CN113599492B

CN113599492B - Anti-aging medicine or cosmetic and preparation method thereof

Info

Publication number: CN113599492B
Application number: CN202110936213.5A
Authority: CN
Inventors: 王京; 张然
Original assignee: Yangmei Biotechnology Beijing Co ltd
Current assignee: Yangmei Biotechnology (Beijing) Co.,Ltd.
Priority date: 2021-08-16
Filing date: 2021-08-16
Publication date: 2022-03-22
Anticipated expiration: 2041-08-16
Also published as: CN113599492A

Abstract

The invention relates to an anti-aging medicine or cosmetic and a preparation method thereof. According to the invention, the high-activity stem cell exosome is prepared by adding FGF (fibroblast growth factor) and high oxygen in stem cell culture, after the exosome is loaded with the active peptide with antioxidant activity, experiments prove that the exosome loaded with the polypeptide has the characteristic of promoting skin fibroblast anti-aging, and mouse experiments also prove that the exosome can improve the anti-aging performance of skin and has good market application value.

Description

Anti-aging medicine or cosmetic and preparation method thereof

Technical Field

The application relates to the field of biological medicine, in particular to an anti-aging medicine or cosmetic and a preparation method thereof.

Background

Aging is a process in which the functions of various tissues and organs of the body undergo degenerative changes with age. Aging can reduce the body's ability to maintain homeostasis in the face of environmental stress, thereby increasing the likelihood of the body becoming ill and dying. Aging is closely related to hypertension, type 2 diabetes, atherosclerosis, senile dementia and other diseases. Aging of the body is related to reduction of regenerative cells of tissues, deficiency of viscera, increase of free radicals in the body, body poisoning, eating arrhythmicity, etc., and is the result of combined action of a plurality of factors (environmental pollution, mental stress, heredity, etc.) in and out of the body.

Aging is an irreversible life process, and the most common mode of aging resistance is the use of anti-aging drugs. The synthetic drugs are substances with preventive, therapeutic and diagnostic effects prepared by chemical or biological methods, and are widely used in clinical practice. At present, most of the anti-aging drugs used clinically are synthetic drugs, for example, vitamin E can promote cell division and inhibit the generation of oxygen free radicals, a procaine preparation can prolong the cell life, amidopirone can delay the brain aging, and the like. Aspirin delays age-related decline in body function by combating oxidative stress, thereby prolonging nematode life; metformin is used as an adenosine activated protein kinase (AMPK) activator, can also improve cognitive impairment, and has a certain effect of delaying aging. The anti-aging effects of PAL-12, resveratrol analogs, etc. are favored recently.

Currently, active biological polypeptide has the advantages of safety, stability, easy water solubility, easy absorption, small molecular weight and the like, simultaneously has various biological activities of free radical removal, oxidation resistance, aging resistance and the like, can promote the proliferation of skin cells, can provide nutrition for skin, delay skin aging and promote skin wound repair, and is widely applied to skin cosmetics and anti-aging cosmetics. Nowadays, the proportion of anti-aging skin care products in the cosmetic market is also increasing year by year due to the increasing aging degree of society and the continuous pursuit of consumers for youthful beauty. Consumer demand for safety and effectiveness of anti-aging cosmetics has also prompted continued improvement and perfection in research and development techniques for anti-aging cosmetics. According to the survey of anti-aging products that are currently on the market, there are several international well-known brands that have introduced peptide anti-aging cosmetics, such as: yulan oil, elegant fragrance, lanocao, Cardamom, Irelaya, rhizoma Kaempferiae, the Ordinary, Dio, SK-II and other international first-line famous brands can be called as the hot tide of peptide beauty skin care products.

In addition, research on exosomes for skin anti-aging is also increasing. Exosomes (exosomes) are vesicle-like substances secreted by cells and having a diameter of 40-150 nm and a density range of 1.09-1.18 g/ml and a double-layered phospholipid membrane structure. Exosomes such as neuronal cells, mesenchymal stem cells, fibroblasts, endothelial cells, megakaryocytes, etc. have been extracted from various species and types of cell culture supernatants. It has been shown that depletion of mouse hypothalamic neural stem cells (htnscs) results in the symptoms of aging and a shortened life cycle of the mouse. The injection of the neural stem cell exosome into the ventricles of the aged mice can delay the aging speed of the brain and prolong the life of the mice. Research also shows that miRNA carried by exosome can partially regulate anti-aging effect of htNSC and play corresponding role after htNSC enters cerebrospinal fluid. In the aspect of skin aging resistance, studies by Kim et al in korea have found that exosomes derived from umbilical cord mesenchymal stem cells can easily penetrate the stratum corneum to reach the dermis, promote the generation of type i collagen and elastin in human skin tissues, and improve the skin texture. These studies indicate that exosomes have anti-aging application prospects. In the aspect of early detection of aging signs of organisms, exosomes also have clinical application value. Saliva contains many molecular substances and microorganisms, which can be used as effective biological indicators of local or systemic, and blood-derived molecules enter salivary glands through transcellular or cellular bypass to affect saliva components. Researchers at the university of okangshan speculate that miRNA of salivary exosomes may be a biomarker for aging. Research shows that miRNA screening and verification are carried out on saliva exosomes of young people and old people, and the expression of miR-24-3p is found to have obvious difference, so that miR-24-3p is considered to be a new aging-related biomarker. This result indicates that saliva exosome detection holds promise as a means to label aging.

Although the research on anti-aging mechanisms and anti-aging drugs is developed rapidly, the research is not deep enough, and the anti-aging effect of most drugs is not ideal. The clinical medicines directly used for treating the aging have a few varieties, and further research on aging molecular mechanisms and development of anti-aging medicines are needed.

Disclosure of Invention

The invention overcomes the defects of the prior art and provides an anti-aging cosmetic.

In one aspect of the present invention, an exosome derived from a high-activity bone marrow mesenchymal stem cell is provided.

In another aspect, the invention further provides a preparation method of the exosome, which specifically comprises the following steps:

mixing bone marrow with equal amount of PBS, slowly injecting into 1.073g/ml Percoll lymphocyte separation solution along tube wall with needle to obtain single cell suspension, centrifuging at 2000r/minAnd (3) 30 min. Gently sucking out the white turbid mesenchymal stem cell layer, putting the mesenchymal stem cell layer into another centrifuge tube, adding PBS, centrifuging at 1500r/min for 15min, and washing 3 times in the same way. PBS was discarded, and the cell suspension prepared in L-DMEM medium containing 10% FCS was counted on a counting plate at 1X10⁹The cell count/L was inoculated in L-DMEM medium containing 10000U/100ml each of penicillin and streptomycin and 10% FCS. After 48h of culture, adherent cells become fusiform when the first liquid change is observed, and the cells are subcultured in 25ml when the cell fusion reaches 85 percent⁶⁰Co irradiation dose 5Gy treated cell culture flask, placed in 5% CO₂Culturing in a carbon dioxide incubator at 37 ℃; and after 48h, changing the solution for the first time, sucking the suspension cells which are not attached to the wall, adding a fresh L-DMEM culture solution containing 10% FCS, and continuously incubating, wherein the solution is changed every 3-4 days. After 80% of adherent cells are fused, discarding the culture solution, washing the bottle wall with PBS, digesting with 0.25% pancreatin, observing and controlling for 2-3 min under the mirror, stopping digestion with serum-containing culture solution, repeatedly blowing the bottle wall to prepare single cell suspension, and performing single cell suspension treatment according to the proportion of 1X10⁹L, subculturing again, and harvesting cells when the third generation is reached;

resuspending bone marrow mesenchymal stem cells in serum-free MSC culture medium, inoculating to bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20ml of culture medium; when the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20ml of α -MEM basal medium supplemented with 5mg/L FGF was added to 1% O₂、3％CO₂Culturing for 3 days under the culture condition of (1), collecting culture supernatant, and extracting exosomes;

respectively placing the collected cell supernatants into 50ml centrifuge tubes, centrifuging at room temperature of 3500rpm for 25min, and removing cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding 5% of 5 × PEG 6000 reagent, and standing at 4 deg.C overnight; centrifuging the mixture of PEG 6000 cell supernatant standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the tube, discarding the supernatant, and turning the tube over on absorbent paper, and air drying for 15 min. Adding a proper amount of PBS according to the weight/volume ratio of 1g to 200mL for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with PBS according to the weight/volume ratio of 1g to 100mL to obtain the mesenchymal stem cell exosome.

The invention adds FGF and 1% O in stem cell culture₂、3％CO₂When the culture is carried out under the culture condition, the improvement of an exosome product can be effectively promoted, and the content of the prepared exosome protein is also obviously improved.

In another aspect of the invention, the inventor previously separated polypeptide components from salmon skin by collagen hydrolysis process and identified and separated 2 antioxidant peptides which are DYT-1 polypeptide and DYT-4 polypeptide respectively, have good scavenging ability to DPPH free radicals, hydroxyl free radicals and the like and good antioxidant property.

In yet another aspect of the invention, an exosome loaded with the polypeptide is provided.

Specifically, the preparation method of the polypeptide-loaded exosome comprises the steps of respectively adding 100 mu g of exosome and 20 mu l of polypeptide DMSO solution of 1mg/mL SEQ ID NO:1 into 1mL 50mM trehalose PBS solution; after mixing, adding the mixture into a 4mm electroporation cuvette, putting the cuvette into an electroporator, and reacting according to the following reaction conditions: voltage 120V, capacitance 155 μ F, discharge time 1ms, number of discharges: 2 times; after perforation, the suspension can be put into a cell culture box for incubation for 1 hour, then the suspension is ultracentrifuged twice at 100000g, each time is 80min, the supernatant is carefully discarded, and the suspension is resuspended and precipitated by PBS liquid to obtain the polypeptide-loaded exosome.

In another aspect of the present invention, there is provided an anti-aging cosmetic comprising exosomes loaded with polypeptides.

Further, the cosmetic is in the form of a gel, and in particular, the gel is prepared according to the following formula by adopting a preparation method which is conventional in the field: 0.65% of acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer (U-21), 0.05% of EDTA disodium, 3% of glycerol, 3% of butanediol, 0.25% of PhenenIP, 1% of exosome loaded with polypeptide, 4% of molecular sodium hyaluronate, and then adjusting the pH to 5.6 by using 10% potassium hydroxide solution, and the balance being water.

In some cases, the cosmetic may be those used for treatment or care, or somehow moisturize, improve, accelerate renewal, protect, prevent damage, or clean the skin.

A cosmetic can be used as cream, patch for external use, hydrogel patch, transdermal patch, ointment, gel, liquid, powder, lotion, serum, emulsion, oil, clay, moisturizer, foam, pack, mousse, aerosol, spray, cleanser, toner or shampoo.

In some cases, the cosmetic product may be in the form of an adhesive, a bandage, an exfoliating agent, a toothpaste, a moisturizer, a lotion, a primer, a lipstick, an anhydrous occlusive moisturizer, an antiperspirant, a deodorant, a personal cleansing product, an occlusive drug delivery patch, nail polish, a powder, a paper towel, a wipe, a conditioner, or a shaving cream.

In some cases, the cosmetic may comprise a skin conditioning agent (e.g., a humectant, exfoliant, emollient, or hydrate). In some cases, emollients are agents that prevent water loss and have a softening and soothing effect on the skin. In some embodiments, the emollient may comprise at least one of vegetable oil, mineral oil, shea butter, cocoa butter, petrolatum, fatty acids (animal oils including EMU, mink, and lanolin), triglycerides, benzoates, myristates, palmitates, stearates, glycolipids, phospholipids, squalene, glycerol, rosehip oil, andioba oil, grape seed oil, avocado oil, plum oil, Pracaxi oil, cyanobacterial oil, almond oil, Argan oil, caprylic/capric triglyceride, jojoba oil, Spectrastat G2, ceramides, and algae extract.

In some cases, the cosmetic additionally includes, but is not limited to, glycerin, squalene, sorbitol, hyaluronic acid derivatives, sodium hyaluronate crosspolymer.

Advantageous effects

According to the invention, the high-activity stem cell exosome is prepared by adding FGF (fibroblast growth factor) and high oxygen in stem cell culture, after the exosome is loaded with the active peptide with antioxidant activity, experiments prove that the exosome loaded with the polypeptide has the characteristic of promoting skin fibroblast anti-aging, and mouse experiments also prove that the exosome can improve the anti-aging performance of skin and has good market application value.

Drawings

FIG. 1 is a graph showing the results of the antioxidant activity test

FIG. 2 is a graph showing the results of cell proliferation

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 culture of high Activity Stem cells

Mixing 1ml bone marrow with equal amount of PBS, slowly injecting into 1.073g/ml Percoll lymphocyte separation solution along the tube wall with needle to obtain single cell suspension, and centrifuging at 2000r/min for 30 min. Gently sucking out the white turbid mesenchymal stem cell layer, putting the mesenchymal stem cell layer into another centrifuge tube, adding PBS, centrifuging at 1500r/min for 15min, and washing 3 times in the same way. PBS was discarded, and the cell suspension prepared in L-DMEM medium containing 10% FCS was counted on a counting plate at 1X10⁹The cell count/L was inoculated in L-DMEM medium containing 10000U/100ml each of penicillin and streptomycin and 10% FCS. After 48h of culture, adherent cells become fusiform when the first liquid change is observed, and the cells are subcultured in 25ml when the cell fusion reaches 85 percent⁶⁰Co-irradiated (irradiation dose 5Gy) treated cell culture flasks were placed in 5% CO₂And culturing in a carbon dioxide incubator at 37 ℃. And after 48h, changing the solution for the first time, sucking the suspension cells which are not attached to the wall, adding a fresh L-DMEM culture solution containing 10% FCS, and continuously incubating, wherein the solution is changed every 3-4 days. After 80% of adherent cells are fused, discarding the culture solution, washing the bottle wall with PBS, digesting with 0.25% pancreatin, observing and controlling for 2-3 min under the mirror, stopping digestion with serum-containing culture solution, repeatedly blowing the bottle wall to prepare single cell suspension, and performing single cell suspension treatment according to the proportion of 1X10⁹The cells were harvested by passaging/L again until the third passage.

Cell identification: (1) pre-treated coverslips were pre-applied to the bottom of the plate toPreparing cell climbing tablets. The 3 rd generation well-grown cells were taken, 1X10⁴The seed/ml was inoculated into 16-well plates. (2) After 2d of culture, the culture medium was removed when 70% of the cells had fused. (3) PBS (0.01M, pH7.4) washed 5minX 3. (4) Fixing with 10% formaldehyde solution for 10 min. (5) PBS rinse 5min × 3. (6) 3% hydrogen peroxide was incubated at room temperature for 10min to eliminate endogenous peroxidase. (7) PBS rinse 5min × 3. (8) Human CD29, CD44, CD34 and CD45 monoclonal antibodies were added, and PBS was added to the negative control group. (9) Incubate at 4 ℃ overnight. (10) The next day, the primary antibody was removed from the wells of the cell culture plate and washed 5minX3 with PBS. (11) Adding horseradish peroxidase to mark goat anti-rabbit secondary antibody. (12) Incubate at 4 ℃ for 30 min. (13) PBS wash 5min X3, add DAB color: and (4) dropwise adding DAB color developing agent diluted in a proper proportion, observing the color development condition under a microscope, and fully washing with tap water to stop the color development.

The morphology of the MSCs transmitted to the 3 rd generation and the primary cell sample are still in a long fusiform shape, the cells are stained by immunocytochemistry, the expression of CD34 and CD45 is negative, the expression of CD29 and CD44 is positive, the positive rate of CD29 is (97.8 +/-2.9)%, the positive rate of CD44 is (99.1 +/-3.1)%, the cell uniformity is high, and the prepared stem cells are better.

Example 2 preparation of Stem cell exosomes

The bone marrow stem cells identified in example 1 were resuspended in serum-free MSC medium and plated on a bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20ml of culture medium. When the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20ml of α -MEM basal medium supplemented with 5mg/L FGF was added to 1% O₂、3％CO₂Culturing for 3 days under the culture condition of (2), and collecting culture supernatant for exosome extraction.

After cell counting, the collected cell supernatants are respectively filled in 50ml centrifuge tubes, and the centrifuge tubes are centrifuged at 3500rpm at normal temperature for 25min to remove cells and cell debris. The centrifuged supernatant was collected, filtered through a 0.22 μm membrane to remove large particles and vesicles, and 5 × PEG 6000 reagent was added to the supernatant to a final concentration of 5%, and the mixture was allowed to stand overnight at 4 ℃. Centrifuging the mixture of PEG 6000 cell supernatant standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the tube,the supernatant of the centrifuge tube was discarded, and the centrifuge tube was inverted on absorbent paper and air-dried for 15 min. Adding a proper amount of PBS according to the weight/volume ratio of 1g to 200mL for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with PBS according to the weight/volume ratio of 1g to 100mL to obtain an exosome solution. The exosome is a small vesicle with a round shape and the diameter of the vesicle is about 50-110nm through the detection of a projection electron microscope. The total protein content of the extracted exosomes is determined according to the BCA kit instruction, and the total protein content of the exosomes is determined to be (916.9 +/-47.2) mu g/L multiplied by 10⁷And (4) cells. The expression of the marker in the exosome is detected by flow cytometry, wherein CD63 and CD44 are highly expressed, and the isolated mesenchymal stem cell exosome is shown.

Example 3 preparation of control Stem cell exosomes

The bone marrow stem cells identified in example 1 were resuspended in serum-free MSC medium and plated on a bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20ml of culture medium. When the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20ml of α -MEM basal medium was added in 3% CO₂Culturing for 3 days under the culture condition of (2), and collecting culture supernatant for exosome extraction.

After cell counting, the collected cell supernatants are respectively filled in 50ml centrifuge tubes, and the centrifuge tubes are centrifuged at 3500rpm at normal temperature for 25min to remove cells and cell debris. The centrifuged supernatant was collected, filtered through a 0.22 μm membrane to remove large particles and vesicles, and 5 × PEG 6000 reagent was added to the supernatant to a final concentration of 5%, and the mixture was allowed to stand overnight at 4 ℃. Centrifuging the mixture of PEG 6000 cell supernatant standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the tube, discarding the supernatant, and turning the tube over on absorbent paper, and air drying for 15 min. Adding a proper amount of PBS according to the weight/volume ratio of 1g to 200mL for resuspension, centrifuging again at 5000rpm for 20min, discarding the supernatant, and resuspending with PBS according to the weight/volume ratio of 1g to 100mL to obtain an exosome solution. The exosome is a small vesicle with a round shape and the diameter of the vesicle is about 50-110nm through the detection of a projection electron microscope. The total protein content of the extracted exosomes was determined according to the BCA kit instructions, and the total protein content in the exosomes was determined to be (803.4 + -53.7) μ g/L × 10⁷And (4) cells. Using a streamThe expression of the marker in the exosome is detected by the cytology, wherein CD63 and CD44 are highly expressed, and the isolated mesenchymal stem cell exosome is shown. From the results, it can be seen that the total protein content in exosomes is lower than that of example 2.

Example 4 preparation of exosomes loaded with polypeptide nanomaterials

100 mu g of the exosome of example 2 or example 3 and 20 mu l of 1mg/mL DMSO solution of the polypeptide of SEQ ID NO:1DYT-1 are respectively added into 1mL 50mM trehalose PBS solution; after mixing, the mixture was put into a 4mm (750. mu.l capacity) electroporation cuvette and placed in an electroporator under the following reaction conditions: voltage 120V, capacitance 155 μ F, discharge time 1ms, number of discharges: 2 times. After perforation, the suspension can be put into a cell culture box for incubation for 1 hour, then the suspension is ultracentrifuged twice at 100000g, each time is 80min, the supernatant is carefully discarded, the suspension is resuspended and precipitated by PBS liquid to obtain the polypeptide-loaded exosome, and the polypeptide-loaded exosome is stored for standby at 4 ℃.

Example 5 antioxidant Capacity test

Measuring polypeptide, exosome and polypeptide-loaded exosome sample solution with different concentrations, adding 0.3mL of 8mmol/L ferrous sulfate solution and 1mL of 20mmol/L salicylic acid solution into a centrifuge tube by taking Vc as a control, mixing, carrying out water bath by using warm water at 37 ℃, taking out the centrifuge tube for cooling, adding 0.45mL of distilled water, fixing the volume to 3mL, centrifuging for 10min by using the condition of rotating speed of 3000r/min, and measuring the absorbance of the supernatant of the solution at 510 nm. Each solution sample was prepared in 3 parallel groups, and the hydroxyl radical scavenging rate of the sample was calculated as follows. Hydroxyl radical clearance (%) ((a 0- (a 1-a 2))/a0) × 100 in the formula: a0 is the absorbance of the solution without sample; a1 is the absorbance of the solution added with sample, ferrous sulfate, salicylic acid-ethanol and hydrogen peroxide solution; a2 is the absorbance of the solution without hydrogen peroxide. The results are shown in FIG. 1.

The efficiency of the natural antioxidant in neutralizing and eliminating hydroxyl free radicals can show the activity of the natural antioxidant and the antioxidant function of the medicine. As can be seen from fig. 1, compared with the control Vc, both the DYT-1 polypeptide and exosome of the present invention have the ability to scavenge hydroxyl radicals, and although there is no strong clearance ability of Vc at the same concentration, the clearance ability of hydroxyl radicals is significantly improved when the polypeptide is loaded on exosome, and at a concentration of 250ug/mL, 95% of the clearance ability of hydroxyl radicals can be reached at most.

Example 6 human aging skin fibroblast assay

The skin specimen is primarily cultured according to the planting block method in the 'primary culture of human aging skin fibroblasts by the planting block method', and an aging human skin fibroblast line is established. The 4 th generation cells were selected for the experiment. Dividing the cell proliferation into 0, 50, 100, 200mg/L polypeptide groups, exosome groups and exosome groups loaded with polypeptides, taking a Vc group as a control, and detecting the cell proliferation condition by a tetramethylazozolium (MTT) colorimetric method after treating the cells for 72 hours; the results are shown in FIG. 2.

As can be seen from the results of FIG. 2, after the cells of each group cultured for 72h are added with the corresponding drugs, the cells all have the effect of promoting growth under the condition of 0mg/L without adding, and compared with a control group, the exosome loaded with the polypeptide has the better effect of promoting the proliferation of the senescent cells, and the absorbance of the exosome can reach 0.76 after the exosome is acted for 72h at the concentration of 200 mg/L.

The antioxidant enzyme activity of the cells cultured for 72h is measured on human aged skin fibroblasts without administration of a blank and on each group of cells treated at a drug concentration of 200 mg/L: collecting each group of cells, breaking by ultrasound, centrifuging, taking supernatant, and measuring SOD, CAT, GSH-Px activity according to the method in the kit instruction. The results are shown in Table 1.

TABLE 1 SOD, CAT, GSH-Px assay results

The results in table 1 show that the polypeptide group, the exosome group loaded with the polypeptide and the Vc control group can improve the activities of SOD, CAT and GSH-Px compared with the blank group, and compared with the control group, the exosome group loaded with the polypeptide can obviously improve the activities of SOD, CAT and GSH-Px of the corresponding components, wherein the SOD is (23.46 ± 2.87) KU/L, the CAT is (2.67 ± 0.95) KU/L, and the GSH-Px is (21.06 ± 3.04) KU/L, and the effects are better.

Example 7 preparation and validation of gel anti-aging cosmetic

Preparation of the gel was prepared according to the following formulation using preparation methods conventional in the art: 0.65% of acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer (U-21), 0.05% of EDTA disodium, 3% of glycerol, 3% of butanediol, 0.25% of PhenenIP, 1% of polypeptide-loaded exosomes, 4% of molecular sodium hyaluronate, and then adjusting the pH to 5.6 by using 10% potassium hydroxide solution, and the balance being water.

Firstly, the female mice are bred adaptively for 7d, and the experiment is started after the mice are ensured to adapt to the current environment. Dividing 3 female mice into 5 groups according to random number table grouping method, wherein 4 groups of mice are used as model group, and injecting D-galactose 1.0 g.kg subcutaneously^-1·d^-1A total injection of 30 d; the remaining group of mice served as a blank group and were injected daily with the same volume of physiological saline. The skin appearance of the model group and the blank group after 30d were compared, the model group had obvious skin laxity and fine wrinkles, and the blank group was opposite. 4 groups of skin aging model mice were divided into: a model group, a polypeptide group, an exosome group and a polypeptide-loaded exosome group. The hair on the back of the mouse is cut short by a pair of scissors and then shaved by a shaver for standby. Selecting a 4cm × 4cm area at the center of the back of the mouse, applying each group of gel on the surface of the selected skin area corresponding to each group of mice, applying 0.2g of gel to each mouse every day, cleaning the skin after 24h, applying again, continuously applying for 21d, and manually depilating for 4 times.

0.5g of the skin tissue at the application part is taken, rinsed with ice-bath precooled normal saline, wiped by filter paper, added with ice-bath precooled normal saline, and ground into homogenate with the concentration of 10 percent by a glass homogenizer. The resulting homogenate was centrifuged at 3000r/min at 0 ℃ for 10min to obtain the supernatant. According to the method shown in the HYP kit specification, an ultramicro microplate spectrophotometer is used for measuring OD values, and the HYP content of the mouse skin of each experimental group is calculated.

TABLE 2 mouse skin tissue HYP content results

Group of	Drug concentration (g)	HYP(μg/mg)
			Blank group	0	4.17±0.05
Polypeptide group	0.2g/d	3.99±0.08
			Exosome group	0.2g/d	3.80±0.11
Polypeptide-loaded exosome group	0.2g/d	4.21±0.23
			Model set	0	3.59±0.12

From the results in table 2, it can be seen that the skin tissue HYP content of the mice in the model group is significantly lower than that of the blank group, indicating that the model of skin aging of the mice is successful. The polypeptide group, the exosome group and the exosome group loaded with the polypeptide can have improved HYP effect relative to a model group, and particularly, the HYP of the exosome group loaded with the polypeptide reaches (4.21 +/-0.23) mu g/mg, so that the polypeptide has a better effect.

It should be understood that the above describes only some embodiments of the present invention and that various other changes and modifications may be affected therein by one of ordinary skill in the related art without departing from the scope or spirit of the invention.

Sequence listing

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<120> an anti-aging medicine or cosmetic and a method for preparing the same

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Claims

1. An anti-aging medicament, which is characterized in that: the medicament contains exosomes loaded with polypeptides and is in the form of a gel; the exosome is prepared by resuspending bone marrow mesenchymal stem cells in serum-free MSC culture medium, inoculating to bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20mL of culture medium; when the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20mL of α -MEM basal medium supplemented with 5mg/L FGF was added to 1% O₂、3％CO₂Culturing for 3 days under the culture condition of (1), collecting culture supernatant, and extracting exosomes; respectively placing the collected cell supernatants into 50mL centrifuge tubes, centrifuging at room temperature of 3500rpm for 25min, and removing cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding 5% of 5 × PEG 6000 reagent, and standing at 4 deg.C overnight; centrifuging the mixture of PEG 6000 cell supernatant standing overnight at 3000rpm for 30min to obtain white flocculent precipitate on the bottom and wall of the tubePrecipitating, discarding supernatant of the centrifuge tube, reversely buckling the centrifuge tube on absorbent paper, air-drying for 15min, adding a proper amount of PBS (phosphate buffer solution) according to the weight/volume ratio of 1g:200mL for resuspension, centrifuging at 5000rpm for 20min again, discarding supernatant, and resuspending with PBS according to 1g:100mL to obtain mesenchymal stem cell exosome;

the preparation method of the exosome loaded with the polypeptide comprises the steps of respectively adding 100 mu g of exosome and 20 mu L of 1mg/mL 1DYT-1 polypeptide DMSO solution in 1mL 50mM trehalose PBS solution; after mixing, adding the mixture into a 4mm electroporation cuvette, putting the cuvette into an electroporator, and reacting according to the following reaction conditions: voltage 120V, capacitance 155 μ F, discharge time 1ms, number of discharges: 2 times; after perforation, putting the suspension into a cell culture box to incubate for 1 hour, ultracentrifuging twice by adopting 100000g for 80min each time, carefully discarding the supernatant, and resuspending the precipitate with PBS (phosphate buffer solution) to obtain the exosome loaded with the polypeptide;

preparing the polypeptide-loaded exosome into gel by adopting a preparation method which is conventional in the field according to the following formula: 0.65% of acrylic acid (ester)/C10-30 alkanol acrylate cross-linked polymer, 0.05% of EDTA disodium, 3% of glycerol, 3% of butanediol, 0.25% of PHENONIP, 1% of exosome loaded with polypeptide and 4% of molecular sodium hyaluronate, and then adjusting the pH to 5.6 by using 10% potassium hydroxide solution, and the balance being water.

2. An anti-aging cosmetic characterized by: the cosmetic contains exosomes loaded with polypeptides, and is in the form of a gel; the exosome is prepared by resuspending bone marrow mesenchymal stem cells in serum-free MSC culture medium, inoculating to bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20mL of culture medium; when the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20mL of α -MEM basal medium supplemented with 5mg/L FGF was added to 1% O₂、3％CO₂Culturing for 3 days under the culture condition of (1), collecting culture supernatant, and extracting exosomes; respectively placing the collected cell supernatants into 50mL centrifuge tubes, centrifuging at room temperature of 3500rpm for 25min, and removing cells and cell debris; the supernatant after centrifugation is collected and the supernatant is collected,filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding 5% of 5 × PEG 6000 reagent, and standing at 4 deg.C overnight; centrifuging the PEG 6000 cell supernatant mixed solution which is kept stand overnight, centrifuging at 3000rpm for 30min to see that white flocculent precipitates are formed on the bottom and the wall of a centrifugal tube, discarding the supernatant of the centrifugal tube, reversely buckling the centrifugal tube on absorbent paper, air-drying for 15min, adding a proper amount of PBS (phosphate buffer solution) according to the weight/volume ratio of 1g to 200mL for resuspension, centrifuging at 5000rpm again for 20min, discarding the supernatant, and resuspending with PBS according to 1g to 100mL to obtain the mesenchymal dry cell exosome;

3. The cosmetic according to claim 2, wherein the mesenchymal stem cell is prepared by: adding equal amount of PBS into bone marrow, mixing, injecting into 1.073g/mL Percoll lymphocyte separation solution along the tube wall with a needle to obtain single cell suspension, centrifuging at 2000r/min for 30min, sucking out the white turbid mesenchymal stem cell layer, placing into another centrifuge tube, adding PBS, centrifuging at 1500r/min for 15min, and washing for 3 times; PBS was discarded, and the cell suspension prepared by L-DMEM medium containing 10% FCS was used for countingCount at 1 × 10⁹The cell number of the cells/L is inoculated in L-DMEM culture solution containing 10% FCS and 10000U/100mL of each of penicillin and streptomycin, adherent cells become fusiform when the first solution is changed after 48 hours of culture, the cells are subcultured in a 25mL cell culture bottle when the cell fusion reaches 85%, and the cell culture bottle is placed in a 5% CO culture bottle₂Culturing in a carbon dioxide incubator at 37 ℃; after 48h, changing the solution for the first time, sucking and removing the suspension cells which are not attached to the wall, adding a fresh L-DMEM culture solution containing 10% FCS, and continuously incubating, wherein the solution is changed every 3-4 days; after 80% of adherent cells are fused, discarding the culture solution, washing the bottle wall with PBS, digesting with 0.25% pancreatin, observing and controlling for 2-3 min under the mirror, stopping digestion with serum-containing culture solution, repeatedly blowing the bottle wall to prepare single cell suspension, and processing into single cell suspension according to the proportion of 1 × 10⁹and/L, subculturing again, and harvesting cells when the cells reach the third generation, namely the bone marrow mesenchymal stem cells.

4. The cosmetic of claim 2, further comprising a humectant, exfoliant, emollient.

5. A cosmetic product according to claim 4, characterized in that the emollient comprises at least one of vegetable oils.

6. The cosmetic of claim 4, wherein the emollient comprises one of mineral oil, shea butter, cocoa butter, petrolatum, fatty acids, triglycerides, benzoates, myristates, palmitates, stearates, glycolipids, phospholipids, squalene, glycerol, rosehip oil, pomace oil, grapeseed oil, avocado oil, plum oil, bacea oil, cyanobacterial oil, almond oil, argan oil, and jojoba oil.

7. The application of the exosome loaded with the polypeptide in preparing anti-aging drugs or cosmetics; wherein the preparation method of the exosome loading the polypeptide comprises the steps of respectively adding 100 mu g of exosome and 20 mu L of polypeptide DMSO solution of 1mg/mL SEQ ID NO:1 into 1mL 50mM trehalose PBS solution; after mixing, adding the mixture into a 4mm electroporation cuvette, putting the cuvette into an electroporator, and reacting according to the following reaction conditions: voltage 120V, capacitance 155 μ F, discharge time 1ms, number of discharges: 2 times; after perforation, putting the suspension into a cell culture box to incubate for 1 hour, ultracentrifuging twice by adopting 100000g for 80min each time, carefully discarding the supernatant, and resuspending the precipitate with PBS (phosphate buffer solution) to obtain the exosome loaded with the polypeptide;

the preparation method of the exosome comprises the following steps: resuspending bone marrow mesenchymal stem cells in serum-free MSC culture medium, inoculating to bottom area of 25cm²The culture dish (2) is seeded with 1X10 cells⁶20mL of culture medium; when the cells proliferated to 90%, the cell surface was washed with sterile PBS solution, repeated 2 times, and 20mL of α -MEM basal medium supplemented with 5mg/L FGF was added to 1% O₂、3％CO₂Culturing for 3 days under the culture condition of (1), collecting culture supernatant, and extracting exosomes; respectively placing the collected cell supernatants into 50mL centrifuge tubes, centrifuging at room temperature of 3500rpm for 25min, and removing cells and cell debris; collecting the centrifuged supernatant, filtering with 0.22 μm filter membrane to remove large particles and vesicles, adding 5% of 5 × PEG 6000 reagent, and standing at 4 deg.C overnight; centrifuging the PEG 6000 cell supernatant mixed solution which is kept stand overnight, centrifuging at 3000rpm for 30min to see that white flocculent precipitates are formed on the bottom and the wall of a centrifugal tube, discarding the centrifugal tube supernatant, reversely buckling the centrifugal tube on absorbent paper, air-drying for 15min, adding a proper amount of PBS (phosphate buffer solution) according to the weight/volume ratio of 1g to 200mL for resuspension, centrifuging at 5000rpm again for 20min, discarding the supernatant, and resuspending with PBS according to 1g to 100mL to obtain the mesenchymal dry cell exosome.