-wrinkle activity and antioxidant characteristics of skin.

Drawings

FIG. 1 is a graph showing elution of polypeptide from a column

FIG. 2Y2-2-T3 polypeptide pair H ₂ O ₂ Protective effect pattern of HepG2 cells inducing oxidative damage

FIG. 3 graph of the effect of Y2-2-T3 polypeptide on skin epidermis moisture content

Detailed Description

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Example 1 preparation and identification of antioxidant polypeptide Components

Weighing 10g of pelteobagrus fulvidraco, cutting into small pieces, adding 0.05mol/L NaOH solution (W: V=1:5) for soaking for 40min, and washing with water to neutrality. Soaking in 0.2% sulfuric acid solution (W: v=1:5) for 30min, washing with water to neutrality, extracting with hot water overnight, centrifuging at 4000r/min for 30min, discarding precipitate insoluble substances to obtain crude gel product, rotary evaporating and concentrating the crude gel, and lyophilizing in a freeze dryer.

The prepared collagen was weighed and dissolved in 100mL of buffer (pH 9.0,0.02 mol/L) to prepare a 1% collagen solution. Adding alkaline protease properase in the ratio of enzyme to substrate of 1:50, hydrolyzing in water bath at 45 ℃ for 3h, inactivating enzyme in boiling water bath for 5min, centrifuging for 12min at 5000 r/min. Sequentially ultrafiltering the supernatant with ultrafiltration membranes with cutoff molecular weights of 10000D,5000D and 1500D to obtain polypeptides with different components, wherein the molecular weight (Mr) ranges are as follows: y is Y>10，Mr>10000D；5-Y-10，5000D<Mr≤10000D；1.5-Y-5，1500D<Mr<≤5000；Y<1.5，Mr<1500D, the protein content of which is determined for the above products, and the protein content of which is kept the same for the various components, in vitro, by measuring the para-hydroxy radical (. OH) and the superoxide anion radical (O) _2· ^- ) Is effective in scavenging activity of (a). The results are shown in Table 1.

Table 1 analysis of antioxidant Properties of the Components

As can be seen from Table 1, the products of different molecular weights are para-hydroxy radicals (. OH) and superoxidesAnionic radical (O) _2· ^- ) Has a difference in scavenging activity with respect to Y having the smallest molecular mass<1.5，Mr<The highest inhibition rate of 1500D to two free radicals exceeds 65%, and has a good inhibition effect.

EXAMPLE 2 isolation and identification of antioxidant Polypeptides

The pretreated SephadexG-25 was packed. Slowly adding 4mL of peptide solution with molecular mass less than 1500D, eluting with distilled water at room temperature at a flow rate of 0.4mL/min, collecting eluate by a distribution collector, measuring OD value of each 4mL of the eluate at 280nm wavelength by a nucleic acid protein meter, and drawing an elution curve. The results are shown in FIG. 1.

As can be seen from FIG. 1, the alkaline proteolytic liquid has a molecular mass less than 1500D and two peaks are obtained after the separation of SephadexG-25. The components separated by SephadexG-25 are respectively collected and concentrated, and the antioxidant activity of each component is detected, and the results show that the clearance rate of the Y1 component and the Y2 component to the hydroxyl radical is 19.48 percent and 83.51 percent respectively.

The Y2 fraction was further purified using SephadexG-15. The pretreated SephadexG-15 is loaded on a column. 3mL of the Y2 component solution was slowly added, elution was performed with distilled water at a flow rate of 0.3mL/min at room temperature, the eluate was collected by a distribution collector, the OD value of each 4mL of the eluate was measured at a wavelength of 280nm by a nucleic acid protein meter, and an elution curve was drawn. The Y2 component is separated and purified by SephadexG-15 to respectively obtain three peaks Y2-1, Y2-2 and Y2-3, and the eluent of each peak is collected for antioxidant activity analysis. The results showed that the clearance of the peaks Y2-1, Y2-2 and Y2-3 to hydroxyl radicals was 40.57%, 88.32% and 37.56%, respectively.

The Y2-2 component is entrusted to Beijing Baitai park to carry out mass spectrometry, polypeptides with high abundance and the matching coefficient Xcorr larger than 1.5 in database retrieval results are shown in table 2, and the polypeptide is artificially synthesized and then the clearance of hydroxyl free radicals of the corresponding polypeptide is detected.

Table 2 amino acid sequences identified by isolation in Y2 component

As can be seen from Table 2, the Y2-2-T3 polypeptide has the highest antioxidant properties, and the PP is a conserved amino acid sequence with corresponding antioxidant properties, as analyzed initially.

Example 3Y2-2-T3 polypeptide pair H ₂ O ₂ Protection of HepG2 cells against oxidative damage

HepG2 cells were seeded in 96-well plates at a density of 1.0X10 ⁴ cells/mL, 80. Mu.L of cell suspension was added to each well. After 12h incubation in the incubator, 10 μLDMEM medium or polypeptide solution was added to each well and incubated for 12h. Respectively adding DMEM culture medium or H according to experimental groups ₂ O ₂ After 6h incubation (500. Mu.M concentration), 20. Mu.LMTS was added to each well and incubated for 1h, and absorbance of 96-well plate samples was measured at 490nm wavelength. The experimental groupings were set as follows: 1) Blank group: 100 mu LDMEM medium was added to each well; 2) Control group: mu.L of cell suspension and 20. Mu.LDMEM medium were added to each well; 3) Model group: mu.L of cell suspension, 10. Mu.L of DMEM medium and 10. Mu. L H were added to each well ₂ O ₂ Solution (final concentration 500. Mu. Mol/L); 4) Positive control group: mu.L of cell suspension, 10. Mu.L of Vc solution (final mass concentration 0.1 mg/mL) and 10. Mu.L of DMEM medium and 10. Mu. L H were added to each well ₂ O ₂ Solution (final concentration 500. Mu. Mol/L); 5) Experimental group: mu.L of the cell suspension, 10. Mu.L of peptide solution (final mass concentration 0.01 (low dose), 0.1 (medium dose), 1mg/mL (high dose)) and 10. Mu. L H were added to each well ₂ O ₂ The solution (final concentration 500. Mu. Mol/L) was set up with 6 multiplex wells.

As can be seen from FIG. 2, the model set adds H ₂ O ₂ In the case of injury, the relative survival of cells decreased by half relative to the control group (significant difference, P<0.01). In the experimental group, the relative cell survival rate is obviously improved along with the increase of the concentration of the polypeptide, and in the high-dose experimental group, the relative cell survival rate reaches (93.17+/-2.46)%; compared with a positive control group with the same concentration, the effect of similar relative cell survival rate is also maintained, which shows that the polypeptide obtained by screening has better protection effect on oxidative damage of HepG2 cells.

EXAMPLE 4 Effect of polypeptide on rat skin Properties

SD rats are raised in special mouse cages, environmental facilities meet SPF grade requirements of test animals, the room temperature is 20-25 ℃, the humidity is 40-70%, and natural day and night alternation is realized. Animal experiments obey the ethical requirements of experimental animals, and after 1 week of adaptive feeding, healthy SD rats were selected and randomly divided into 14 groups, namely, a normal control group, a positive control group, a model group and a peptide sample group (peptide samples are divided into groups with low, medium and high doses), and 10 animals in each group. The normal control group and the model group use clean tap water as the only drinking water, peptide samples of low, medium and high dosage groups are respectively added with peptide with final concentration of 0.1, 0.5 and 1g/L in the clean tap water, the positive control group is Vc with 0.5g/L, and the feeds are all basic feeds and can be ingested freely. The normal control group was not irradiated, and the other groups were irradiated with the same amount of ultraviolet light. The back spine was then stroked with markings to a 5.0cm x 5.0cm area on both sides and shaved with an electric hair clipper, shaved prior to each irradiation and Na2S chemical dehairing, the dehaired rats were placed in a home-made holder to expose the back shaved skin, and the holder and rats were placed 35cm directly under an ultraviolet light source (UVA+UVB) for ultraviolet irradiation, where UVA intensity was 199.3mW/cm, measured by an ultraviolet irradiation meter ² UVB intensity was 68.4mW/cm ² . The irradiation frequency is 1 time every other day, the irradiation time is 1 week 10min/d, 2 weeks 20min/d, 3 weeks 30min/d, then the irradiation time is 30min/d, the appearance of the skin at 18 weeks is in a typical aging state to indicate the end of molding, and after the ultraviolet irradiation is ended, each group of rats are observed and photographed. Skin appearance wrinkle measurement score: the photo-aging degree and the improvement effect are scored according to the condition of the skin transverse wrinkles, and the scoring is based on sensory score 5: normal fine skin lines parallel to the long axis of the body are visible on the skin surface, appear and disappear along with the movement, and have no wrinkles; sensory score 4: the small dermatoglyph distributed along the spine disappears, a small amount of transverse shallow wrinkles appear, and the small wrinkles can appear and disappear along with the movement; sensory score 3: all the fine skin lines disappear, a small amount of transverse deep wrinkles appear, and the fine skin lines continuously exist; sensory score 2: the occurrence of more transverse coarse wrinkles persists and persists; sensory score 1: the surface of the skin is covered with a large number of transverse rough deep wrinklesIs continuously present. The results are shown in Table 3.

Table 3 sensory score results for each group

Group of	Sensory score
		Normal control group	4.85±0.17
Model group	1.63±0.09
		Positive control group	3.49±0.13
Low dose group	2.68±0.08
		Medium dose group	3.52±0.11
High dose group	4.23±0.16

As can be seen from Table 3, the sensory scores of the model groups were lower than 2.0, significantly lower than those of the blank group and each experimental group (P < 0.05), while the scores of the photoaged skin wrinkles after the polypeptide dry prognosis were substantially improved in the quantity-effect relationship, and the differences could reach the significance level especially at the medium and high doses. The effect is most remarkable in improving photo-aged skin wrinkles at high doses.

Skin barrier function assay: the skin barrier function was evaluated by measuring the moisture content of the epidermis of the skin and quantifying it using an FC1502 field kel skin analyzer, specifically according to the instructions of the instrument, and taking an average value (the unit of measurement is denoted as A.U) 3 times using a point touch measurement, and the result is shown in fig. 3.

As can be seen from fig. 3, the skin of SD rats in the model group showed significantly reduced surface water content (P < 0.05) during photoaging molding compared to the normal control group, indicating severely impaired skin water barrier function. The positive control group and the low, medium and high dose experimental groups recovered the water barrier function of the polypeptide oral dry process to a different extent compared with the model group, and the water content overall showed an increase in the quantity-effect relationship. Under the condition of high-dose experiment group, the A.U value reaches (32.6+/-1.03)%, and the skin moisture increasing effect is good. By increasing the moisture content, the skin anti-wrinkle effect can be better achieved.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims

1. An antioxidant polypeptide Y2-2-T3 for improving skin water retention, which is characterized in that the amino acid sequence is shown in SEQ ID NO: 1.

2. Use of the antioxidant, skin water retention enhancing polypeptide Y2-2-T3 according to claim 1 for the preparation of a pharmaceutical composition for enhancing skin antioxidant and skin water content anti-wrinkle.

3. Use of the antioxidant, skin water retention enhancing polypeptide Y2-2-T3 according to claim 1 for the preparation of a cosmetic for reducing skin uv irradiation and skin wrinkle resistance.

4. A pharmaceutical composition comprising the antioxidant skin water retention enhancing polypeptide Y2-2-T3 of claim 1 and a pharmaceutically acceptable carrier.