Use of umbilical cord stem cells in the preparation of anti-aging drugs and related cosmetic products

Abstract

This invention provides an application of umbilical cord stem cells in the preparation of anti-aging drugs and related cosmetic products. More specifically, it demonstrates that exosomes prepared from umbilical cord stem cells, when used together with or alone with the anti-aging peptides screened and identified in this invention, can effectively promote fibroblast proliferation, reduce cellular aging characteristics, effectively promote skin wound healing, and promote skin collagen production and increased hydration, showing promising application prospects.

Description

Technical Field

[0001] This application relates to the field of biology, specifically to the application of umbilical cord stem cells in the preparation of anti-aging drugs and related cosmetic products. Background Technology

[0002] Skin is the most obvious indicator of age. As the body ages, the skin also ages. Externally, skin aging manifests as dry, rough skin, sagging, increased and deepened wrinkles, loss of elasticity, and pigmentation and depigmentation. Internally, the fundamental mechanism is the damage and aging of dermal fibroblasts. Skin aging is the outward manifestation of changes in tissue structure. The epidermis, dermis, and skin appendages all change with age, but the most characteristic change is in the composition of the dermis. The internal cause of these changes is fibroblast aging. Fibroblasts are important cellular components of the dermis, responsible for synthesizing structural components, including collagen fibers, elastic fibers, reticular fibers, and the extracellular matrix. Therefore, delaying or inhibiting fibroblast aging is key to treating and preventing skin aging.

[0003] In recent years, the demand for facial rejuvenation and anti-aging treatments has been increasing, leading to a proliferation of various methods for treating facial skin aging. Currently, clinically applied facial rejuvenation techniques mainly include: facelift surgery, hyaluronic acid fillers, botulinum toxin injections, chemical peels, and physical abrasion, all of which can achieve certain effects in facial skin rejuvenation. However, these methods only achieve aesthetic enhancement and do not truly address the physiological aspects of skin aging. With the widespread development of stem cell research, adipose-derived stem cells (ADSCs) have become a very effective anti-aging material. However, using intact stem cells for anti-aging applications faces technical obstacles, and they are difficult to store and inconvenient to use. Therefore, developing suitable stem cell anti-aging components is an important research direction.

[0004] Furthermore, the use of peptides in skin anti-aging has become an important research direction, and many peptides have already been applied to anti-aging. For example, L-carnosine, composed of β-alanine and histidine, is mainly used in skin beauty and anti-aging cosmetics to regulate skin pH, scavenge excess oxygen free radicals and their metabolites, and chelate heavy metal ions. Adding L-carnosine to cosmetics can effectively delay skin aging. Simultaneously, it can regulate the growth of human fibroblasts and repair aging human cells, making it a good natural anti-aging and antioxidant agent 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 in its molecular structure has a particularly significant integrative effect on copper and iron ions, giving it a certain skin whitening effect.

[0005] In addition, glutathione, one of the earliest products used in skin beauty and anti-aging cosmetics, is a polypeptide composed of glutamic acid, cysteine, and glycine condensed through peptide bonds. It contains active sulfhydryl groups and is the best broad-spectrum small-molecule antioxidant discovered to date. It can effectively remove excess free radicals and peroxides produced during skin tissue metabolism and defend against lipid peroxidation in mitochondria, thus protecting skin tissue and cells and delaying skin aging. Furthermore, adding glutathione to skin beauty and anti-aging cosmetics can effectively inhibit dopaminergic production and prevent the formation and aggravation of lipofuscin—age spots. This achieves the effects of preventing or reducing skin aging, reducing pigmentation, and improving the skin's antioxidant capacity. It has beautifying, skin-care, and anti-allergic effects, making it suitable for various skin types, especially sensitive skin. Some small-molecule functional peptides synthesized using chemical methods, known as 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 well-known international cosmetic brands and are widely used in skin care and anti-aging cosmetics, hence the name "beauty peptides." These beauty peptides are actually a type of bioactive biological peptide, composed of a specific sequence of amino acids linked by amide bonds. Typically, peptides consisting of two amino acids linked by amide bonds are called dipeptides, and those consisting of three amino acids are called tripeptides. Generally, dipeptides, tripeptides, and pentapeptides help promote collagen production in skin tissue; tetrapeptides help with anti-inflammatory and antioxidant effects; and hexapeptides help prevent the release of neurotransmitters in facial skin, similar to the effects of botulinum toxin. The broad application prospects of peptides in skin care and anti-aging cosmetics are closely related to their mechanisms of action. The mechanisms of action of these bioactive biological peptides are mainly manifested in their antioxidant function and free radical scavenging effect. Studies have shown that active peptides can effectively scavenge not only cationic free radicals such as DPPH, but also superoxide anion free radicals, hydroxyl free radicals, and lipid free radicals. Their antioxidant mechanism is likely related to the fact that during the enzymatic hydrolysis process of these active biopeptides, antioxidant amino acid residues that were originally embedded inside the molecule are exposed, releasing small molecule peptides and free amino acids with antioxidant activity. This exposes the side chain groups of amino acids that can participate in the reaction, which helps them to insert into the lipid interior, thus making it easier for the antioxidant amino acid residues to exert their antioxidant effect.

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

[0007] This invention provides an application of umbilical cord stem cells in the preparation of anti-aging drugs and related cosmetic products.

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

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

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

[0011] Specifically, the present invention also provides an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, the amino acid sequence of which 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 the peptide activity is still maintained.

[0013] Specifically, the modification or substitution, in other embodiments, also includes variants 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 shown in the corresponding template sequence of 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, typically 1-10 amino acids in length, preferably 1-5 amino acids. N-terminal truncations are generally not preferred. For example, as is well known to those skilled 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 “nonpolar” side chains. The substitution of one amino acid for another 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, a dressing, or a tablet.

[0015] More specifically, in addition to the active or therapeutic ingredient, tablets may contain a number of inert materials known as excipients. If desired, the pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. The term "excipient" as used herein means any substance that is not itself a therapeutic agent but can be used as a carrier or medium for delivering a therapeutic agent to a subject or with a therapeutic agent (e.g., to produce a pharmaceutical composition) to improve its handling or storage properties or to allow or facilitate the formation of dosage units of the composition. By way of illustration and not limitation, excipients include binders, disintegrants, flavor enhancers, solvents, thickeners or gelling agents (and any neutralizers, if desired), penetration enhancers, solubilizers, wetting agents, antioxidants, lubricants, emollients, emulsifiers, surfactants, substances added to mask or counteract unpleasant odors, flavors, or tastes, and substances added to improve the appearance or texture of the composition. Any such excipient may be used in any dosage form according to this disclosure. The types of excipients described above are not exhaustive but merely illustrative, as those skilled in the art will recognize that other types and combinations of excipients can be used to achieve the desired release and stability of the eltrombopagolamine tablet compositions.

[0016] Excipients can be classified according to their role 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 acquired through pretreatment steps such as wet granulation, spray drying spheroidization, or crystallization.

[0017] Typically, a disintegrant is included to ensure that the tablet has an acceptable disintegration rate. In another aspect of the invention, the disintegrant is selected from starch, cellulose, gums, cross-linked polymers, and effervescent agents, such as corn starch, potato starch, pregelatinized starch, modified corn starch, croscarmellose sodium, crospovidone, glycolic acid starch sodium, Veegum HV, methylcellulose, microcrystalline cellulose, cellulose, modified cellulose gum, agar, bentonite, montmorillonite clay, natural sponge, cation exchange resin, ion exchange resin (e.g., polyadenosine potassium), alginate and alginate, guar gum, citrus pulp, carboxymethyl cellulose and its salts such as sodium dodecyl sulfate, magnesium aluminum silicate, hydrated aluminum silicate, and a mixture of sodium bicarbonate and an acidifier such as tartaric acid or citric acid. In one embodiment of the invention, the disintegrant is a combination of glycolic acid starch sodium and crospovidone.

[0018] Binders are agents that impart cohesive properties to powdered materials. In another aspect of the invention, the binder is selected from starch (e.g., pastes, pregelatinized forms, mucilage), gelatin, sugars (e.g., sucrose, glucose, dextrose, molasses, lactose, dextrin, xylitol, sorbitol), polymethyl methacrylates, natural and synthetic gums (e.g., gum arabic, alginate and its salts such as sodium alginate, tragacanth gum, Irish moss extract, panwa gum, Indian gum, guar gum, zein), cellulose derivatives [e.g., carboxymethyl cellulose and its salts, methyl cellulose (MC), hydroxypropyl methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxyethyl cellulose (HEC), and ethyl cellulose (EC)], polyvinylpyrrolidone, magnesium silicate, larch arabinogalactan, polyethylene glycol, waxes, water, alcohols, magnesium aluminum silicate, and bentonite. In one embodiment of the invention, the binder comprises polyvinylpyrrolidone (PVP-polyvinyl ether).

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

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

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

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

[0023] The compressed tablets are further coated with a film by non-aqueous coating, aqueous coating, or hydroalcohol coating. The coating composition contains film-forming substances such as hydroxypropyl methylcellulose, hydroxypropyl cellulose, methylcellulose, and polyvinyl alcohol; solvents; colloidal silica; and optional excipients such as plasticizers, lubricants, and colorants.

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

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

[0026] Furthermore, the cosmetic of the present invention can be a skin cleanser. A mixture of water, oil, and other ingredients is formulated to form the skin cleanser to provide a homogeneous mixture, such as an emulsion. Additional ingredients include those that can be described by their function in the emulsion, such as diluents, thickeners, moisturizers, preservatives, neutralizers, emulsifiers, co-emulsifiers, emmollients, occlusives, fragrances, and colorants. Some ingredients may have more functions in the skin lotion. The topical composition can be prepared by any suitable method for preparing a topical composition. The topical composition can be a fully formulated topical emulsion for reducing exposure of gaseous pollutants to a patient's skin.

[0027] The cosmetics may also contain cationic surfactants, specific examples of which include behentrimethylammonium chloride; N,N,N-trimethyldocosane-1-ammonium chloride; docosyltrimethylammonium chloride; benzalkonium chloride; N-alkyl-N-benzyl-N,N-dimethylammonium chloride; BZK; bKC; BAC; benzyl chloride; N-benzyl-N,N-dimethyl-2-{2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy}acetyl chloride; benzyldimethyl(2-{2-[4 ... -(2,4,4-trimethylpentane-2-yl)phenoxy]ethoxy}ethyl)azaium chloride; benzododecaneammonium bromide; benzyl-dodecyl-dimethylammonium bromide; bronidox; 5-bromo-5-nitro-1,3-dioxane; cabozantrone bromide; (1-ethoxy-1-oxohexadecanyl)trimethylnitrogen bromide; carboxyethoxydecane base; [1-(ethoxycarbonyl)pentadecanyl]trimethylammonium bromide; citochlor; benzylhexadecyldimethylammonium chloride; benzyldimethylhexadecane 16-BAC; benzyl dimethyl-n-hexadecyl ammonium chloride; hexadecyl dimethyl benzyl ammonium chloride; cetrimonium bromide; hexadecyl-trimethyl-ammonium bromide; cetrimonium chloride; hexadecyl-trimethyl ammonium chloride; cetrimonium chloride; 1-hexadecylpyridinium chloride; acetylhydrochloride CPC; exadecylpyridinium chloride; didecyl dimethyl ammonium chloride; didecyl-dimethyl ammonium chloride; DDAC; dimethyl didecyl ammonium chloride; 1-decanoic acid; didecyl dimethyl ammonium chloride; Decyl dimethyl ammonium chloride; Quaternary ammonium salt-12; Dimethyl dioctadecyl ammonium bromide; Dimethyl dioctadecyl ammonium bromide; Distearyl dimethyl ammonium bromide; Dimethyl dioctadecyl ammonium chloride; N,N-Dimethyl-N-octadecyloctadecane-1-ammonium chloride; Dimethyl dioctadecyl ammonium chloride; Distearyl dimethyl ammonium chloride; Aliquot 207; DDAC; Distearyl dimethyl ammonium chloride; DSDMAC; Domiphen bromide; Lauryl methyl glucose ethyl-10-hydroxypropyl dimethyl ammonium chloride; Glucquat 125; Otinibidine dihydrochloride; N-octyl-1-[10-(4-octyliminopyridin-1-yl)decyl]pyridine-4-imine dihydrochloride; N,N'-(decane-1,10-diyldipyridin-1-yl-4-ylidene)dioctyl-1-amine dihydrochloride; N,N'-(decane-1,10-diyldi-1(4H)-pyridinyl-4-ylidene)bis(octylammonium) dichloride; Ola Fluorine; {3-[octadecyl(2-hydroxyethyl)amino]propyl}bis(2-hydroxyethyl)amine difluoride; fluorinated amine 297; N-oleo-1,3-propanediamine; N-[(9Z)-9-octadecen-1-yl]-1,3-propanediamine; stearyl chloride; benzyl dimethyl octadecyl ammonium chloride; dimethyl benzyl octadecyl ammonium chloride; benzyl dimethyl octadecyl ammonium chloride; benzyl dimethyl stearyl ammonium chloride;Benzylstearyl dimethylammonium chloride; N,N-dimethyl-N-octadecylbenzamide chloride; thonzoniumbromide; n-{2-[(4-methoxybenzyl)(pyrimidin-2-yl)amino]ethyl}-N,N-dimethylhexadecane-1-ammonium bromide; or mixtures thereof.

[0028] Beneficial effects

[0029] This invention provides an application of umbilical cord stem cells in the preparation of anti-aging drugs and related cosmetic products. More specifically, it demonstrates that exosomes prepared from umbilical cord stem cells, when used together with or alone with the anti-aging peptides screened and identified in this invention, can effectively promote fibroblast proliferation, reduce cellular aging characteristics, effectively promote skin wound healing, and promote skin collagen production and increased hydration, showing promising application prospects. Attached Figure Description

[0030] Figure 1 Graph showing the percentage of positive cells in each group relative to the total number of cells. Detailed Implementation

[0031] For illustrative purposes, the principles of the invention are described with reference to various exemplary embodiments thereof. Although certain embodiments of the invention have been specifically described herein, those skilled in the art will readily recognize that the same principles are equally applicable to other devices and methods, and may be employed in other devices and methods. Before explaining the embodiments disclosed herein in detail, it should be understood that the application of the invention is not limited to the details of any particular embodiment shown. The terminology used herein is for descriptive purposes and not for limitation. Furthermore, although a particular method is described with reference to specific steps presented in a particular order herein, in many cases these steps may be performed in any order that would 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., catalog number HZR-5302. After thawing the cryopreserved stem cell tubes, the cells were centrifuged at 1000 rpm for 3 min, and the supernatant was discarded. The cells were resuspended in 5 ml of PBS, centrifuged again at 900-1000 rpm for 3 min, and then resuspended in fresh complete culture medium and seeded into new culture flasks. The complete culture medium consisted of 89% H-DMEM + 10% FBS + 1% penicillin-dextrose antibody. When the cells reached approximately 80% confluence with the bottom of the culture flask, they were trypsinized to resuspend the adherent cells, centrifuged, and passaged at a 1:3 ratio. The medium was changed every 3 days, and the supernatant was collected during passage and medium change. The supernatant was stored at -20°C. After collecting a sufficient amount, the supernatant was thawed and filtered through a 0.22 μm filter to remove impurities. Transfer the supernatant into centrifuge tubes, balance them with a balance, and centrifuge for 75 min (100,000 g, 4 °C) using a sub-ultra-low temperature centrifuge. Discard the supernatant, wash the bottom and side walls of the centrifuge tubes with 1 ml PBS, resuspend the precipitate, and determine the protein concentration using the BCA method. Adjust the concentration to 5 mg / mL and store at -80 °C.

[0034] Electron microscopy of exosomes: At room temperature, 10 μl of the resulting precipitate was added to a copper grid. After 60 seconds, excess liquid was blotted off around the grid with filter paper. Then, 15 μL of 2% phosphotungstic acid solution was added to the grid. After 60 seconds, the grid was blotted off and baked under an incandescent lamp for 10 minutes. The grid was then placed under a transmission electron microscope (TEM) with the operating voltage adjusted to 120 kV. The exosomes were observed to be disc-shaped vesicles with diameters ranging from 55 to 110 nm.

[0035] In addition, the precipitate obtained by centrifugation was added to 100 μL of WB-IP cell lysis buffer for lysis, and the protein concentration was measured by the BCA method. Then, 10 μL of 5×SDS loading buffer was added to the lysis buffer, and the mixture was heated to boiling for 10 min before loading onto a gel. The gel was then separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The membrane was blocked at room temperature with blocking buffer (5% skim milk) for 50 min, followed by elution with 1×TBST buffer. CD63, CD81, Tsgl01, and Calnexin were added and incubated overnight. Elution was then performed again with 1×TBST buffer. Horseradish peroxidase-labeled secondary antibody was added to the loading wells, and the mixture was incubated for 120 min before development with a luminescent substrate. The results showed that the exosome marker proteins CD63, CD81, and Tsgl01 were expressed, while Calnexin was not expressed, indicating that the precipitate obtained in this experiment was an exosome.

[0036] Example 2: Screening and Identification of Anti-aging Peptides

[0037] 100g of Ganoderma lucidum spore powder was placed in a 1L supercritical fluid extraction vessel. The temperature and pressure of the extraction vessel and the separation vessel were set to 35MPa, 40℃ and 6MPa, 50℃, respectively, and run for 1 hour each. Then, the 1-hour run sample was subjected to high-pressure static aging for 12 hours. After rapidly releasing the critical gas, the spore powder with broken cell walls was obtained. Deionized water was added according to the optimized material-to-liquid ratio of 1:18, and the mixture was stirred evenly to ensure that the powder and water were fully mixed. 1mol / L sodium hydroxide solution was added dropwise to adjust the pH to the optimal extraction pH of 10.5. The mixture was then placed in a water bath at a certain temperature for 2 hours. The supernatant was collected by suction filtration, and the pH was adjusted to the isoelectric point with 1mol / L hydrochloric acid. The mixture was allowed to stand at room temperature for a period of time, and then centrifuged at 8000r / min for 20 minutes to remove the supernatant and obtain the protein. The protein was dissolved in a small amount of deionized water and transferred to a petri dish. The crude protein was obtained by freeze-drying. 1g of crude protein was taken, and the material-to-liquid ratio was adjusted to 1:50 with deionized water. 1% alkaline protease was added, and the mixture was enzymatically hydrolyzed at 50°C and pH 8.0 for 12 hours. After hydrolysis, the protein was inactivated in a boiling water bath for 10 minutes to obtain an enzymatically hydrolyzed polypeptide solution. The polypeptide powder was then lyophilized to obtain polypeptide powder. The polypeptide powder sample was subjected to DEAE-32 ion exchange chromatography, yielding five main peaks. The DPPH radical scavenging method identified the third main peak as having the best antioxidant properties, with a scavenging rate of 86.5%. The third main peak was further separated using a Superdex 30 column to obtain three fractions: LZ-3-1, LZ-3-2, and LZ-3-3. The ABTS radical scavenging method determined that fraction LZ-3-2 had the strongest antioxidant properties, with a scavenging rate of 90.4%. This fraction was further detected by LC-MS / MS. The amino acid sequence of the polypeptide was determined by comparison with the uniprot database, as shown in SEQ ID NO: 1.

[0038] The LZ-3-2 polypeptide was synthesized by Ketai 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 (catalog number: IM-H032, source: IMMOCELL) were seeded 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 + 0.005 mg / ml insulin).

[0041] +5ng / ml bfGF +1ug / ml hydrocortisone +7.5ml-Gln). Once most fibroblasts are visible to adhere to the wall, and when the fibroblasts reach 70-80% confluence, digest them with 0.25% trypsin (containing 0.01% EDTA) and passage them at a 1:3 ratio.

[0042] The exosomes prepared in Example 1 and the LZ-3-2 peptide prepared in Example 2 were co-cultured with fibroblasts indirectly through Transwell chambers with micropores of 0.4 μm diameter to establish a non-contact cell co-culture.

[0043] The experiment was conducted in the following groups:

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

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

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

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

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

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

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

[0051] In each group, fibroblasts were seeded in the lower layer of the Transwell chamber at a cell suspension concentration of 2 × 10⁻⁶. 5 / mL.

[0052] After 3 days of cell culture, the viability of each component fibroblast was detected using a CCK-8 assay kit, and the absorbance of each well at 450 nm was measured using a microplate reader. 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 all higher than those of the blank control group, and the differences were statistically significant (P < 0.05). Table 1 also shows that the peptide of this invention promotes fibroblast proliferation in a dose-dependent manner, with a better promoting effect than the positive control group at the same concentration. Furthermore, the combined use of the peptide and exosomes exhibits a synergistic effect in promoting cell proliferation.

[0056] Subsequently, cells from each group were collected and stained with SA-β-Gal using a β-galactosidase staining kit. The percentage of positive cells relative to the total cell count was calculated by counting the total number of cells and the number of β-galactosidase-positive cells, thus determining the cellular senescence status. The results are as follows: Figure 1 As shown.

[0057] from Figure 1 The results showed that the proportion of positively stained 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 the differences were statistically significant (P<0.01). Figure 1 The results also show that the peptides of this invention have a dose-dependent effect in reducing cellular senescence, and the promoting effect is better than that of the positive control group at the same concentration. Furthermore, the combined use of the peptides and exosomes has a synergistic effect in reducing cellular senescence, with the percentage of positive staining being only (10.32±1.83)%.

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

[0059] Preparation of a subacute aging model: After acclimatizing mice for one week, the model was prepared according to conventional methods for preparing subacute aging models in this field. Specifically, mice were subcutaneously injected with 1000 mg / (kg·d) of 15% D-galactose in the neck and back for 8 consecutive weeks to obtain mice identified as aging models. The blank control group mice were given an equal volume of physiological saline subcutaneously injected in the neck and back daily.

[0060] Hair was removed from the backs of mice in the model group and the blank control group. The back skin was then lifted and clamped between the two sides with a 1.2 cm diameter circular magnet for 12 hours, and then released for 12 hours. This constituted one cycle. After three ischemia-reperfusion injuries, a significant ulcer was formed after waiting for one week, thus preparing pressure ulcer injury.

[0061] After successful initiation of pressure ulcers in mice in both the control and model groups, local debridement was performed. In the control group (Op.S.), 100 μL of sterile PBS was applied topically to the wounds once daily. In the model group (Op.S.), aged mice received topically applied sterile PBS. In the model exosome treatment group (Op.S.), aged mice received topically applied 100 μL of 1×10⁻⁶ PBS. 10 To ensure the effectiveness of exosome therapy, exosomes were administered at a concentration of particles / 100 μL, once daily. In the model peptide treatment group (aged mice), 100 μL of LZ-3-2 peptide at a concentration of 50 mg / mL was applied topically to the wound once daily. In the model peptide combined with exosome therapy group (aged mice), 100 μL of LZ-3-2 peptide at a concentration of 50 mg / mL was applied topically to the wound, followed by a 2-hour interval, 100 μL of LZ-3-2 peptide at a concentration of 1 × 10⁻⁶ ppm was applied topically to the wound. 10 The treatment regimen consisted of exosomes at a concentration of particles / 100 μL, administered once daily. In the model-positive treatment group (aged mice), 100 μL of vitamin C at a concentration of 50 mg / mL was applied topically to the wounds once daily. After 14 days of treatment, the wound area in each group was measured. The results are shown in Table 2.

[0062] Table 2. Therapeutic effects 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 untreated group, the wounds in the untreated group exhibited significantly delayed healing. Local treatment in all treatment groups and the positive treatment group significantly accelerated the healing of skin wounds in aging mice. In particular, the combined treatment with the model peptide and exosomes accelerated the healing of pressure ulcers in aging skin. These results indicate that the peptides of this invention have a good healing-promoting effect, and the combination of the peptides and exosomes results in even better therapeutic effects.

[0065] In addition, the content and water content of type I collagen in the healed skin tissue of each group were tested. It was found that the content and water content of type I collagen in the skin treated with peptides were higher than those in the model positive treatment group. Moreover, the detection of enzyme activity in mouse blood and liver showed no obvious toxic side effects, indicating good safety.

[0066] The invention described illustratively herein may be practiced without any limitation or restriction on any one or more elements not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” “including,” etc., should be read expansively and without limitation. Furthermore, the terms and expressions used herein have been used as descriptive terms but are not limiting, and the use of such terms and expressions does not imply exclusion of any equivalents of the shown and described features or portions thereof, but it should be recognized that various modifications can be made within the scope of the claimed invention. Therefore, it should be understood that although the invention is specifically disclosed by preferred embodiments and optional features, modifications and variations of the invention in the embodiments disclosed herein are available to those skilled in the art, and such modifications and variations are considered to be within the scope of the invention. The invention is described herein in a broad and general sense.

Claims

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

1.

2. Use of the anti-aging peptide LZ-3-2 as described in claim 1 in the preparation of a medicament for anti-skin aging.

3. The use of the anti-aging peptide LZ-3-2 as described in claim 1 in the preparation of anti-skin aging cosmetics.

4. A pharmaceutical composition for skin anti-aging and promoting fibroblast proliferation, comprising exosomes derived from umbilical cord mesenchymal stem cells and an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, the amino acid sequence of said anti-aging peptide LZ-3-2 being shown in SEQ ID NO:

1.

5. A cosmetic product for anti-aging skin, characterized in that, It contains exosomes derived from umbilical cord mesenchymal stem cells and an anti-aging peptide LZ-3-2 extracted from Ganoderma lucidum, the amino acid sequence of which is shown in SEQ ID NO: 1.

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