CN113750216A

CN113750216A - Anti-aging cosmetic or medicine

Info

Publication number: CN113750216A
Application number: CN202111233721.3A
Authority: CN
Inventors: 张冬久; 钟春秀
Original assignee: Beijing Yuanshengda Biotechnology Development Co ltd
Current assignee: North American Life Sciences Shanghai Co ltd
Priority date: 2021-10-22
Filing date: 2021-10-22
Publication date: 2021-12-07
Anticipated expiration: 2041-10-22
Also published as: CN113750216B

Abstract

The invention relates to an anti-aging cosmetic or medicine. The invention prepares the outer vesicle from the fibroblast, and in addition, the polypeptide RO-3 with the antioxidant and anti-aging activities is obtained by screening, identifying and preparing, the two have better antioxidant activities respectively, and the combined use of the two has a synergistic effect, so the invention has good application prospect in the field of anti-aging beauty treatment or medicines.

Description

Anti-aging cosmetic or medicine

Technical Field

The invention relates to the field of medicines and cosmetics, in particular to an anti-aging cosmetic or medicine.

Background

Aging is a continuous change process, and international mainstream believes that free radicals damage human cells, thereby causing a plurality of diseases and accelerating human aging. The free radicals from generation to quenching deeply affect the formation of lipofuscin, the mutation of mitochondrial DNA, the induction of apoptosis and the synthesis of protein, the relationship between the free radicals and aging and diseases caused by the free radicals, thereby providing a certain scientific basis for slowing down the aging of human beings. The various biomolecules in the system have a large number of active groups which must interact with each other to generate chemical reaction, so that the biomolecules are slowly crosslinked to tend to be stable in chemical activity. With the lapse of time, the crosslinking degree is increased continuously, the active groups of the biomolecules are consumed and reduced continuously, the original molecular structure is changed gradually, and the aging phenomenon of the biological tissues can be generated gradually due to the accumulation of the changes. Oxygen radicals are the first killers of skin and body to accelerate aging.

In the cosmetic industry, how to effectively prevent skin aging is an important direction of current research.

Extracellular Vesicles (EVs), including exosomes, are particulate matter derived from cells, contain large amounts of proteins and small nucleic acids, and can regulate local or remote target cells to exert their biological effects.

Exosomes may be released into the extracellular environment from a variety of cells, for example: tumor cells, dendritic cells, lymphoid cells, mesothelial cells, epithelial cells, or cells from different tissues or organs. It contains proteins, mRNAs, mirnas and signal molecules that reflect the physiological state of the cell and are also a rich source of potential biomarker molecules. Up to now, exosomes have been detected in body fluids such as urine, serum, saliva and breast milk, and their wide distribution and strong function have been observed.

Research has found that human induced pluripotent stem cell exosomes can significantly reduce the expression level of aging-related beta-galactosidase, and up-regulate the expression of collagen in HDFs, thereby providing a theoretical basis for the beauty treatment and anti-aging of stem cell exosomes. At present, stem cell skin care products released by various large beauty institutions: oxidants, retinoic acid, peptides, growth factors, etc. have been used to protect or repair the skin, and studies have shown that skin fillers are more effective in smoothing facial contours and last longer than topical treatments.

Research shows that extracellular vesicles (UC-EVs) derived from umbilical cord mesenchymal stem cells (UC-MSCs) of newborns can reverse aging of adult bone marrow mesenchymal stem cells (AB-MSCs) and remarkably enhance osteogenesis and injury repair capacity of the adult bone marrow mesenchymal stem cells, particularly, the umbilical cord mesenchymal stem cells (UC-EVs) used in vivo can remarkably delay aging of naturally aged mice and have remarkable influence on degeneration of various tissues and organs such as bones, kidneys, vascular smooth muscles and the like. Stem cell exosomes have also been reported in the treatment of age-related diseases. Cardiovascular diseases are high in incidence in the elderly, and the cardiac extracellular secretion can treat acute myocardial infarction through specific macrophage polarization.

Polypeptides are another important direction currently used for skin anti-aging. Peptides such as Signal peptides are very related to collagen, which stimulates collagen production. Such peptides can promote the production of elastin, hyaluronic acid, glycosaminoglycans and fibronectin, in addition to collagen. The best named is palmitoyl pentapeptide-4, which has been monopolized by Baojie because it was experimentally confirmed to reduce the generation of wrinkles. Rosemary, the name of Latin (Rosmarinus officinalis), is a shrub of the genus Rosmarinus, the family Labiatae, the class Dicotyledoneae. Antioxidant and rosemary essential oil with excellent antioxidant activity can be extracted from flower and leaf of rosemary. At present, the research on the rosmarinus officinalis antioxidant polypeptide is not much, so that the separation and preparation of the corresponding antioxidant and anti-aging polypeptide from the plant is also an important research direction.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides a high-activity fibroblast extracellular vesicle and a preparation method thereof, and an anti-aging cosmetic product or medicament prepared from antioxidant polypeptide.

On one hand, the invention provides a high-activity fibroblast outer vesicle and a preparation method thereof, and specifically, the foreskin of an infant is fully washed by PBS containing 100U/ml penicillin and 100U/ml streptomycin on a super clean bench, D-hanks is washed once, a skin specimen is cut into small pieces by sterile ophthalmic scissors after subcutaneous tissues are removed, the small pieces are placed in type II collagenase digestive juice for digestion overnight at 4 ℃, and the epidermis and the dermis are separated aseptically in the super clean bench after 24 hours. Discarding epidermis, cutting dermis, transferring to 0.25% protease (without EDTA) for about 15min, stopping digestion with high-sugar-DMEM culture solution containing 10% calf serum, centrifuging at 2000r/min for 5min, inoculating precipitate to 25cm²Adding about 4ml of high-sugar DMEM culture solution containing 10% calf serum into a plastic culture bottle, taking care not to float the tissue, and placing the culture bottle in37℃,50％CO₂In the incubator, 5ml of the culture solution was supplemented the next day. The primary cells can be passaged after growing over about 80 percent, the cells are digested by 0.25 percent pancreatin (without EDTA) during passage, the culture bottle is tapped when the cell bodies retract under an inverted microscope, then DMEM-high-sugar culture solution containing 10 percent calf serum is added to stop digestion, the cells on the wall of the bottle are blown by a suction tube lightly, and the cells are collected in a 10ml centrifuge tube. Centrifuging at 2000 r.min for 5min, discarding the supernatant, and performing centrifugation according to the ratio of 1: 3 inoculating, subculturing and conventionally culturing to obtain cells, namely fibroblasts; culturing fibroblast until about 80% of fibroblast is fused, removing supernatant, washing with PBS for 3 times, replacing with serum-free low-sugar culture medium, and culturing for 24 hr. The supernatant was collected, centrifuged at 3000g for 10min, and cells and cell debris were removed. The centrifuged supernatant was collected and passed through filters of 450nm and 220nm in order to remove large-diameter vesicles. The filtered culture broth was collected, added to 100000MWCO Millipore ultrafiltration tubes, and concentrated and filtered by centrifugation at 3000g for 60 min. PBS was added to the ultrafiltration tube for washing and centrifuged again by the above-mentioned centrifugation method until the volume of the ultrafiltration tube was 100-. Extracellular vesicles were collected from the ultrafiltration tube and stored in a-80 ℃ freezer for use.

On the other hand, the invention carries out enzymolysis from the antioxidant plant rosewood by a specific two-step enzymolysis method, namely a first-stage enzymolysis, adopts neutral protease, and has the enzymolysis temperature of 45 ℃, the solution pH value of 6, the enzymolysis time of 3h and the enzyme adding amount of 6 percent; and in the second stage of enzymolysis, papain is added, the enzymolysis temperature is 60 ℃, the pH value is 6, the enzymolysis time is 7h, and the enzyme adding amount is 3%, so that the specific antioxidant polypeptide RO-3 with antioxidant activity is separated, wherein the sequence of the polypeptide RO-3 is shown as SEQ ID NO: 1 is shown.

The two-step enzymolysis method adopted by the invention is through optimized design,

furthermore, the invention provides an anti-wrinkle and anti-aging pharmaceutical composition, which contains fibroblast outer vesicles and an antioxidant polypeptide, wherein the sequence of the polypeptide RO-3 is shown as SEQ ID NO: 1 is shown.

Furthermore, the invention provides an anti-wrinkle and anti-aging cosmetic, which contains fibroblast outer vesicles and an antioxidant polypeptide, wherein the sequence of the polypeptide RO-3 is shown as SEQ ID NO: 1 is shown.

Further, the composition or cosmetic further comprises a corresponding carrier.

Advantageous effects

The outer vesicles are prepared from fibroblasts, and the polypeptides with antioxidant and anti-aging activities are obtained through screening, identifying and preparing, have good antioxidant activities respectively, have a synergistic effect when being used in combination, and have good application prospects in the field of anti-aging beauty treatment or medicines.

Drawings

FIG. 1 graph of the results of percentage of senescent cells

Detailed Description

The technical scheme of the invention is described by combining specific embodiments. The experimental materials not particularly emphasized in the following examples are all conventional experimental materials, and are not particularly required, and are all conventional materials readily available to those skilled in the art.

EXAMPLE 1 Primary Screen of Rosmarinus officinalis antioxidant fraction

(I) preparation of polypeptide mixture

Freezing 10g of herba Rosmarini officinalis in liquid nitrogen, grinding, crushing, mixing with deionized water at a ratio of 1: 6, pulping, heating at 90 deg.C for 20min, cooling, performing first-stage enzymolysis with neutral protease at 45 deg.C, adjusting pH of solution 6, enzymolysis time 3h, and enzyme adding amount 6%; and in the second stage of enzymolysis, papain is added, the enzymolysis temperature is 60 ℃, the pH value is 6, the enzymolysis time is 7h, and the enzyme adding amount is 3%. After the reaction is finished, putting the mixture into boiling water to inactivate enzyme for 10min, cooling, filtering with four layers of gauze to remove filter residue, centrifuging the filtrate for 10min at 8000r/min to obtain supernatant which is the polypeptide mixture, and taking the supernatant for later use.

And (3) determining the hydrolysis degree in the supernatant by adopting a formaldehyde titration method, and specifically comprising the following steps:

(1) respectively taking 5mL of the solution before and after enzymolysis, placing the solution in a 100mL volumetric flask, and adding ultrapure water to a constant volume to reach a scale mark.

(2) 20mL of the diluted solution was taken out and placed in a 250mL Erlenmeyer flask, and 60mL of ultrapure water was added thereto and mixed.

(3) The solution in the flask was titrated with a calibrated concentration of about 0.05mol/L NaOH to pH 8.2, 10mL of formaldehyde was added to the flask, mixed well and titrated with the same NaOH to pH 9.2. The NaOH consumption volume was recorded.

(4) A blank of 80mL of ultrapure water was used in the test, and three replicates of each sample were run. The calculation formula is as follows: DH (%) - (AN-AN0)/N × 100

AN-free amino nitrogen content (g/100mL) in the enzymolysis solution;

AN 0-content of amino nitrogen in raw material solution before enzymolysis (g/100 mL);

n-total protein nitrogen content in the stock solution (g/100 mL);

the content determination formula of amino acid nitrogen in the solution to be detected is as follows:

N＝{【(V1-V2)×C×0.014】/(5×V3÷100)}×100

n-amino nitrogen content (g/mL);

v1-volume (mL) of standard solution that consumes NaOH after formaldehyde is added to the sample diluent;

v2-volume (mL) of standard solution that consumes NaOH after formaldehyde is added to the blank control solution;

v3 volume dosage (mL) of the solution to be detected;

c, the concentration (mol/L) of the prepared NaOH standard solution;

0.014-mass (g) of nitrogen equivalent to 1.00mL of NaOH calibration solution;

the result shows that the hydrolysis degree of the polypeptide supernatant prepared by the two-step enzymolysis method is 65.8 percent, and the polypeptide supernatant has a better hydrolysis effect.

(II) primary purification

SephadexG-25 Sephadex: and preparing the treated supernatant sample into a solution with the mass concentration of 50mg/mL, and separating and purifying on a SephadexG-25 gel column, wherein the sample loading amount is 10 mL. Using deionized water as eluent, with an elution flow rate of 0.5mL/min, collecting the eluent (3 mL/tube) with an automatic fraction collector, detecting with an ultraviolet detector at 280nm, collecting each elution peak, vacuum concentrating, and freeze drying each stage of elution components; and (3) screening the component with the highest antioxidant activity by taking the DPPH free radical clearance rate as a detection index. The DPPH clearance rate determination method comprises the following steps: adding the elution peak and an isovolumetric 0.2mmol/L DPPH solution into the same test tube with a plug, shaking up, and measuring the absorbance after 30min by using absolute ethyl alcohol as a reference. And simultaneously measuring the absorbance of a mixed solution of 0.2mmol/L DPPH solution and the equal volume of absolute ethyl alcohol and the absorbance of a mixed solution of the solution to be measured and the equal volume of absolute ethyl alcohol. The clearance was calculated according to the following formula: clearance (%) - (a1-A3)/a2) X100%, wherein: a3 is the absorbance of DPPH solution when no solution to be detected is added; a1: the absorbance of the DPPH solution when the solution to be detected is added; a2 is the absorbance of the solution to be detected at the detection wavelength; the measurement wavelength was 517 nm.

And separating the enzyme hydrolysate by a Sephadex G-25 gel column to obtain three elution peaks of RO-1/RO-2/RO-3. Samples were collected 3.

TABLE 1 elution peaks antioxidant Activity

As can be seen from Table 1, both RO-2 and RO-3 have a good DPPH scavenging effect, and RO-3 polypeptides were identified and further studied.

EXAMPLE 2 identification of RO-3 antioxidant Polypeptides

The RO-3 sample is prepared into 0.5mg/mL solution and then is detected by C18 reversed phase high performance liquid chromatography. The experimental conditions were: sample loading amount: 10uL, volume flow: 0.8mL/min, wherein the eluent is A liquid: 5% acetonitrile, 0.05% trifluoroacetic acid; and B, liquid B: 80% acetonitrile, 0.05% trifluoroacetic acid (all volume fractions). The linear elution procedure was: o-1min, V (A): v (b) 85: 15, 1-40min, V (A): v (b) ═ 50: 50, the temperature is 3O ℃, and the detection is carried out at 220 nm.

Reverse phase high performance liquid chromatography (RP-HPLC) was used to identify the purity of RO-3. The result shows that the RO-3 reverse high performance liquid chromatography obtained after SephadexG-25 column chromatography purification is a three-main-peak mixture, wherein the DPPH clearance of the second main peak is the highest, the purity is about 52.4 percent by calculating according to the peak area, and the second main peak component is collected and used for sequence identification.

Amino acid sequence analysis:

detecting parameters: acceleration voltage of 20kV and laser wavelength of 355 min; reagent and sample injection operation: reagent and configuration: samples were formulated with phi 0.1% trifluoroacetic acid to 10^-4mol/L solution. Matrix 4-HCCA was prepared using a solution of 0.1% trifluoroacetic acid in acetonitrile in a volume ratio of 2: 1 to prepare a solution of 7 mg/mL. Sample introduction operation: 1. mu.L of the prepared sample was mixed with 4. mu.L of a base (4-HCCA) solution. Dripping 1 mu L of the mixed solution on a sample table, drying at room temperature, and then conveying the sample table to an ion source for determination; method for analyzing amino acid sequence: the amino acid sequence of the polypeptide is SEQ ID NO: 1. the amino acid sequence is prepared by artificial synthesis.

Example 3 preparation of fibroblast extracellular vesicles

Taking foreskin of the infant, fully washing the foreskin of the infant on a superclean bench by PBS containing 100U/ml penicillin and 100U/ml streptomycin, cleaning D-hanks once, removing subcutaneous tissues, shearing a skin specimen into small pieces by using sterile ophthalmic scissors, placing the small pieces in II type collagenase digestive juice for digestion overnight at 4 ℃, and performing sterile separation on epidermis and dermis in the superclean bench after 24 hours. Discarding epidermis, cutting dermis, transferring to 0.25% protease (without EDTA) for about 15min, stopping digestion with high-sugar-DMEM culture solution containing 10% calf serum, centrifuging at 2000r/min for 5min, inoculating precipitate to 25cm²Adding about 4ml of high-sugar DMEM culture solution containing 10% calf serum into a plastic culture flask, taking care not to float the tissue, placing the flask at 37 deg.C and 50% CO₂In the incubator, 5ml of the culture solution was supplemented the next day. The primary cells can be passaged after growing over about 80 percent, the cells are digested by 0.25 percent pancreatin (without EDTA) during passage, the culture bottle is tapped when the cell bodies retract under an inverted microscope, then DMEM-high-sugar culture solution containing 10 percent calf serum is added to stop digestion, the cells on the wall of the bottle are blown by a suction tube lightly, and the cells are collected in a 10ml centrifuge tube. Centrifuging at 2000 r.min for 5min, discarding the supernatant, and performing centrifugation according to the ratio of 1: and 3, inoculating, subculturing and conventionally culturing.

Take P4 generationCells were cultured at 5X10⁴Inoculating each cell/ml in 6-well plate with sterile cover glass, making cell slide at 37 deg.C and 50% CO₂After 4h of incubation, the slide was removed and washed 3 times with PBS. Cells were fixed in 4% paraformaldehyde (pH7.2) for 30min and washed 3 times with PBS. Then, the cell identification is carried out according to the kit operation instruction by adopting an immunochemical staining S-ABC method, wherein the primary antibody is a vimentin rabbit anti-monoclonal antibody, the monoclonal antibody is diluted according to the proportion of 1 to 100, DAB color developing solution is prepared freshly, the color development is about 3min at room temperature, and the counterstaining and the mounting are carried out. And (4) observing and counting the positive cells under a visual field of 100 times of an optical microscope, wherein the percentage of the number of the positive cells is calculated to be 100% Vimentin expression positive, which indicates that pure fibroblasts are prepared by passage.

Culturing fibroblast until about 80% of fibroblast is fused, removing supernatant, washing with PBS for 3 times, replacing with serum-free low-sugar culture medium, and culturing for 24 hr. The supernatant was collected, centrifuged at 3000g for 10min, and cells and cell debris were removed. The centrifuged supernatant was collected and passed through filters of 450nm and 220nm in order to remove large-diameter vesicles. The filtered culture broth was collected, added to 100000MWCO Millipore ultrafiltration tubes, and concentrated and filtered by centrifugation at 3000g for 60 min. PBS was added to the ultrafiltration tube for washing and centrifuged again by the above-mentioned centrifugation method until the volume of the ultrafiltration tube was 100-. Collecting extracellular vesicles in the ultrafiltration tube, wherein the diameter of the extracellular vesicles is about 80-120nm, and the concentration of the extracellular vesicles is adjusted to 20mg/mL for later use.

Example 4 anti-aging test

The male Kunming mice are divided into six groups randomly, namely a normal control group, a D-galactose-induced oxidative damage model group, a polypeptide treatment group, an outer vesicle treatment group, a polypeptide combined outer vesicle treatment group and a positive control group, wherein the weight of the male Kunming mice is 20-25 g. Except for a normal control group, the mice of other groups are injected with 400mg/kg of D-galactose subcutaneously every day for 32 days, the mice of the control group are injected with the same amount of normal saline subcutaneously every day, the polypeptide treatment group is injected subcutaneously every day at a dose of 10mg/kg, the outer vesicle treatment group is injected subcutaneously every day at 10 mg/D, and the combination group is injected subcutaneously every day at a dose of 10mg/kg plus 10 mg/D; the positive control group is the subcutaneous injection/d of the dose of vitamin C10 mg/kg; the normal control group and the model group were injected subcutaneously with 10mg of distilled water and weighed 1 time every 3 days.

After injecting D-galactose and each group of reagents for 2h at 32D, taking the liver, crushing, preparing into 10% w/v liver tissue homogenate, centrifuging at 3000r/min, taking the supernatant, and measuring by using a measuring method (a TBA method, a DTNB method and a Folin-phenol method) for measuring SOD and GSH-Px activity and MDA content which are conventional in the field. The results are shown in Table 1.

TABLE 1 Effect of the activity of each component in the serum of each group of mice

Each group of	MDA(nmol/ml)	SOD(U/ml)	GSH-PX(U/0.1ml)
				Normal control group	14.6±1.1	522.1±34.2	620.3±34.5
Model set	23.1±1.5	461.3±29.7	430.1±34.7
				Polypeptide treatment group	17.0±0.8	495.1±22.0	583.1±26.3
Outer vesicle treatment group	19.1±1.7	489.7±19.5	553.9±30.1
				Polypeptide combined outer vesicle processing group	14.3±0.7	530.1±20.2	609.4±19.7
Positive control group	20.1±1.6	472.4±18.3	243.2±17.9

MDA is a lipid peroxide, and the measurement of the content of MDA can reflect the damage degree of cells. As can be seen from FIG. 1, compared with the control group, the MDA content in the liver of the mice in the oxidative damage model group is remarkably increased, and the SOD activity and the GSH-Px activity are remarkably reduced. Compared with the model group, the mouse liver MDA content of each experimental group is reduced, the GSH-Px activity is improved, and in addition, the SOD activities of the polypeptide group, the outer vesicle group and the combined treatment group are obviously improved. The RO-3 polypeptide and the outer vesicle are used as exogenous antioxidant substances, so that the activity of endogenous antioxidant enzymes of an organism can be improved, free radicals can be eliminated, and lipid peroxidation damage can be inhibited.

Example 5 anti-aging test

After 24h adherent culture of the human fibroblasts prepared in example 4, absorbing the culture solution, adding a small amount of PBS, respectively adding outer vesicles or polypeptide or a combination of the two to the cells for pretreatment for 2h, placing the cells under a UVB lamp tube for irradiation, placing the UVB lamp tube on a test tube rack with the height of 6cm, detecting the ultraviolet energy by an ultraviolet energy detector, wherein the irradiation dose is 100mj/cm²The experiment was divided into 4 groups, and the control group: no UVB irradiation and no pretreatment; UVB group:UVB irradiation, no pretreatment; outer vesicle addition pretreatment group: UVB irradiation, and treating the outer vesicle with 0.5 mu g/ml; polypeptide pretreatment group: UVB irradiation, 1 mu g/ml treatment of polypeptide, and combination of polypeptide and outer vesicle pretreatment group: UVB irradiation, and treatment of polypeptide 1. mu.g/ml.

Culturing for 72h after UVB irradiation, fixing cells by SA-beta-gal staining fixing solution, washing by PBS, adding prepared staining working solution, incubating overnight at 37 ℃, observing and counting the aged cells under a common optical microscope on day 2, and calculating the percentage of the aged cells. The results are shown in FIG. 1.

Compared with the control group, the SA-p-gal staining positive cells in the UVB group are higher than those in the blank control group and the treatment group, which indicates that the number of the aged cells is remarkably increased after the ultraviolet irradiation for 72 hours; after the treatment of the outer vesicles or the polypeptide, staining positive cells are obviously reduced, and particularly the pretreatment group of the polypeptide combined with the outer vesicles is as low as (3.5 +/-0.4)%, which shows that the number of aged cells can be reduced through the treatment.

The above embodiments are only used for explaining the technical solution of the present invention, and it should be understood by those skilled in the art that the scope of the present invention is not limited to the above embodiments.

Sequence listing

<110> Beijing Yunshenda Biotechnology development Co., Ltd

<120> an anti-aging cosmetic or medicine

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Thr Ser Gly Arg Asp Gly Arg Tyr Asn Cys Lys Ile Ser

1 5 10

Claims

1. An anti-wrinkle and anti-aging pharmaceutical composition, which comprises fibroblast outer vesicles and an antioxidant polypeptide, wherein the polypeptide RO-3 has a sequence shown in SEQ ID NO: 1 is shown.

2. An anti-wrinkle anti-aging cosmetic, which contains fibroblast outer vesicles and an antioxidant polypeptide, wherein the sequence of the polypeptide RO-3 is shown in SEQ ID NO: 1 is shown.

3. The pharmaceutical composition according to claim 1 or the cosmetic according to claim 2, characterized in that: the preparation method of the fibroblast outer vesicle comprises the following steps: culturing fibroblast till about 80% fusion, removing supernatant, washing with PBS for 3 times, replacing with serum-free low-sugar culture medium, and continuously culturing for 24 hr; collecting supernatant, centrifuging at 3000g for 10min, and removing cells and cell debris; collecting the centrifuged supernatant, and sequentially passing through 450nm and 220nm filters to remove vesicles with large diameters; collecting the filtered culture solution, adding into an ultrafiltration centrifugal tube, centrifuging at 3000g for 60min, concentrating and filtering; adding PBS into the ultrafiltration tube for cleaning, centrifuging again by the centrifugation method until the liquid in the ultrafiltration tube is 100-.

4. The application of the fibroblast outer vesicle and the antioxidant polypeptide in the preparation of anti-wrinkle and anti-aging pharmaceutical compositions is provided, wherein the polypeptide RO-3 sequence is shown as SEQ ID NO: 1 is shown in the specification; wherein the outer vesicle is prepared by the following method: culturing fibroblast till about 80% fusion, removing supernatant, washing with PBS for 3 times, replacing with serum-free low-sugar culture medium, and continuously culturing for 24 hr; collecting supernatant, centrifuging at 3000g for 10min, and removing cells and cell debris; collecting the centrifuged supernatant, and sequentially passing through 450nm and 220nm filters to remove vesicles with large diameters; collecting the filtered culture solution, adding into an ultrafiltration centrifugal tube, centrifuging at 3000g for 60min, concentrating and filtering; adding PBS into the ultrafiltration tube for cleaning, centrifuging again by the centrifugation method until the liquid in the ultrafiltration tube is 100-.

5. The use according to claim 4, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

6. Use according to claim 4, characterized in that further antioxidant active substances are added.