Application of umbilical cord stem cells in preparation of anti-aging medicines and related beauty products

Abstract

The invention provides application of umbilical cord stem cells in preparation of anti-aging drugs and related beauty products. More specifically, after the exosome prepared from the umbilical cord stem cells and the anti-aging peptide screened and identified by the invention are used together or independently, the proliferation of fibroblasts can be effectively promoted, and the aging characteristic of the cells can be reduced; the skin wound healing, the skin collagen generation and the water content increase are effectively promoted, and the application prospect is good.

Description

Technical Field

[0001] The present application relates to the biological field, and specifically to the application of umbilical cord stem cells in the preparation of anti-aging drugs and related beauty products. Background Art

[0002] Skin is the most obvious indicator of age. When the body ages, the skin also ages. Skin aging can be manifested externally as dry and rough epidermis, sagging skin, increased and deepened wrinkles, lack of elasticity, skin pigmentation and loss, etc.; internally, its fundamental mechanism is the damage and aging of skin fibroblasts. Skin aging is the external manifestation of changes in tissue structure. The epidermis, dermis and skin appendages of the skin will change accordingly with the increase of body age, but the most characteristic change is the change in the composition of the dermis. The internal cause of the change in the composition of the dermis is the aging of fibroblasts. Fibroblasts are important cell components of the dermis of the skin, responsible for synthesizing structural components in the dermis, including collagen fibers, elastic fibers, reticular fibers and extracellular matrix. Therefore, delaying or inhibiting the aging of fibroblasts is the key to treating and preventing skin aging.

[0003] In recent years, people's demand for facial beauty and anti-aging has been increasing, and various methods for treating facial skin aging have emerged one after another. The facial rejuvenation technologies currently used in clinical practice mainly include: face lifting surgery, hyaluronic acid filling, botulinum toxin injection, chemical peeling and physical grinding, which can achieve certain effects in the treatment of facial skin rejuvenation. However, these methods only achieve the effect of beautifying the appearance of the skin, and cannot truly achieve the effect of anti-aging from a physiological perspective. With the widespread development of stem cell research, adipose-derived stem cells (ADSCs) have become a very effective anti-aging material. However, there are technical barriers to the use of intact stem cells for anti-aging applications, and they are not easy to store and inconvenient to use. Therefore, the development of suitable stem cell anti-aging ingredients is an important research direction.

[0004] In addition, the use of peptides for skin anti-aging has become an important research direction, and many peptides have been used for anti-aging. For example, L-carnosine is composed of β-alanine and histidine. It is added to skin beauty and anti-aging cosmetics mainly to regulate the pH of the skin, remove excessive oxygen free radicals and their metabolites, and can be used to complex heavy metal ions. Adding L-carnosine to beauty cosmetics can effectively delay skin aging. At the same time, it can also regulate the growth of human fibroblasts and repair aging human cells. It is a good natural anti-aging and antioxidant for the skin. In particular, it has a significant inhibitory effect on lipid peroxidation caused by oxygen free radicals and metal ions. The imidazole structure contained in its molecular structure is particularly effective in integrating copper ions and iron ions, giving it a certain skin whitening effect.

[0005] In addition, glutathione, which has been used earlier in skin beauty and anti-aging cosmetics, is a polypeptide containing active hydrophobic groups and is composed of glutamic acid, cysteine ​​and glycine through peptide bond condensation. It is the best broad-spectrum small molecule antioxidant discovered so far. It can effectively remove excessive free radicals and peroxides produced in the metabolism of skin tissues, and can defend against lipid peroxidation in mitochondria in cells, etc., to protect skin tissues and cells. It has the effect of delaying skin aging. In addition, adding glutathione to skin beauty and anti-aging cosmetics can not only effectively inhibit the production of dopa pigment, but also prevent the formation and aggravation of lipofuscin-age spots, thereby preventing or alleviating skin aging, reducing pigmentation and improving the skin's antioxidant capacity. It has the effects of beauty, beauty, skin care, and anti-allergy, and is suitable for various types of skin, especially for people with sensitive skin. Some small molecule functional peptides synthesized by chemical methods, namely beauty peptides, such as palmitoyl pentapeptide-3, acetyl hexapeptide-3, acetyl tetrapeptide-5, palmitoyl oligopeptide (palmitoyl tripeptide-1), palmitoyl tetrapeptide-3 and carnosine, have been adopted by many famous foreign cosmetic brands and widely used in skin beauty and anti-aging cosmetics, and are called beauty peptides. This type of beauty peptide is actually also a type of active biological peptide, which is composed of a certain sequence of amino acids connected by amide bonds. Usually, two amino acids connected by amide bonds are called dipeptides, and three amino acids are called tripeptides. Usually, dipeptides, tripeptides, and pentapeptides help promote the accelerated production of collagen in skin tissue, tetrapeptides help the skin to resist inflammation and oxidation, and hexapeptides help prevent the release of neurotransmitters in the facial skin. Similar to the effect of botulinum toxin. Peptides show their broad application prospects in skin beauty and anti-aging cosmetics, which is mainly closely related to their mechanism of action. The mechanism of action of these active biological peptides is mainly manifested in antioxidant function and free radical scavenging effect. Studies have shown that active peptides can not only effectively scavenge cationic free radicals such as DPPH, but also effectively scavenge superoxide anion free radicals, hydroxyl free radical activity, lipid free radicals, etc. The antioxidant mechanism is likely to be related to the fact that during the enzymatic hydrolysis process of these active biological peptides, the antioxidant amino acid residues originally embedded in the molecules are exposed, small molecule peptides and free amino acids with antioxidant activity are released, and the side chain groups of amino acids that can participate in the reaction are exposed, which helps to insert into the lipid interior, thereby making it easier for the antioxidant amino acid residues to exert their antioxidant effects.

[0006] However, there are not enough types of highly effective anti-aging peptides at present, especially peptides with independent intellectual property rights with good efficacy, which need further development, especially the combination with stem cell extracts for anti-aging applications, which need further development. Summary of the invention

[0007] The present invention provides an application of umbilical cord stem cells in the preparation of anti-aging medicines and related beauty products.

[0008] More specifically, the present invention provides exosomes from umbilical cord stem cells for skin anti-aging.

[0009] More specifically, the present invention provides a pharmaceutical composition for anti-aging of the skin, comprising exosomes from umbilical cord mesenchymal cells.

[0010] More specifically, the present invention provides a cosmetic for skin anti-aging, comprising exosomes from umbilical cord mesenchymal cells.

[0011] Specifically, the present invention also provides an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0012] The anti-aging peptide LZ-3-2 of the present invention can also be modified or conservatively substituted, but still maintain the activity of the polypeptide.

[0013] The modification or substitution is specifically in another embodiment, and also includes variants of these sequences having at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, at least 99% or at least 99.5% sequence identity with the amino acid sequence sequence shown in the corresponding template sequence in SEQ ID NO.1. These variants include truncated forms of the sequence shown in SEQ ID NO.1, such as N- or C-terminal truncations, preferably C-terminal truncations, and the lengths of these truncations are generally 1-10 amino acids, preferably 1-5 amino acids. N-terminal truncation is generally not preferred. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids that are similar to each other, and / or have "polar" or "non-polar" side chains. The substitution of one amino acid for another amino acid of the same type can generally be considered a "homologous" substitution.

[0014] Furthermore, the pharmaceutical composition of the present invention can be an oral preparation, or a dressing or a tablet.

[0015] More specifically, in addition to active or therapeutic ingredients, tablets may contain many inert materials known as excipients. If necessary, pharmaceutical compositions as described herein may include one or more pharmaceutically acceptable excipients. The term "excipient" herein means any substance, which is not a therapeutic agent itself, which can be used as a carrier or vehicle for delivering a therapeutic agent to a subject or with a therapeutic agent (e.g., to produce a pharmaceutical composition) to improve its processing or storage properties or to allow or promote the formation of a dosage unit of a composition. As an illustration and not limitation, excipients include adhesives, disintegrants, flavor enhancers, solvents, thickeners or gelling agents (and any neutralizers, if necessary), penetration enhancers, solubilizers, wetting agents, antioxidants, lubricants, emollients, emulsifiers, surfactants, additions to mask or offset unpleasant odors, spices or tastes, and additions to improve the appearance or texture of a composition. Any such excipient can be used in any dosage form according to the present disclosure. The above types of excipients are not meant to be exhaustive, but merely illustrative, as one of ordinary skill in the art will recognize that other types and combinations of excipients may be used to achieve the desired goals of release and stability of the eltrombopag tablet composition.

[0016] Excipients can be classified according to the role they play in the final tablet. The primary composition includes fillers / diluents, binders, lubricants and disintegrants. Typically, excipients are added to the formulation to impart good flow and compression properties to the compressed material. These properties are imparted to these excipients by pre-treatment steps, such as wet granulation, spray drying spheronization or crystallization, etc.

[0017] Disintegrants are usually included to ensure that the tablet has an acceptable disintegration rate. In another aspect of the present invention, disintegrants are selected from starch, cellulose, gum, cross-linked polymers and effervescent agents, such as corn starch, potato starch, pregelatinized starch, modified corn starch, cross-linked sodium carboxymethylcellulose, crospovidone, sodium starch glycolate, Veegum HV, methylcellulose, microcrystalline cellulose, cellulose, modified cellulose gum, agar, bentonite, montmorillonite clay, natural sponge, cation exchange resin, ion exchange resin (such as potassium polyadenosine), alginic acid and alginates, guar gum, citrus pulp, carboxymethyl cellulose and its salts such as sodium lauryl sulfate, magnesium aluminum silicate, hydrated aluminum silicate, sodium bicarbonate and acidulant such as tartaric acid or citric acid mixture. In one embodiment of the present invention, the disintegrant is a combination of sodium starch glycolate and cross-linked polyvidone.

[0018] Binder is the reagent that gives powder material cohesive property.In another aspect of the present invention, binder is selected from starch (for example paste, pregelatinization, mucus), gelatin, sugar (for example sucrose, glucose, dextrose, molasses, lactose, dextrin, xylitol, sorbitol), polymethacrylate, natural and synthetic gum (for example,, arabic gum, alginic acid and its salt such as sodium alginate, tragacanth gum, Irish moss extract, panva gum, India gum, guar gum, zein), cellulose derivative [such as carboxymethyl cellulose and its salt, methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC) and ethyl cellulose (EC)], polyvinyl pyrrolidone, magnesium silicate, larch arabinogalactan, polyethylene glycol, wax, water, alcohol, magnesium aluminum silicate and bentonite.In one embodiment of the present invention, binder comprises polyvinyl pyrrolidone (PVP-polyvidone).

[0019] Filler / diluent is added to increase the bulk weight of the blend, thereby obtaining the actual size for compression. In another aspect of the present invention, filler / diluent is selected from erythritol, isomalt, maltitol, xylitol, microcrystalline cellulose, powdered cellulose, pregelatinized starch, starch, lactitol, mannitol, sorbitol, maltodextrin. In one embodiment of the present invention, filler / diluent is a combination of microcrystalline cellulose and mannitol.

[0020] Mannitol is widely used in pharmaceutical preparations and foods. In pharmaceutical preparations, it is mainly used as a diluent in tablet formulations (10-90% w / w), where it is of particular value because it is not hygroscopic and can therefore be used with active ingredients that are sensitive to moisture. Granules containing mannitol have the advantage of being easy to dry.

[0021] Lubricants are usually added to prevent the tabletting material from sticking to the punch, minimize friction during tablet compression, and allow compressed tablets to be taken out from the die. Such lubricants are usually included in the final tablet mixture in an amount usually less than 1% by weight. In another aspect of the present invention, the lubricant is selected from talc, stearates (e.g., magnesium stearate, calcium stearate, zinc stearate, palmitic acid stearate), stearic acid, hydrogenated vegetable oil, glyceryl behenate, polyethylene glycol, ethylene oxide polymers (e.g., carbowax), liquid paraffin, sodium lauryl sulfate, magnesium lauryl sulfate, sodium oleate, sodium stearyl fumarate, DL-leucine and silicon dioxide derivatives (e.g., colloidal silicon dioxide, colloidal silicon dioxide, pyrogenic silicon dioxide and hydrated sodium aluminosilicate). In one embodiment of the present invention, the lubricant comprises magnesium stearate.

[0022] The solvent used for granulation and coating can be aqueous or non-aqueous solvent. Suitable non-aqueous solvents include but are not limited to isopropanol, ethanol, dichloromethane, acetone, etc. Aqueous solvents include water.

[0023] The compressed tablets are further film-coated by non-aqueous coating or aqueous coating or by hydroalcoholic coating. The coating composition contains film-forming substances such as hydroxypropyl methylcellulose (hydroxypropyl methylcellulose), hydroxypropyl cellulose, methylcellulose, polyvinyl alcohol, etc.; solvent, colloidal silicon dioxide, and optional other excipients such as plasticizers, lubricants and colorants.

[0024] Specifically, the present invention provides a pharmaceutical composition for skin anti-aging, which comprises exosomes from umbilical cord interstitial cells and an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0025] More specifically, the present invention provides a cosmetic for skin anti-aging, which comprises exosomes from umbilical cord interstitial cells and an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, and the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

[0026] Further, cosmetics of the present invention can be skin lotion. The mixture of water, oil and other ingredients of the skin lotion is prepared to provide uniform mixture, for example emulsion. Other ingredients include ingredients that can be described by their functions in the emulsion, for example diluent, thickener, humectant, preservative, neutralizer, emulsifier, co-emulsifier, emollient, sealant, fragrance and colorant. Some ingredients may have more functions in skin care lotion. The topical composition can be prepared by any suitable method for preparing the topical composition. The topical composition can be a topical emulsion prepared completely for reducing gaseous pollutants and being exposed to the patient's skin.

[0027] Cationic surfactants may also be added to the cosmetics, and specific examples include behentrimethylammonium chloride; N,N,N-trimethylbehenyl-1-ammonium chloride; behenyltrimethylammonium chloride; benzalkonium chloride; N-alkyl-N-benzyl-N,N-dimethylammonium chloride; BZK; bKC; BAC; benzethonium chloride; N-benzyl-N,N-dimethyl-2-{2-[4-(2,4,4-trimethylpentane-2-yl)phenoxy]ethoxy}ethylammonium chloride; benzyldimethyl (2-{2-[4 -(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}ethyl)azonium chloride; benzododecanium bromide; benzyl-dodecyl-dimethylammonium bromide; bronidox; 5-bromo-5-nitro-1,3-dioxane; carbopentylamine bromide; (1-ethoxy-1-oxohexadecan-2-yl)-trimethylnitrogen bromide; carboethoxypentadecanium bromide; [1-(ethoxycarbonyl)pentadecanyl]trimethylammonium bromide; cimetropium chloride; benzylhexadecyldimethylammonium chloride; benzyldimethylhexadecane ammonium chloride; 16-BAC; benzyldimethyl-n-hexadecyl ammonium chloride; hexadecyldimethylbenzyl ammonium chloride; cetrimonium bromide; hexadecyl-trimethyl-ammonium bromide; cetrimonium chloride; hexadecyl-trimethylammonium chloride; cetylpyridinium chloride; 1-hexadecylpyridinium chloride; acetothamide CPC; exadecylpyridinium chloride; didecyldimethylammonium chloride; didecyl-dimethylammonium chloride; DDAC; dimethyl didecyl ammonium chloride; 1-decyl ammonium; didecyldimethylammonium chloride; di Decyldimethylammonium chloride; Quaternium-12; Dimethyldioctadecylammonium bromide; Dimethyldioctadecylammonium bromide; Distearyldimethylammonium bromide; Dimethyldioctadecylammonium chloride; N,N-dimethyl-N-octadecyloctadecyl-1-ammonium chloride; Dimethyldioctadecylammonium chloride; Distearyldimethylammonium chloride; Aliquot 207; DDAC; Distearyldimethylammonium chloride; DSDMAC; Domiphenium bromide; Laurylmethylglucoseethyl-10-hydroxypropyldimonium chloride; Glucquat 125; Octenidine dihydrochloride; N-octyl-1-[10-(4-octyliminopyridin-1-yl)decyl]pyridin-4-imine dihydrochloride; N,N'-(Decan-1,10-diyldipyridin-1-yl-4-ylidene)dioctyl-1-amine dihydrochloride; N,N'-(Decan-1,10-diyldi-1(4H)-pyridin-4-ylidene)bis(octylammonium) dichloride; Ola Fluorine; {3-[octadecyl(2-hydroxyethyl)amino]propyl}bis(2-hydroxyethyl)amine dihydrogen fluoride; Amine fluoride 297; N-oleyl-1,3-propanediamine; N-[(9Z)-9-octadecene-1-yl]-1,3-propanediamine; Stearoyl chloride; Benzyl dimethyl octadecyl ammonium chloride; Dimethyl benzyl octadecyl ammonium chloride; Benzyl dimethyl octadecyl ammonium chloride; Benzyl dimethyl stearyl ammonium chloride;Benzylstearyldimethylammonium chloride; N,N-dimethyl-N-octadecylbenzyl ammonium chloride; thonzoniumbromide; n-{2-[(4-methoxybenzyl)(pyrimidin-2-yl)amino]ethyl}-N,N-dimethylhexadecane-1-ammonium bromide; or a mixture thereof. ;

[0028] Beneficial Effects

[0029] The present invention provides an application of umbilical cord stem cells in the preparation of anti-aging drugs and related beauty products. More specifically, it provides that exosomes prepared from umbilical cord stem cells can effectively promote the proliferation of fibroblasts, reduce the aging characteristics of cells, effectively promote the healing of skin wounds, and promote the production of skin collagen and the increase of water content when used together with the anti-aging peptides screened and identified by the present invention or alone, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The percentage of positive cells in each group to the total number of cells DETAILED DESCRIPTION

[0031] For the purpose of illustration, the principles of the present invention are described by reference to various exemplary embodiments of the present invention. Although certain embodiments of the present invention are specifically described herein, it will be readily appreciated by those of ordinary skill in the art that the same principles are equally applicable to and can be adopted in other devices and methods. Before explaining the embodiments disclosed in the present invention in detail, it should be understood that the application of the present invention is not limited to the details of any particular embodiment shown. The terms used herein are for the purpose of description rather than limitation. In addition, although specific methods are described with reference to specific steps presented in a specific order herein, in many cases, these steps can be performed in any order that can be understood by those skilled in the art, and the method is not limited to the specific arrangement of the steps disclosed herein.

[0032] Example 1 Preparation of stem cell extract

[0033] Rat umbilical cord mesenchymal stem cells were purchased from Shanghai Huzhen Industrial Co., Ltd. with the item number HZR-5302. After thawing the stem cell cryopreservation tube, centrifuge at 1000rpm for 3 minutes and discard the supernatant. Add 5ml PBS to resuspend the cells, centrifuge at 900-1000rpm for 3 minutes, resuspend with fresh complete medium and inoculate into a new culture bottle. The complete medium is 89% H-DMEM + 10% FBS + 1% double antibody. When the cells cover about 80% of the bottom of the culture bottle, digest with trypsin to suspend the adherent cells and then centrifuge, and pass them at a ratio of 1:3. Change the liquid once every 3 days, and collect the supernatant of the culture medium while passing and changing the liquid. Store the supernatant in a -20℃ refrigerator. After collecting a sufficient amount, thaw the supernatant and filter with a 0.22μm filter to remove impurities. Pour the supernatant into a centrifuge tube, balance it with a balance, and centrifuge it in a sub-ultraspeed low-temperature centrifuge for 75 minutes (100,000 g, 4°C). Pour away the supernatant, wash the bottom and side walls of the centrifuge tube with 1 ml of PBS, resuspend the precipitate, detect the protein concentration with the BCA method, adjust the concentration to 5 mg / mL, and store it in a -80°C refrigerator.

[0034] Exosome electron microscopy detection: At room temperature, add 10μl of the obtained precipitate to the sample-loading copper grid. After 60s, use filter paper to absorb the excess liquid around the sample-loading copper grid, and then drop 15μL of 2% phosphotungstic acid solution onto the sample-loading copper grid. After 60s, absorb and dry, place the sample-loading copper grid under an incandescent lamp and bake for 10min before taking it out. Place it under a transmission electron microscope and adjust the working voltage to 120kV. It is observed that the morphology of the exosomes is disc-shaped vesicles with a diameter range of 55 to 110nm.

[0035] In addition, the precipitate obtained by centrifugation was lysed by adding 100 μL WB-IP cell lysate, and the protein concentration was measured by the BCA method. Then, 10 μL 5×SDS loading buffer was added to the lysate, heated and boiled for 10 minutes, and then loaded on the gel, and then separated by SDS-polyacrylamide gel electrophoresis and electrotransferred to the nitrocellulose membrane. At room temperature, the blocking solution (5% skim milk) was used for 50 minutes, and then eluted with 1×TBST buffer, and then CD63, CD81, Tsgl01 and Calnexin were added overnight, and eluted again with 1×TBST buffer. The secondary antibody labeled with horseradish peroxidase was added to the loading well, incubated for 120 minutes, and developed with a luminescent substrate. The results showed that the exosome marker proteins CD63, CD81 and Tsgl01 were all expressed, and Calnexin was not expressed, indicating that the precipitate mentioned in this experiment was exosomes.

[0036] Example 2 Screening and identification of anti-aging peptides

[0037] 100g of Ganoderma lucidum spore powder was placed in a 1L supercritical extraction kettle. The temperature and pressure of the extraction kettle and the separation kettle were set to 35MPa, 40℃ and 6MPa, 50℃, respectively. They were run for 1h, and then the 1h running sample was kept at high pressure for 12h. The spore powder after the wall was broken was obtained after the critical gas was quickly released. Deionized water was added according to the optimized 1:18 solid-liquid ratio, and the mixture was stirred evenly to make the powder and water fully contact and mix. 1mol / L sodium hydroxide solution was added dropwise, and the pH was adjusted to the optimal extraction pH of 10.5. Then, the mixture was placed in a water bath at a certain temperature for 2h, and the supernatant was filtered out. The pH value was adjusted to the isoelectric point with 1mol / L hydrochloric acid, and the mixture was kept at room temperature for a period of time. The supernatant was removed by centrifugation at 8000r / min for 20min to obtain the protein. The protein was dissolved with a small amount of deionized water and transferred to a plate, and the crude protein was obtained by freeze drying. Take 1g of crude protein, adjust the material-liquid ratio to 1:50 with deionized water, add 1% alkaline protease and perform enzymolysis for 12h at 50 degrees Celsius pH 8.0, after hydrolysis, inactivate in boiling water bath for 10min to obtain enzymolysis polypeptide solution, and obtain polypeptide powder after freeze-drying. The polypeptide powder sample is subjected to DEAE-32 ion exchange chromatography column to obtain five main peaks of absorption, and the third main peak is identified by DPPH free radical scavenging method to have the best antioxidant property and a clearance rate of 86.5%, and the third main peak is further separated by Superdex 30 column to obtain three components, LZ-3-1, LZ-3-2, and LZ-3-3. The antioxidant property of LZ-3-2 component is the strongest and the clearance rate is 90.4% determined by ABTS free radical scavenging method, and the component is further detected by mass spectrometry using LC-MS / MS method, and the result is compared with the uniprot database to determine the amino acid sequence of the polypeptide as shown in SEQ ID NO: 1.

[0038] The LZ-3-2 polypeptide was commissioned to be synthesized by Keti Biotechnology with a purity of 98.5% and the concentration was adjusted to 2 mg / mL for later use.

[0039] Example 3 Activity Identification of Peptides and Stem Cell Extracts

[0040] After activation and culture, HSF human skin fibroblasts (Cat. No.: IM-H032 Source: IMMOCELL) were inoculated into T25 culture flasks and cultured in an incubator at 37°C and 5% CO2. The culture medium used was: fibroblast basal culture medium (DMEM / F12+10% FBS+1% p / s+0.005mg / ml insulin

[0041] +5ng / ml bfGF+1ug / ml hydrocortisone+7.5mM L-Gln). When most of the fibroblasts were observed to be attached to the wall and when the fibroblasts reached 70-80% fusion, they were digested with 0.25% trypsin (containing 0.01% EDTA) and passaged at a ratio of 1:3.

[0042] The exosomes prepared in Example 1 and the LZ-3-2 polypeptide prepared in Example 2 were separately or jointly co-cultured with fibroblasts through a Transwell chamber with a micropore diameter of 0.4 μm to establish non-contact cell co-culture.

[0043] The experiments were grouped as follows:

[0044] Experimental group 1: low concentration exosomes prepared in Example 1 (final concentration of 50 μg / mL) + fibroblasts;

[0045] Experimental group 2: high concentration exosomes prepared in Example 1 (final concentration of 100 μg / mL) + fibroblasts;

[0046] Experimental group 3: low concentration LZ-3-2 polypeptide prepared in Example 2 (final concentration of 10 μg / mL) + fibroblasts;

[0047] Experimental group 4: high concentration LZ-3-2 polypeptide prepared in Example 2 (final concentration of 50 μg / mL) + fibroblasts;

[0048] Experimental group 5: high concentration exosomes prepared in Example 1 (final concentration of 100 μg / mL) + high concentration LZ-3-2 polypeptide prepared in Example 2 (final concentration of 50 μg / mL) + fibroblasts;

[0049] Blank control group: only fibroblasts; the upper layer of the Transwell chamber is blank culture medium.

[0050] Positive control group: vitamin C (final concentration of 10 μg / mL) + fibroblasts;

[0051] In each group, fibroblasts were inoculated in the lower layer of the Transwell chamber, and the cell suspension concentration was 2×10 5 / mL.

[0052] After 3 days of cell culture, the activity of fibroblasts in each group after co-culture was detected using a CCK-8 kit, and the absorbance of each well at a wavelength of 450 nm was measured using an enzyme marker. The results are shown in Table 1.

[0053] Table 1 CCK-8 results

[0054] Group OD results Experimental Group 1 1.124±0.012# Experimental Group 2 1.238±0.031# Experimental Group 3 1.379±0.029# Experimental Group 4 1.548±0.041# Experimental Group 5 1.984±0.047# Blank control group 0.835±0.021 Positive control group 1.214±0.019#

[0055] The results showed that the OD values ​​of experimental groups 1-5 and the positive control group were higher than those of the blank control group, and both were statistically significant (P < 0.05). It can also be seen from the results in Table 1 that the polypeptide of the present invention has a dose-dependent effect on promoting the proliferation of fibroblasts, which is better than the positive control group with the same concentration. At the same time, the combined use of polypeptides and exosomes has a synergistic effect on promoting cell proliferation.

[0056] Subsequently, cells from each group were taken and stained with SA-β-Gal using a β-galactosidase staining kit. The total number of cells and the number of β-galactosidase-positive cells were counted, and the percentage of positive cells in the total number of cells was calculated to understand the senescence of the cells. The results are as follows: Figure 1 shown.

[0057] from Figure 1 The results show that the percentage of positive cells in experimental groups 1-5 and the positive control group was lower than that in the blank control group (83.45±3.12)%, and both were statistically significant (P<0.01). Figure 1 The results also show that the polypeptide of the present invention has a dose-dependent effect of reducing the aging state of cells, which is better than the positive control group with the same concentration. At the same time, the combination of polypeptide and exosomes has a synergistic effect of reducing cell aging, and the proportion of positive staining is only (10.32±1.83)%.

[0058] Example 4 Animal experiments with polypeptides and stem cell extracts

[0059] Preparation of subacute aging model: After the mice were adapted for 1 week, the model was prepared according to the conventional subacute aging model preparation method in the art, specifically, 15% D-galactose 1000 mg / (kg·d) was subcutaneously injected at the back of the neck for 8 consecutive weeks to obtain mice identified as aging models. The blank control group mice were given an equal amount of physiological saline subcutaneously injected at the back of the neck every day.

[0060] The back skin of the mice in the model group and the blank control group was depilated, the back skin was lifted, and the skin was clamped on both sides with a circular magnet with a diameter of 1.2 cm for 12 hours and then released for 12 hours, which was one cycle. After three ischemia-reperfusion injuries, obvious ulcers were formed after waiting for another week, thus preparing pressure ulcer injuries.

[0061] After the pressure ulcer was successfully established in the blank control group and the model group, the wound was debrided locally. The wounds of the blank untreated group (Control) were topically smeared with 100 μL sterilized PBS, and the treatment frequency was once a day; the wounds of the model untreated group (i.e., aged mice) were topically smeared with sterilized PBS; the wounds of the model exosome treatment group (i.e., aged mice) were topically smeared with 100 μL of 1×10 10 In order to ensure the effectiveness of exosome treatment, the treatment frequency was once a day. In the model peptide treatment group, 100 μL of LZ-3-2 peptide with a mass concentration of 50 mg / mL was locally dripped onto the wound surface of aged mice, and the treatment frequency was once a day. In the model peptide combined with exosome treatment group, 100 μL of LZ-3-2 peptide with a mass concentration of 50 mg / mL was locally dripped onto the wound surface of aged mice. After an interval of 2 hours, 100 μL of 1×10 10 The exosomes of the model positive treatment group, i.e., the aged mice, were topically dripped with 100 μL of vitamin C with a mass concentration of 50 mg / mL, and the treatment frequency was once a day. After 14 days of treatment, the wound area of ​​each group was detected. The results are shown in Table 2.

[0062] Table 2 The therapeutic effect of each group on wound healing

[0063] Group Healing rate (%) Blank untreated group 86.75±0.32 Model untreated group 78.31±0.23 Model exosome treatment group 84.19±0.44 Model peptide treatment group 88.76±0.29 Model peptide combined with exosome treatment group 94.56±0.32 Model positive treatment group 85.17±0.35

[0064] The results in Table 2 show that compared with the blank untreated group, the wounds in the model untreated group showed a significant delayed healing phenomenon, while the wounds in the aging mice skin were significantly accelerated after local treatment in each treatment group and the positive treatment group, especially the combined treatment of the model polypeptide and exosomes can accelerate the healing of pressure ulcers in aging skin. These results show that the polypeptide of the present invention has a good effect of promoting healing, and the combination of the polypeptide and exosomes has a better therapeutic effect.

[0065] In addition, the corresponding type I collagen content and water content of the healed skin tissue of each group were tested, and it was found that the type I collagen content and water content in the skin after polypeptide treatment were also higher than those in the model-positive treatment group. In addition, by testing the blood and liver enzyme activity of mice, it was found that there were no obvious toxic side effects, and the safety was good.

[0066] The invention illustratively described herein may be appropriately implemented without any one or more elements, limitations or restrictions not specifically disclosed herein. Thus, for example, the terms "comprises," "includes," "comprising," and the like should be read expansively and without restriction. In addition, the terms and expressions used herein have been used as descriptive terms, but are not limited thereto, and the use of such terms and expressions does not mean that any equivalents of the features shown and described or portions thereof are excluded, but it should be recognized that various modifications may be made within the scope of the claimed invention. Therefore, it should be understood that, although the present invention is specifically disclosed by preferred embodiments and optional features, modifications and variations of the invention in the embodiments disclosed herein may be adopted by those skilled in the art, and such modifications and variations are considered to be within the scope of the present invention. The present invention is described broadly and generally herein.

Claims

1. An anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum spore powder, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. Use of the anti-aging peptide LZ-3-2 as claimed in claim 1 in the preparation of drugs for resisting skin aging.

3. Use of the anti-aging peptide LZ-3-2 as claimed in claim 1 in the preparation of cosmetics for resisting skin aging.

4. A pharmaceutical composition for anti-aging of the skin and promoting fibroblast proliferation, comprising exosomes from umbilical cord interstitial cells and the anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

5. A cosmetic for skin anti-aging, characterized in that The invention comprises exosomes from umbilical cord intercellular cells and anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO: 1.

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