CN117700487A

CN117700487A - Application of extracellular matrix protein in cosmetics for repairing skin

Info

Publication number: CN117700487A
Application number: CN202311732328.8A
Authority: CN
Inventors: 张凤彩; 李光顺
Original assignee: Beijing Yuesi Biotechnology Co ltd
Current assignee: Shenzhen Helen Sea Biotechnology Co ltd
Priority date: 2023-12-16
Filing date: 2023-12-16
Publication date: 2024-03-15
Anticipated expiration: 2043-12-16
Also published as: CN117700487B

Abstract

The invention relates to application of extracellular matrix protein in cosmetics for repairing skin. According to the invention, polypeptide HS-SOD with good antioxidant property is screened and identified from finless eel skin by a molecular docking technology, and the polypeptide HS-SOD has good antioxidant property, so that proliferation of human epidermis keratinocytes is promoted, and collagen and ECM secretion of the human epidermis keratinocytes is promoted. The polypeptide can synergistically and effectively promote the antioxidant property of the skin of a mouse after being combined with ECM. The polypeptide can be prepared into cosmetics by combining with ECM, and has good application prospect.

Description

Application of extracellular matrix protein in cosmetics for repairing skin

Technical Field

The application relates to the biological field and the cosmetic field, in particular to application of extracellular matrix protein in cosmetics for repairing skin.

Background

Skin aging includes both exogenous and endogenous factors. Endogenous aging is mainly related to genes, age, hormones, etc. While exogenous factors are mainly derived from adverse effects of environmental factors such as: regional and climate, bad lifestyle, mental stress, pollution, etc. In the case of skin, the organ which is in direct contact with the outside world, the aging of the skin caused by solar radiation becomes the most important factor. Even experts believe that 80-90% of skin aging is derived from exogenous light radiation. Senile plaque (sunburn) is a representative of exogenous aging. It can be seen that the aging of the skin is advanced compared to the aging rate of other tissues and organs of the body, and is not synchronized with the other tissues and organs. But ultimately, both endogenous and exogenous causes of aging are essentially the result of skin aging by both oxygen ion Radicals (ROS) and ECM protein glycation, resulting in damage to the ECM.

Extracellular matrix ECM is a macromolecular substance synthesized and secreted by cells to the outside of cells, distributed on the cell surface or between cells, supporting and connecting tissue structures, regulating cell growth and physiological activities, and playing an important role in body development, tissue homeostasis and wound repair. Fibroblasts are the most important cells in the ECM. They are responsible for the production of all components of the ECM, such as collagen fibers, proteoglycans, and hundreds of proteins, including growth factors and other bioactive molecules. Thus, the behavior and status of fibroblasts has a direct relationship with the youthful status of the skin.

It is well known that maintaining the function of the extracellular matrix (ECM) is critical to skin health, and it has four main functions in the skin: providing a skin elastic, firm, strong support structure, promoting communication between cells, properly spacing cells, and promoting dynamic changes between cells (including movement, growth, differentiation, etc.). With the aging and the influence of external environmental factors, the extracellular matrix structure composition can be gradually decomposed, so that the skin is loose, wrinkles appear and elasticity is lost.

Among them, collagen, fibronectin and glycosaminoglycans play a vital role in maintaining skin health and younger age. Collagen provides a sufficient supporting framework for skin, and avoids wrinkles caused by tissue collapse; fibronectin can maintain intercellular adhesion and tighten skin; the glycosaminoglycan has the function of attracting water molecules, can help skin retain moisture and keep skin elasticity, and hyaluronic acid (hyaluronic acid) is one of the glycosaminoglycans; to maintain the smoothness, bulking and tightening of the skin, it is critical to maintain sufficient levels of these proteins and basal media in the extracellular matrix.

In recent years scientists have found an interesting phenomenon in research on ECM. If a weak, senescent fibroblast is implanted into the dermis (ECM) in the youthful state, the "senescent" fibroblast will gradually rejuvenate and exhibit a similar viability to youthful fibroblasts; in contrast, if a young fibroblast is implanted into the aged dermis, the cell quickly loses its viability, becomes atrophic, retarded, decaying, and loses regenerative capacity. The rising application of stem cells has been found since the 21 st century, and it has also been found that, in the case of ECM abnormality, stem cells are directly implanted, not only the survival rate is low, but also reliable effects cannot be obtained. In contrast, with healthy ECM, higher therapeutic effects are exhibited in similar situations. For example, in recent years PRP has been used to treat hair regrowth and skin aging, in fact, growth factors in the self plasma (ECM) inventory have been used to promote skin regeneration (anti-aging) and hair regrowth.

The application of ECM in wound and chronic wound healing has also been found to provide healthy ECM that accelerates the healing of wounds that otherwise have a healing disorder, accelerates and increases the survival rate, healing rate and quality of skin grafts. The above examples all alert us that the major problem of skin aging and repair disorders is probably not in the fibroblasts themselves, but rather in direct relation to their living ECM.

CN106794227B provides extracellular matrix (ECM) compositions prepared using placental tissue (e.g., human placental tissue). The ECM composition comprises about 30% to about 60% collagen and about 10% to about 35% elastin. CN113164645a method for obtaining extracellular matrix from the skin of tilapia (nile tilapia), the method comprising the steps of: chemical and enzymatic decellularization, detoxification, chemical sterilization, crosslinking, bleaching, dehydration, and sterilization by gamma radiation, more specifically, each of the procedures includes steps and the extracellular matrix is disrupted in the treated tissue; dermatitis is treated; acute, chronic and traumatic injury; a battlefield wound; necrotic wounds; a lacerated wound; scratching; bruise; and other lesions and pathologies. The present invention is in the fields of pharmacy, medicine and veterinary medicine, dentistry, chemistry, tissue engineering, molecular biology and biotechnology.

CN115634169a provides an application of extracellular matrix beta-glucan protein in cosmetics for repairing hormone-dependent skin, relates to the technical field of maintenance products, and aims to solve the problems that in the prior art, patients are prone to generating hormone-dependent and poor in effect of delaying skin aging. Although there are prior art methods for preparing pharmaceuticals or cosmetics using extracellular matrices, effective cosmetics for skin repair have yet to be further developed and studied.

Disclosure of Invention

The finless eel contains rich antioxidant substances, the inventor discovers that the polypeptide with better antioxidant property is screened and identified from finless eel skin by a molecular docking technology, the polypeptide is named as HS-SOD, and the amino acid sequence of the polypeptide is shown as SEQ ID NO: 1.

The polypeptides of the invention may also be conservatively substituted while still retaining the corresponding activity.

The polypeptide HS-SOD has good antioxidant property, and can promote proliferation of human epidermis keratinocytes and promote collagen and ECM secretion of human epidermis keratinocytes. The polypeptide and the ECM can effectively promote the oxidation resistance of the skin of the mice after being combined for use, and has better application prospect.

In one aspect, the invention provides a composition for promoting skin repair comprising the polypeptide HS-SOD of the antioxidant property of the invention and extracellular matrix protein ECM.

Further, the invention also provides a cosmetic for promoting the antioxidant effect of skin, which contains the polypeptide HS-SOD with antioxidant property and extracellular matrix protein ECM.

Further, the ECM is prepared by growing human epidermal keratinocytes.

The preparation process is to culture human epidermis keratinocyte in the culture medium containing the antioxidant polypeptide.

Wherein the ECM protein comprises alpha-1-antiprotease, alpha-2-HS-glycoprotein precursor, alpha-2-macroglobulin, alpha-actin-1, annexin A2, biglycan, trapezin-1, collagen alpha-1 (I), collagen alpha-1 (II), collagen alpha-1 (III), collagen alpha-1 (VI), collagen alpha-1 (XII), collagen alpha-1 (XIV), collagen alpha-2 (I), collagen alpha-2 (V), collagen alpha-2 (VI), collagen alpha-3 (VI), type I collagen, type III collagen, type IV collagen type V collagen, type VI collagen, decorin, elongation factor 1-alpha, EMILIN-1, endoplasmin, fibrinogen, fibronectin, fibrin-1 (Fibulin-1), fibrin-2 (Fibulin-2), galectin-1-homo (human), interferon-induced GTP-binding protein, laminin-A/C, laminin, LIM domain and actin-binding protein 1, penetratin-related protein, periostin (Periostin), periostin Precursor (PN), monin (Perlecan), plasminogen, reticulin, inhibin-1, one or more of rubber elongation factor protein, serine protease inhibitor protein H1, serum albumin, cohesin-proteoglycan-1, tenascin precursor (TN) (human), platelet response protein-1, transforming growth factor-beta induction protein, transcgglutinin, vimentin. Embodiment 18 is the ECM protein extract of embodiment 1 or 5, wherein all or part of the soluble proteins originally present in the ECM that have been removed include alpha-1-antiprotease, alpha-2-HS-glycoprotein precursor, alpha-2-macroglobulin, alpha-auxiliary actin-1, annexin A2, biglycan, trapin-1, collagen alpha-1 (I), collagen alpha-1 (II), collagen alpha-1 (III), collagen alpha-1 (VI), collagen alpha-1 (XII), collagen alpha-1 (XIV), collagen alpha-2 (I), collagen alpha-2 (V), collagen alpha-2 (VI), collagen alpha-3 (VI) type I collagen, type III collagen, type IV collagen, type V collagen, type VI collagen, decorin, elongation factor 1-alpha, EMILIN-1, endoplasmin, fibrinogen, fibronectin-1, fibrin-2, galectin-1-homo (human), interferon-induced GTP-binding protein, laminin-A/C, laminin, LIM domain and actin-binding protein 1, penetratin-related proteins, periostin Precursor (PN), leuconostoc, plasminogen, reticulin, inhibitor protein-1, rubber elongation factor protein, serine protease inhibitor protein H1, serum albumin, adhesive proteoglycan-1, tenascin precursor (TN) (human), platelet response protein-1, transforming growth factor-beta inducer protein, trans-coagulin, vimentin, etc.

Advantageous effects

According to the invention, polypeptide HS-SOD with good antioxidant property is screened and identified from finless eel skin by a molecular docking technology, and the polypeptide HS-SOD has good antioxidant property, so that proliferation of human epidermis keratinocytes is promoted, and collagen and ECM secretion of the human epidermis keratinocytes is promoted. The polypeptide can synergistically and effectively promote the antioxidant property of the skin of a mouse after being combined with ECM. The polypeptide can be prepared into cosmetics or medicines by combining with ECM, and has good application prospect.

Drawings

FIG. 1 is a graph showing the effect of polypeptides on cell viability

FIG. 2 is a graph showing the effect of polypeptides on collagen secretion by human epidermal keratinocytes

Detailed Description

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is contemplated that any of the embodiments discussed in this specification may be implemented according to any of the methods or compositions of the present invention and vice versa. Furthermore, the compositions of the present invention may be used to carry out the methods of the present invention.

Example 1 screening and identification of Monopteri albi antioxidant Polypeptides

Taking 10g of fresh finless eel skin, freezing and crushing, dissolving the fresh finless eel skin by using PBS, and carrying out mathematical analysis on a response surface by using 0.5% trypsin and 0.5% neutral protease under the condition that the mass fraction of a substrate is 12%, wherein the optimal hydrolysis condition is that the enzymolysis time is 2h, the enzymolysis temperature is 50 ℃, and the hydrolysis degree result reaches 78.4%;

the enzymatic hydrolysate was assayed using MALDI-TOFMS/MS. The acceleration voltage of the ion source is 20kV, the laser with the laser wavelength of 337nm, the energy of 5800, the ion delay extraction time of 390ns, the mass spectrum signal is accumulated for 2000 times in a single scanning mode, and the mass scanning range of 500-3000 u is corrected by using the ion peak of a standard polypeptide calibrator (peptideIIstandardkit (Bruker)) (m/z is 500-3000). MALDI-TOFMS/MS tandem mass spectrometry results were retrieved by querying the NCBInr database with local MASOT software. The resulting polypeptide amino acid sequence was imported into the Discovery Studio 2017 by module Build and Edit Protein to obtain an untreated polypeptide 3D structure and energy optimized by function Full Minimization in the minimizer links module to obtain the optimal energy polypeptide 3D structure. Keap1, whose ID number is 5DAD, is retrieved and downloaded from the PDB database. After 5DAD is opened in DS, water is removed for hydrogenation, and the protein is pretreated by Prepareprotein, including protonation state under different pH values, loop missing supplementation and the like. The defined Receptor in Tools Explorer defines the treated 5DAD protein molecule as a Receptor molecule, and the active site is designated at the small molecule ligand self-contained in the Receptor protein by expansion Define and Edit Binding Site. The molecules are docked in the Tools Explorer, the Receptor-Ligand Interactions module is unfolded, the CDOCKER is clicked, and the corresponding parameter panel is opened. In the parameter panel, the Input Receptor is set to 5DAD-prep 5DAD and then run. The HS-SOD polypeptide (the amino acid sequence of which is shown as SEQ ID NO: 1) obtained by screening has the lowest energy required for combining with receptor protein, the highest combination, can combine with Keap1 protein mutually and inhibit the activity of the Keap1 protein, thereby blocking Nrf2 signal path and achieving the antioxidation effect.

EXAMPLE 2 identification of HS-SOD polypeptide Activity

The active peptide HS-SOD polypeptide of the invention is diluted to 5,10, 50, 100 and 200 mug/mL in a 96-well cell culture plate, and each concentration is 5 multiple wells. All of the above operations are performed under aseptic conditions.

The human epidermal keratinocytes (product number: ORCH267, aujersey organism) were passaged by first pouring out the cell culture solution, washing the cells twice with 2mLPBS, discarding the liquid, adding 1mL of 0.25% trypsin digestion solution by mass fraction for 3min until the cells are completely disaggregated, transferring the cell suspension to a centrifuge tube, adding 2mL of 10% superior fetal bovine serum DMEM/F12 to terminate digestion, and centrifuging for 5min at 1000 r/min. After centrifugation, the supernatant was discarded, 2mL of 10% high-quality bovine serum was added and the mixture was thoroughly blown with DMEM/F12, and the cell concentration was controlled to 5.0X10 per 1mL ⁵ Individual cells were cultured at 37℃under conditions of 5% carbon dioxide saturation. Inoculated in 96-well cell culture plates at 100. Mu.L per well and cultured at 37℃under 5% carbon dioxide and saturated humidity for 24 hours. The prepared cell culture plate was added with 100. Mu.L of active peptide solution per well, and a blank control group (100. Mu.L of maintenance medium was added only) and a positive control group (100. Mu.L of bFGF maintenance medium was added at a mass concentration of 10 ng/mL) were set at 37℃with 5% carbon dioxide, saturated humidityCulturing for 48h under the condition of degree. 10. Mu.L of MTT solution was added to each well, and the mixture was incubated at 37℃under 5% carbon dioxide and saturated humidity for 5 hours. All of the above operations are performed under aseptic conditions. After the liquid in the culture plate was discarded, 100. Mu.L of dimethyl sulfoxide was added to each well, the absorbance was measured at 570nm using 630nm as a reference wavelength on an enzyme-labeled instrument after mixing, and the measurement result was recorded, whereby the cell viability was calculated as cell viability= (absorbance A570 of experimental group/absorbance A570 of blank group) ×100%. The results are shown in FIG. 1.

As can be seen from FIG. 1, the active peptide gradually increases in the concentration range of 0-200 mug/mL along with the increase of the action concentration, so that the proliferation promoting effect of human fibroblasts is also gradually enhanced, and the active peptide is dose-dependent. The growth of the fibroblast can be obviously promoted when the mass concentration is 200 mug/mL, the survival rate is (148.27 +/-6.23)%, the proliferation effect is much stronger than that of a control, and the difference is obvious (P is less than 0.05) compared with a blank control group.

Example 3 Effect of active peptides on collagen secretion by human epidermal keratinocytes

Hydroxyproline is relatively high in collagen, so that the amount of hydroxyproline can reflect collagen metabolism. The hydroxyproline content determination is realized by using a hydroxyproline content detection kit. The results of the measurement of the cells of each group cultured in example 2 were quantified to have the same density, and are shown in FIG. 2.

As can be seen from FIG. 2, the production of hydroxyproline gradually increased with increasing concentration of active peptide in the concentration range of 0-200. Mu.g/mL, and the production was dose-dependent. The growth of the fibroblast can be obviously promoted when the mass concentration is 200 mug/mL, the proliferation rate is (3.31+/-0.17) mug/mL, the proliferation effect is much stronger than that of a control, and the difference is obvious compared with a blank control group (P is less than 0.05).

Since collagen and elastin, which are important components of extracellular matrix cavities, are synthesized and secreted mainly by cells to the outside of the cells, and finally mature and adhere to extracellular matrix layers, a culture medium of cells is collected at 48 hours for WB detection while immunofluorescence observation is performed on the cells. The specific method for WB detection is as follows: equal amounts of cell lysates from each group were separated with 10% gel, and the proteins in the gel were transferred to PVDF membranes and blocked with 5% nonfat milk powder solution. And then cleaning, incubating the 1 antibody, cleaning, incubating the second antibody and cleaning. The antibody used is anti-Col1A1, anti-Elastin and the secondary antibody is goat anti-rabbit IgG. The results are shown in Table 1 as Col1A1, elastin to GAPDH gray scale ratio.

Table 1 collagen and elastin content ratios in each group

Group of	Col1A1/GAPDH	Elastin/GAPDH
			Blank control group	1.13±0.02	1.32±0.03
5 μg/mL polypeptide group	1.20±0.03	1.42±0.05
			10 μg/mL polypeptide group	1.27±0.04	1.51±0.04
50 μg/mL polypeptide group	1.35±0.03	1.59±0.06
			100 μg/mL polypeptide group	1.41±0.05	1.64±0.03
200 μg/mL polypeptide group	1.49±0.04	1.68±0.05
			Positive control group	1.18±0.02	1.34±0.03

From the results in Table 1, it was shown that the collagen and elastin contents in the cells of each group cultured with the polypeptide were significantly increased as compared with the blank group and were also better than those of the positive control group. In particular, the high concentration of 200 μg/mL polypeptide group was significantly different from the blank control group and the positive control group (P < 0.05).

Example 4 preparation of extracellular matrix proteins

Human epidermal keratinocytes were incubated in DMEM/F12 dishes containing 10% of superior bovine serum of active peptide at 100. Mu.g/mL at 37℃under 5% carbon dioxide at saturation humidity for 18h, followed by incubation in serum-free medium for 3-4D until cells were 100% confluent, treatment of cells with 2mM EDTA in D-PBS, washing off residual cells with PBS, and leaving dishes containing underlying ECM. The ECM of the petri dish was scraped mechanically with a putty spatula to loosen the ECM on the surface of the petri dish and the contents were mixed with the spatula. Transfer to a 15ml conical tube. Washed with 0.5ml serum-free medium and the wash was added to a 15ml conical tube containing the ECM/medium mixture. A15 ml conical tube was sonicated 4 times to disperse it, then aspirated into a 1.5ml Eppendorf tube, and centrifuged at 15000g for 5 minutes. Removing supernatant containing soluble protein, leaving insoluble precipitate, and freeze drying to obtain ECM protein extract.

At the same time, a control group, i.e., human epidermal keratinocytes, was cultured under the same culture medium culture conditions without the active peptide, and a control ECM protein extract was obtained in the same manner.

ECM compositions were assayed for elastin content using Fastin Elastin Assay kit, which used 5,10,15, 20-tetraphenyl-21, 23-porphin tetra-sulfonate (TPPS) as dye to stain extracted and solubilized alpha-elastin from test samples. Elastin was extracted from ECM composition in 0.25M oxalic acid at 100 ℃. Elastin was then precipitated with trichloroethanol and hydrochloric acid (TCA/HCl) and subsequently stained with TPPS. The TPPS is then dissociated from the elastin and the level of released TPPS is quantified spectrophotometrically. The elastin level in a sample of ECM composition is determined by interpolation against an elastin standard curve. The results showed that the ECM composition obtained after treatment with the polypeptide had elastin in the range of 27.34% by weight of the ECM composition, whereas the ECM composition obtained without treatment with the polypeptide had elastin in the range of 20.17% by weight of the ECM composition, indicating that the polypeptide was effective in promoting cell production of ECM.

EXAMPLE 5 animal experiments with ECM and active peptides

Healthy male mice were selected, adaptively fed for 7d, randomly grouped, 10 per group. The specific grouping is as follows: HS-SOD polypeptide group (administration dose 0.015g/0.5 mL/d), polypeptide-treated ECM group (administration dose 0.015g/0.5 mL/d), polypeptide-free ECM group (administration dose 0.015g/0.5 mL/d), polypeptide-treated ECM+HS-SOD polypeptide group (both ECM and polypeptide are administration dose 0.015g/0.5 mL/d), aging model group, vc positive control group (administration dose 0.015g/0.5 mL/d), and the other group is blank control group. The polypeptide and ECM were subcutaneously injected once daily, vc was given subcutaneously in the positive control group, and the blank control group and model group were replaced with an equivalent dose of physiological saline. Mice were weighed 1 time a week during the trial to adjust dosing, free feeding, and water intake. After the last administration, the patients were fasted for 24 hours, water was not forbidden, and the body weight was weighed before sacrifice. Wherein, ultraviolet lamp irradiation is carried out after 3 weeks of administration, and specific irradiation conditions are as follows: mice were dehaired with 4% sodium sulfide before the first irradiation, and then dehaired 1 time after every two weeks of irradiation until the end of irradiation. The dehaired mice were exposed to ultraviolet light (uva+uvb, simulated sunlight) 3 times per week. Irradiation at week 1The dosage is 2mJ/cm each time ² And at a rate of 2mJ/cm per week ² Up to 8mJ/cm each time at week 4 ² . This dose was then followed and continued to irradiate for week 8. At the eighth week, the mice all remained healthy, indicating no obvious toxic or side effects of the polypeptide and ECM.

Antioxidant enzyme activity and MDA and Hyp content in skin tissue homogenate. About 0.5g of back dehaired skin tissue is taken, pre-cooled physiological saline is used for rinsing, subcutaneous fat and other connective tissues are removed, filter paper is wiped dry, and the skin dehaired skin tissue is weighed. Pouring into beaker with tissue block, cutting skin, adding 9 times of pre-cooled physiological saline, and homogenizing in ice bath. Centrifuging at 4deg.C at 8000r/min for 15min, collecting supernatant, and respectively measuring SOD, GSH-Px, CAT activity and MDA and Hyp content according to kit specification. The results are shown in Table 2.

TABLE 2 SOD, GSH-Px, CAT, MDA and Hyp content in skin tissue

From Table 2, it can be seen that the activities of SOD, GSH-Px and CAT in the skin tissues of the treatment group are effectively improved, and MDA is effectively reduced, which indicates that the treatment group has the effect of obviously improving the antioxidant capacity of the model mice. At the same time, the treatment group can also significantly increase the total Hyp content in the skin of model mice.

Taking 0.5cm of skin of back irradiation part ² 4% neutral formaldehyde was fixed, frozen and sliced to a slice thickness of 7. Mu.m. The sections are respectively dyed by HE and Van Gieson, and the observation and photographing under an optical microscope show that the skin epidermis layer of the normal control group mice has complete structure and clear cell layering, the skin epidermis layer of the model control group mice has thickening and keratinization, the cell layering is unclear, the dermis layer is damaged, and more inflammatory cells infiltrate. The damage degree of skin tissues of the treatment group, especially the ECM+polypeptide treatment group is obviously reduced, and the treatment group is close to normal tissues, so that the combination treatment has better treatment effect.

When practicing or testing embodiments of the invention, optional methods and materials similar or equivalent to those described in the specification may be used, with the preferred methods, devices, materials described in the specification. However, before describing the materials and methods of the present invention, it is to be understood that the specific size, shape, dimensions, materials, methods, means, etc. described in this specification may be changed according to conventional experimental methods and optimization purposes, and thus the present invention is not limited thereto. And it is to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the scope of the appended claims.

Claims

1. An active polypeptide with antioxidant property, which is named as HS-SOD, and has an amino acid sequence shown in SEQ ID NO: 1.

2. Use of an active polypeptide according to claim 1 for the preparation of cosmetics having antioxidant properties.

3. A composition having antioxidant, anti-aging properties comprising the active polypeptide of claim 1 and an extracellular matrix protein; wherein the extracellular matrix protein is prepared by the following method: an extracellular matrix protein prepared by culturing human epidermal keratinocytes in a medium containing 100. Mu.g/mL of the active peptide of claim 1.

4. Use of an active polypeptide according to claim 1, and an extracellular matrix protein, for the preparation of a composition having antioxidant and anti-aging properties; wherein the extracellular matrix protein is prepared by the following method: an extracellular matrix protein prepared by culturing human epidermal keratinocytes in a medium containing 100. Mu.g/mL of the active peptide of claim 1.

5. The use according to claim 4, wherein the composition is prepared in the form of a cosmetic.

6. The method according to claim 5, wherein the cosmetic further comprises conventional cosmetic adjuvants.