Umbilical cord mesenchymal stem cell active peptide secretion, preparation method, application and skin microecological regulator

Abstract

The present application relates to the field of cosmetic skin care technology, in particular to a umbilical cord mesenchymal stem cell active peptide secretion, a preparation method, an application and a skin microecological regulator. The umbilical cord mesenchymal stem cells secrete many active peptide molecules to the environment, which have diversified regulation functions on cells. The inventors have found by chance that the umbilical cord mesenchymal stem cells can not only regulate skin cells, but also regulate the distribution of microbial colonies on the skin surface. In the research, the inventors have found that the umbilical cord mesenchymal stem cell active peptide secretion can have a positive regulation effect on a probiotic Staphylococcus epidermidis, and in addition, there is no report that other significant regulation colonies are directly related to skin problems. It is determined that the umbilical cord mesenchymal stem cell secretion can regulate the skin surface microecology.

Description

[0001] This invention is a divisional application, with the parent application number being "2021105637228", entitled "Umbilical Cord Mesenchymal Stem Cell Active Peptide Secretion, Preparation Method, Application and Skin Microecological Regulator", and the application date being May 24, 2021. Technical Field

[0002] This invention relates to the field of beauty and skincare technology, and in particular to an active peptide secretion from umbilical cord mesenchymal stem cells, its preparation method, application, and skin microecological regulator. Background Technology

[0003] Skin microbiota are important members of the skin's micro-ecosystem. The flora on the skin surface can generally be divided into resident and transient bacteria. Resident bacteria are a group of microorganisms that reside on healthy skin and are closely related to skin health. They include Staphylococcus, Corynebacterium, Propionibacterium, Acinetobacter, Malassezia, Micrococcus, Enterobacter, and Klebsiella. Transient bacteria refer to microorganisms acquired through contact with the external environment, including Staphylococcus aureus, hemolytic streptococci, and Enterococci. They are the main pathogens causing skin infections.

[0004] A normal skin micro-ecosystem exists in a competitive and restrictive relationship. An abnormal increase, decrease, or even disappearance of skin microbiota can lead to the disruption of the skin micro-ecosystem.

[0005] A growing body of research indicates that certain ingredients in cosmetic formulations can interact with microorganisms, thereby influencing the skin's microecology. Identifying bioactive peptides that promote beneficial bacteria and inhibit harmful bacteria in the skin microecology has significant potential for regulating it. However, current cosmetic research and development rarely focuses on the skin's surface microecology, and no cosmetics demonstrating regulatory effects on this microecology have been reported. Therefore, this invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide an active peptide secretion from umbilical cord mesenchymal stem cells that has the function of regulating the skin surface microecology, and to apply it to the preparation of a skin surface microecological regulator, thereby playing a good regulatory role on the microbial community on the skin surface, maintaining the normal state of the skin surface, giving full play to its natural barrier function, and filling the gap in the current market for products with skin surface microecological regulation function.

[0007] Another objective of this invention is to provide a method for preparing the above-mentioned umbilical cord mesenchymal stem cell active peptide secretion, so as to ensure that the umbilical cord mesenchymal stem cell active peptide secretion prepared by the method has a regulatory effect on the growth of resident and transient bacteria on the skin surface.

[0008] Another objective of this invention is to provide a skin surface microecological regulator based on the above-mentioned umbilical cord mesenchymal stem cell active peptide secretion as the main component, and by rationally selecting excipients, to meet daily needs, while also being suitable for industrial production and commercial promotion.

[0009] To solve the above-mentioned technical problems and achieve the above-mentioned objectives, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides an active peptide secretion from umbilical cord mesenchymal stem cells, the secretion being mainly composed of active peptides, nicotinic acid, fats and carbohydrates.

[0011] In an optional embodiment, the secretion may further include at least one of vitamin C, vitamin A, or vitamin E.

[0012] In an optional embodiment, the secretion, based on 100g of secretion, comprises the following components: 0.1-1g of active peptides, 5-25mg of niacin, 0.2-0.6g of fat, 10-30mg of carbohydrates, 100-500μg of vitamin C, 25-65μg of vitamin A, and 1-5μg of vitamin E.

[0013] Preferably, the sugar includes at least one of glucose, fructose, sucrose, lactose, or maltose.

[0014] Preferably, based on 100g of secretions, the secretions include: 0.24g of active peptides, 17.32mg of niacin, 0.4g of fat, 23.6mg of carbohydrates, 314μg of vitamin C, 47.6μg of vitamin A, and 2.7μg of vitamin E.

[0015] Secondly, the present invention provides a method for preparing the secretion described in any of the foregoing embodiments. The preparation method includes collecting umbilical cord, separating Wharton's jelly, culturing it after disruption to obtain primary stem cells, and continuing to passage and culture the cells after the first passage reaches a fusion rate of 85% to 90%, and collecting the active peptide secretion.

[0016] In an optional implementation, the collected umbilical cord is segmented and blood vessels are removed to obtain Wharton's jelly.

[0017] Preferably, the length of the segment is 2 cm.

[0018] In an optional embodiment, the Wharton's jelly is broken down into a paste and then cultured to obtain primary stem cells. The breaking method is a physical method, including cutting or grinding.

[0019] Thirdly, the present invention provides the application of the secretion described in any of the foregoing embodiments or the secretion prepared by any of the foregoing embodiments in the preparation of skin microecological regulation products, wherein the skin microecological regulation products regulate the distribution of skin surface microbial communities by promoting or inhibiting the growth of skin surface microbial communities.

[0020] Fourthly, the present invention provides a skin microecological regulator, comprising the secretions described in any of the foregoing embodiments or the secretions prepared by the preparation method described in any of the foregoing embodiments.

[0021] In an optional embodiment, the regulator further includes at least one of a humectant, an emulsifier, a thickener, or a preservative.

[0022] Preferably, the moisturizer includes at least one of butylene glycol, glycerin, 1,3-propanediol, pentanediol, isopentanediol, diglycerol, triglycerol, polyglycerol, sorbitol, polyethylene glycol, polypropylene glycol, ethylene glycol, or diethylene glycol.

[0023] Preferably, the emulsifier comprises at least one of stearic acid, cetearyl alcohol, cetearyl glucose, cetearyl alcohol ethylhexanoate, stearyl alcohol polyether-20, potassium cetyl phosphate, lanolin wax, or coconut oil PEG-10 ester.

[0024] Preferably, the thickener includes C 14 ~C 22 alcohols, C 12 ~C 20 Alkyl glucoside, carbomer, xanthan gum, gellan gum, gum arabic, sclerotium gum, hydroxymethyl cellulose, hydroxyethyl cellulose, acrylate / C10-30 alkanol acrylate crosspolymer, polyacrylate-13, ammonium acrylate and acrylamide copolymer, hydroxyethyl acrylate, sodium acryloyl dimethyl taurate copolymer, sodium polyacrylate, ammonium acryloyl dimethyl taurate / VP copolymer or ammonium acryloyl dimethyl taurate / behenol polyether-25 methacrylate crosspolymer, or one or more combinations thereof.

[0025] Preferably, the preservative includes at least one selected from methylparaben, ethylparaben, propylparaben, phenoxyethanol, chlorphenesin, potassium sorbate, sodium benzoate, benzoic acid, salicylic acid, imidazolidinyl urea, p-hydroxyacetophenone, capryloyl hydroxamic acid, pentylene glycol, hexanediol, or ethylhexylglycerin.

[0026] Preferably, the skin microecological regulator comprises, by weight, 15-25 parts of umbilical cord mesenchymal stem cell active peptide secretion, 10-20 parts of stearic acid, 1-3 parts of ethylhexyl palmitate, 0.5-2 parts of lanolin, 0.1-0.4 parts of cetearyl alcohol, 0.1-0.4 parts of cetearyl glucoside, and C 14 -C 22 Alcohol 0.1~0.5 parts, C 12 -C 20 Alkyl glucoside 0.1-0.5 parts, butanediol 5-15 parts, sorbitol 1-5 parts, potassium hydroxide 0.5-2 parts, phenoxyethanol 0.1-0.7 parts, methylparaben 0.1-0.5 parts, fragrance 0.05-0.2 parts, water 40-50 parts.

[0027] Preferably, the skin microecological regulator comprises, by weight, 20 parts of umbilical cord mesenchymal stem cell active peptide secretion, 15 parts of stearic acid, 2 parts of ethylhexyl palmitate, 1 part of lanolin, 0.5 parts of cetearyl alcohol and cetearyl glucoside, and C 14 -C 22 alcohols and C 12 -C 20 The ingredients include 0.6 parts alkyl glucoside, 10 parts butylene glycol, 3 parts sorbitol, 1 part potassium hydroxide, 0.5 parts phenoxyethanol, 0.2 parts methylparaben, 0.1 parts fragrance, and 46.1 parts water.

[0028] In optional embodiments, the dosage form of the skin microecological regulator includes creams, lotions, aqueous solutions, gels, oils, powders, muds, wax-based products, patches, films, or freeze-dried products.

[0029] Umbilical cord mesenchymal stem cells (UCSCs) secrete numerous bioactive peptides into the environment, exhibiting diverse regulatory functions on cells. The inventors unintentionally discovered that UCSCs not only regulate skin cells but also play a role in regulating the distribution of the skin's surface microbiome. In their research, the inventors found that the bioactive peptide secretions from UCSCs have a positive regulatory effect on a probiotic, *Staphylococcus epidermidis*. Furthermore, no other significantly regulated bacterial colonies have been reported to be directly related to skin problems. Therefore, it is determined that UCSC secretions can regulate the skin's surface microecology. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 These are the results of species diversity analysis within the samples in the experimental examples of this invention;

[0032] Figure 2 This is a graph showing the differences in species among the efficacy test groups in the experimental examples of this invention;

[0033] Figure 3 This is a graph showing the differences in species between the placebo group and other experimental groups in the present invention.

[0034] Figure 4 These are the top ten strains with significant changes selected from the experimental examples of this invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The components of the embodiments of the present invention described and shown in the accompanying drawings can generally be arranged and designed in various different configurations.

[0036] Therefore, the following detailed description of the embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the invention without inventive effort are within the scope of protection of the invention.

[0037] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0038] In one specific embodiment, the present invention provides an active peptide secretion from umbilical cord mesenchymal stem cells, the secretion mainly consisting of active peptides, niacin, fats and carbohydrates.

[0039] In an optional embodiment, the secretion may further include at least one of vitamin C, vitamin A, or vitamin E.

[0040] Niacin (CAS Registry No.: 59-67-6), also known as vitamin B3, is one of the 13 essential vitamins for the human body. It is highly water-soluble and can be converted into nicotinamide in the human body, which then participates in the synthesis of coenzyme I and coenzyme II. It plays an important role in lipid metabolism, tissue respiration oxidation, and anaerobic decomposition of carbohydrates in the body.

[0041] Vitamin C (CAS Registry No.: 50-81-7), also known as ascorbic acid, has an enediol structure with a lactone ring and two chiral carbon atoms. Therefore, vitamin C is not only highly reactive but also optically active. It acts as both a hydrogen donor and acceptor, playing a crucial role in various redox processes in the body, such as promoting antibody formation, iron absorption, tetrahydrofolate formation, and free radical scavenging. Furthermore, reports indicate that vitamin C at certain concentrations can exhibit inhibitory effects against Mycobacterium tuberculosis and Pseudomonas aeruginosa.

[0042] Vitamin A (CAS Registry No.: 68-26-8) is a fat-soluble vitamin and a coenzyme that regulates glycoprotein synthesis. It plays a stabilizing role in the cell membrane of epithelial cells, maintaining their morphological integrity and proper function. Vitamin A deficiency can cause dryness of epithelial tissue, with normal columnar epithelial cells transforming into keratinized squamous cells, leading to cellular keratinization.

[0043] Vitamin E (CAS Registry No.: 121854-78-2) is a fat-soluble vitamin and an important antioxidant. It is stable to acid and heat and can protect unsaturated fatty acids in the human body from damage by free radicals.

[0044] In an optional embodiment, the secretion, based on 100g of secretion, comprises the following components: 0.1-1g of active peptides, 5-25mg of niacin, 0.2-0.6g of fat, 10-30mg of carbohydrates, 100-500μg of vitamin C, 25-65μg of vitamin A, and 1-5μg of vitamin E.

[0045] Preferably, the sugar includes at least one of glucose, fructose, sucrose, lactose, or maltose.

[0046] Preferably, based on 100g of secretions, the secretions include: 0.24g of active peptides, 17.32g of niacin, 0.4g of fat, 314μg of vitamin C, 23.6μg of carbohydrates, and 2.7μg of vitamin E.

[0047] Secondly, the present invention provides a method for preparing the secretion described in any of the foregoing embodiments. The method includes collecting an umbilical cord, separating Wharton's jelly, culturing it after disruption to obtain primary stem cells, and after the first passage reaches a fusion rate of 85% to 90%, continuing passage culture and collecting the active peptide secretion. The fusion rate of the first passage includes, but is not limited to, 85%, 86%, 87%, 88%, 89%, or 90%.

[0048] In an optional implementation, the collected umbilical cord is segmented and the blood vessels are removed to obtain Wharton's jelly. Segmenting the cord first and then removing the blood vessels is more efficient than removing the blood vessels first and then segmenting and separating the blood vessels, resulting in a higher yield of mesenchymal stem cells.

[0049] Preferably, the length of the segment is 2 cm.

[0050] In an optional embodiment, the Wharton's jelly is broken down into a paste and then cultured to obtain primary stem cells. The breaking method is a physical method, including cutting or grinding. Compared with chemical methods for breaking Wharton's jelly, physical methods allow mesenchymal stem cells to gradually emerge from the culture medium. This method causes less damage to cells than whole digestion and makes it easier to obtain high-quality mesenchymal stem cells.

[0051] Bioactive peptides are a general term for more than a thousand peptides. According to their functions, they include nearly a dozen types such as conjugated peptides, neuropeptides, antimicrobial peptides, and flavoring peptides. Among them, antimicrobial peptides are mostly derived from microorganisms, such as bacitracin, subtilisin, and nisin. There are fewer types of endogenous antimicrobial peptides from animals and plants. There are no reports on the antimicrobial function of bioactive peptides secreted by mesenchymal stem cells. The inventors unexpectedly discovered that the bioactive peptide secretions obtained by the above preparation method have the effect of regulating the microecology of the skin surface. That is, while promoting the growth of beneficial bacteria on the skin surface, they also show a certain degree of inhibition against harmful bacteria.

[0052] Thirdly, the present invention provides the application of the secretion described in any of the foregoing embodiments or the secretion prepared by any of the foregoing embodiments in the preparation of skin microecological regulation products, wherein the skin microecological regulation products regulate the distribution of skin surface microbial communities by promoting or inhibiting the growth of skin surface microbial communities.

[0053] Fourthly, the present invention provides a skin microecological regulator, comprising the secretions described in any of the foregoing embodiments or the secretions prepared by the preparation method described in any of the foregoing embodiments.

[0054] In an optional embodiment, the regulator further includes at least one of a humectant, an emulsifier, a thickener, or a preservative / antimicrobial agent.

[0055] Preferably, the moisturizer includes at least one of butylene glycol, glycerin, 1,3-propanediol, pentanediol, isopentanediol, diglycerol, triglycerol, polyglycerol, sorbitol, polyethylene glycol, polypropylene glycol, ethylene glycol, or diethylene glycol.

[0056] Preferably, the emulsifier comprises at least one of stearic acid, cetearyl alcohol, cetearyl glucose, cetearyl alcohol ethylhexanoate, stearyl alcohol polyether-20, potassium cetyl phosphate, lanolin wax, or coconut oil PEG-10 ester.

[0057] Preferably, the thickener includes C 14 ~C 22 alcohols, C 12 ~C 20 Alkyl glucoside, carbomer, xanthan gum, gellan gum, gum arabic, sclerotium gum, hydroxymethyl cellulose, hydroxyethyl cellulose, acrylate / C10-30 alkanol acrylate crosspolymer, polyacrylate-13, ammonium acrylate and acrylamide copolymer, hydroxyethyl acrylate, sodium acryloyl dimethyl taurate copolymer, sodium polyacrylate, ammonium acryloyl dimethyl taurate / VP copolymer or ammonium acryloyl dimethyl taurate / behenol polyether-25 methacrylate crosspolymer, or one or more combinations thereof.

[0058] Preferably, the preservative includes at least one selected from methylparaben, ethylparaben, propylparaben, phenoxyethanol, chlorphenesin, potassium sorbate, sodium benzoate, benzoic acid, salicylic acid, imidazolidinyl urea, p-hydroxyacetophenone, capryloyl hydroxamic acid, pentylene glycol, hexanediol, or ethylhexylglycerin.

[0059] Preferably, the skin microecological regulator comprises, by weight, 15-25 parts of umbilical cord mesenchymal stem cell active peptide secretion, 10-20 parts of stearic acid, 1-3 parts of ethylhexyl palmitate, 0.5-2 parts of lanolin, 0.1-0.4 parts of cetearyl alcohol, 0.1-0.4 parts of cetearyl glucoside, and C 14 -C 22 Alcohol 0.1~0.5 parts, C 12 -C 20 Alkyl glucoside 0.1-0.5 parts, butanediol 5-15 parts, sorbitol 1-5 parts, potassium hydroxide 0.5-2 parts, phenoxyethanol 0.1-0.7 parts, methylparaben 0.1-0.5 parts, fragrance 0.05-0.2 parts, water 40-50 parts.

[0060] Preferably, the skin microecological regulator comprises, by weight, 20 parts of umbilical cord mesenchymal stem cell active peptide secretion, 15 parts of stearic acid, 2 parts of ethylhexyl palmitate, 1 part of lanolin, 0.5 parts of cetearyl alcohol and cetearyl glucoside, and C 14 -C 22 alcohols and C 12 -C 20 The ingredients include 0.6 parts alkyl glucoside, 10 parts butylene glycol, 3 parts sorbitol, 1 part potassium hydroxide, 0.5 parts phenoxyethanol, 0.2 parts methylparaben, 0.1 parts fragrance, and 46.1 parts water.

[0061] In optional embodiments, the dosage form of the skin microecological regulator includes creams, lotions, aqueous solutions, gels, oils, powders, muds, wax-based products, patches, films, or freeze-dried products.

[0062] The following detailed description of some embodiments of the present invention is provided in conjunction with the accompanying drawings. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0063] Example 1

[0064] This embodiment provides a method for preparing active peptide secretions from umbilical cord mesenchymal stem cells, including the following steps:

[0065] 1.1 Separation of Wharton's jelly from the umbilical cord

[0066] (1) Collect the umbilical cord. Place the umbilical cord into a sterile disposable umbilical cord collection bottle, immerse it in the preservation solution, tighten the cap, and store it in a 4°C refrigerator. Under sterile conditions, the length of the umbilical cord is measured to be 55cm. Then, cut the umbilical cord into a 2cm long segment, remove the artery and vein of the umbilical cord, and separate Wharton's jelly.

[0067] (2) Collect Wharton's gum, add 1-2 mL of MEM-alpha medium, and cut the Wharton's gum into small pieces to form a paste.

[0068] (3) Take 1 mL of the Wharton's gum shredded in step (2) and add it to the cell culture dish. Then add 2-3 mL of MEM-alpha medium to completely cover the bottom of the culture dish with the Wharton's gum and place it in a cell culture incubator for culture.

[0069] (4) After 7 days of growth of Wharton's gel, observe the cell growth status. Cells slowly begin to crawl out from the edge of the tissue block, and then the cells proliferate rapidly. After about 15 days, remove the tissue to obtain primary stem cells.

[0070] (5) After passage of primary cells to a confluence of 87%, the culture supernatant was aspirated from the culture dish to collect the mesenchymal stem cell secretion polypeptide stock solution in a special collection bottle; then PBS was added to the culture dish for washing, the container was shaken several times, the washing solution was aspirated and discarded, 3 mL of trypsin was added, and the culture flask was shaken to ensure that the trypsin completely covered the cell layer. Under the microscope, the cells were observed to become rounded, and a large number of adherent cells were visible to the naked eye when they fell off. The culture flask was then tilted, and MEM-alpha medium with a volume greater than twice that of the trypsin used was added to stop the digestion. Gently pipette the cell layer surface to disperse the culture medium, aspirate the cell suspension into a 15mL centrifuge tube, centrifuge at 1000rpm for 5 minutes, discard the supernatant, add 5mL of culture medium to resuspend, centrifuge, wash, discard the supernatant, add 7mL of culture medium to resuspend the cells, mix well, add to a T-75 culture flask, and place in an incubator for continued culture. When the confluence reaches 87% again, aspirate the culture supernatant from the culture dish to collect the mesenchymal stem cell secreted peptide stock solution into a special collection bottle. Repeat this process to collect the mesenchymal stem cell secreted peptide stock solution.

[0071] (6) 50 mL of the collected polypeptide mixture from umbilical cord mesenchymal stem cells, i.e., umbilical cord mesenchymal stem cell active peptide secretion, was delivered to Jiaxing Zhongke Testing Technology Service Co., Ltd. for composition testing. The test results are shown in Table 1.

[0072] Table 1. Detection results of the peptide mixed stock solution obtained in Example 1

[0073]

[0074] Example 2

[0075] In this embodiment, a skin microecological regulation cream was formulated using the active peptide secretion of umbilical cord mesenchymal stem cells obtained in Example 1. Its composition is shown in Table 2.

[0076] Table 2. Composition of the skin microecology regulating cream provided in Example 2

[0077]

[0078] Comparative Example 1

[0079] In this comparative example, water was used to replace the umbilical cord mesenchymal stem cell active peptide secretion in Example 2 to obtain a placebo group cream.

[0080] Experimental Example

[0081] This experiment involved recruiting volunteers to apply the creams provided in Example 2 and Comparative Example 1 to the skin surface, and then examining the growth of the skin microbial community at the application site.

[0082] 1. Material preparation

[0083] Prepare the following experimental materials:

[0084] (1) Label;

[0085] (2) A 2mL screw-type tube (external screw) contains 750μL of MoBioBuffer;

[0086] (3) Sterile sampling swabs;

[0087] (4) Sterile SCF-1 solution containing 50 mM Tris buffer (pH 7.6), 1 mM EDTA (pH 8.0) and 0.5% Tween-20 by mass, used to moisten swabs before sampling;

[0088] (5) Sealed bags are used to collect sampling tubes;

[0089] (6) Portable foam box (containing ice).

[0090] 2. Determination of skin type in the population

[0091] (1) Exclusion criteria for subjects:

[0092] ① The subject has used topical antibiotics or steroids on their face, neck, arms, or hands within the past seven days. (Screening and sampling can be postponed if the subject meets the exclusion period for antibiotic or steroid use).

[0093] ② Acne should not appear on the face, chest, back, or shoulders;

[0094] ③ Multiple blisters, pustules, burns, abscesses, erosions or ulcers on the scalp, face, arms, forearms or hands;

[0095] ④ Single blister, abscess, burn, abscess, infection, ulcer, scab, incision, fissure, or pink spot / pigmentation or patch within 4cm of the sampling point;

[0096] ⑤ Multiple pink spots / red scales / patches anywhere on the body (psoriasis or eczema);

[0097] ⑥ The palms and / or soles of the feet show uniformly thickened dead skin, cracking, and excessive dryness;

[0098] ⑦ Using an over-the-counter anti-dandruff shampoo with unknown ingredients every day for up to 2 weeks;

[0099] ⑧ Spreading rashes (appearing on multiple body parts or spreading to most areas of the body).

[0100] (2) Detection conditions

[0101] Skin examinations should be performed under good lighting conditions and include a general survey of local skin (the whole body) and a detailed examination of the sampling area.

[0102] (3) Other testing standards:

[0103] ① Carefully examine the back of the forehead, both sides of the cheeks, and the elbow creases (inner elbows) of both arms through visual inspection.

[0104] ② Individuals with any single vesicle, pustules, burns, abscesses, infections, ulcers, scabs, incisions, fissures, or pink spots / pigmented spots or patches, or visible skin lesions, more than 4 cm from the sampling site, are still considered eligible for inclusion in the study.

[0105] (4) Do not clean your face, use any cosmetics, or take a bath with antibacterial active soap 8 hours before sampling.

[0106] Based on the skin characteristics of the aforementioned population, a total of 11 qualified volunteers were recruited.

[0107] 3. Sample collection method

[0108] (1) No sample collection:

[0109] The cubital fossa (inner elbow): refers to the junction of the arm and forearm located at the crease of the inner elbow. Skin sample collection uses two collection swabs / samples, moistened with sterile SCF-1 solution. With one hand, stretch the skin of the elbow; with the other hand, hold the swab, keeping the axis parallel to the skin surface, and rub the swab back and forth along the crease of the elbow approximately 50 times, applying steady pressure (50 swabs completed in approximately 30 seconds). Insert the swab into the tube, immersing the swab head into the collection tube. Aseptically cut the swab head from the handle and screw the cap back into place.

[0110] Precautions:

[0111] a. Key points of sampling technique for obtaining the best skin surface specimens: use a moistened cotton swab, apply steady pressure, and rub consistently (50 back and forth strokes within 30 seconds at the sampling point).

[0112] b. Sample from both sides separately, and label and preserve them separately. Place the tubes in a zip-lock bag and rapidly freeze them in a liquid nitrogen tank for 2 minutes. Transfer them to -80°C for freezing and preservation, and then transport them to the testing agency (with dry ice or ice cubes in a foam box).

[0113] (2) Application of the test substance:

[0114] Volunteers regularly (once a day) applied the cream provided in the comparison ratio to one elbow crease and the cream provided in Example 2 to the same area on the other side.

[0115] (3) Data collection after use:

[0116] Samples were collected on the day of use, and on the 14th and 28th days after use. The collection method and details are the same as "(1) Collection without test substance".

[0117] 4. After sampling, the tubes were rapidly frozen in liquid nitrogen for 2 minutes and then temporarily stored at -80°C. They were then transported to a testing facility for 16-second sequencing. The sequencing steps are as follows:

[0118] (1) 1.1 Extraction of total DNA from the microbiome

[0119] The EZNA® Water DNA Kit was used to extract total DNA from skin samples using the microbiome method. The quality of DNA extraction was detected by agarose gel electrophoresis, and the DNA was quantified using a UV spectrophotometer.

[0120] (2) PCR amplification

[0121] The PCR amplification system is shown in Table 3:

[0122] Table 3 PCR amplification reaction system in the experimental examples

[0123]

[0124] In this experiment, a total of 4 primer pairs were used for PCR amplification reaction. The specific primer sequences are shown in Table 4.

[0125] Table 4 Primer pair information for PCR amplification reactions in the experimental examples

[0126]

[0127] The PCR amplification reaction conditions are shown in Table 5:

[0128] Table 5 PCR amplification reaction conditions in the experimental examples

[0129]

[0130] The sequences were amplified using the primer pairs given in Table 4, and the PCR products were identified by 2% agarose gel electrophoresis. Ultrapure water was used throughout the DNA extraction process to eliminate the possibility of false-positive PCR results serving as negative controls. PCR products were purified using AMPure XT beads (Beckman Coulter Genomics, Danvers, MA, USA) and quantified using Qubit (Invitrogen, USA). Amplicon pools were used for sequencing. The size and number of amplicon libraries were evaluated using an Agilent 2100 bioanalyzer (Agilent, USA) and Illumina (Kapa Biosciences, Woburn, MA, USA) library quantification kits, and then the libraries were sequenced on a NovaSeq PE250 platform.

[0131] (3) Bioinformatics analysis process

[0132] Samples were sequenced on the Illumina NovaSeq platform according to the manufacturer's recommendations, provided by LC-Bio. Paired-end sequences were assigned to samples based on their barcodes, and barcodes and primer sequences introduced during library construction were removed. Matched-end reads were merged using FLASH. The raw reads were quality filtered using fqtrim (v0.94) to obtain high-quality clean tags. Chimeric sequences were filtered using Vsearch software (v2.3.4), and then demodulated using DADA2 to obtain the feature table and feature sequences.

[0133] Diversity was calculated by normalizing to the same random sequences. Then, feature abundance was normalized using the relative abundance of each sample according to the SILVA (release 132) classifier. Alpha diversity was used to analyze the complexity of species diversity in the samples, employing five metrics calculated by QIIME2: Chao1, Observed species, Goodscovery, Shannon, and Simpson. Beta diversity was calculated by QIIME2 and plotted using the R package. Sequence alignment was performed using Blast, and each representative sequence was annotated with the SILVA database.

[0134] In-sample species diversity analysis results (alpha diversity) are as follows: Figure 1As shown, L-D0 / L-D14 / L-D28 represent the skin surface sampling results before, 14 days after, and 28 days after using the test cream containing +20% mesenchymal stem cell active peptide secretion extract. R-D0 / R-D14 / R-D28 represent the skin surface sampling results before, 14 days after, and 28 days after using the placebo control. Alpha diversity analysis measures the number of microbial species in a single sample (i.e., speciesrichness) and the proportion of each microbial species (i.e., evenness). The more microbial species a sample contains, the higher its richness; the more evenly the proportion of each microbial species, the higher the evenness. Chao reflects the evenness of species richness in the sample.

[0135] In practical graphical representations, violin plots combine features of box plots and density plots. The top left corner shows the test method used for the difference analysis and the calculated p-value. When p < 0.01, it is marked with **, indicating a highly significant difference; when p < 0.05, it is marked with *, indicating a significant difference; when p > 0.05, it is marked with ns, indicating no significant difference.

[0136] The results show that, before and after use, the baseline composition of the cream had a significant impact on microbial diversity in both the efficacy test group and the placebo group. However, at 14 and 28 days of use, there was no significant difference in overall microbial diversity between the efficacy test group and the placebo group, indicating that the mesenchymal stem cell active peptide secretion extract does not significantly alter the richness of skin surface microorganisms.

[0137] Based on the relative abundance table of sample species, the Kruskal-Wallistest method was used to compare species differences between two groups for comparisons of multiple groups with biological replicates. The species differences between the efficacy test group and the placebo group are as follows: Figure 2 and Figure 3 As shown, in Figure 2 and Figure 3 The top ten strains showing significant changes in both the efficacy test group and the placebo group were selected separately, such as... Figure 4 As shown.

[0138] Placebo represents the top 10 bacterial species with significant changes in the placebo group, while +20% ARRESO represents the top 10 bacterial species with significant changes in the test cream group containing mesenchymal stem cell active peptide secretion extract. The two groups showed significant differences at this level.

[0139] The following table lists the top 5 strains that showed significant changes and were clearly labeled in the scientific field:

[0140] Table 6. Top 5 strains with significant changes and clearly labeled scientific fields.

[0141]

[0142] As shown in Table 6, among the four strains, Staphylococcus hominis, Pseudomonas fluorescens, and Pseudomonas sp P6 were not reported to be harmful in literature searches and reports. Staphylococcus epidermidis is a probiotic, and its proportion in the mesenchymal stem cell active peptide secretion extract increased significantly over time. It has been reported that Staphylococcus epidermidis can effectively inhibit Propionibacterium acnes, thereby reducing the risk of acne, and can also effectively inhibit Staphylococcus aureus, thereby reducing the risk of sensitive skin. It can also effectively inhibit the risk of skin cancer caused by ultraviolet radiation. It is evident that the mesenchymal stem cell active peptide secretion obtained by the preparation method provided in Example 1 of this invention, when used in a skin microecological conditioning cream, can promote the growth of probiotics under the condition of the overall microbial community growing together. At the same time, it plays a regulatory role in the growth of more than ten kinds of microorganisms, including Staphylococcus hominis, Pseudomonas fluorescens, and Pseudomonas sp P6, thus playing a role in regulating the microecological environment of the skin surface.

[0143] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of umbilical cord mesenchymal stem cell active peptide secretions in the preparation of cosmetics for promoting the growth of Staphylococcus epidermidis strain on the skin surface, characterized in that, Based on 100g of secretions, the secretions include the following components: 0.1-1g of active peptides, 5-25mg of niacin, 0.2-0.6g of fat, 10-30mg of carbohydrates, 100-500μg of vitamin C, 25-65μg of vitamin A, and 1-5μg of vitamin E.

2. The application according to claim 1, characterized in that, The sugars include at least one of glucose, fructose, sucrose, lactose, or maltose.

3. The application according to claim 1, characterized in that, Based on 100g of secretions, the secretions include: 0.24g of active peptides, 17.32mg of niacin, 0.4g of fat, 23.6mg of carbohydrates, 314μg of vitamin C, 47.6μg of vitamin A and 2.7μg of vitamin E.

4. The application according to any one of claims 1 to 3, characterized in that, The method for preparing the secretion includes collecting the umbilical cord, separating Wharton's jelly, culturing it after disruption to obtain primary stem cells, and continuing to passage and culture the cells after the first passage reaches a fusion rate of 85% to 90%, and collecting the active peptide secretion.

5. The application according to claim 4, characterized in that, The collected umbilical cord was cut into segments, and blood vessels were removed to obtain Wharton's jelly.

6. The application according to claim 5, characterized in that, The length of the segment is 2cm.

7. The application according to claim 4, characterized in that, Primary stem cells were obtained by culturing Wharton's gel after it was broken into a paste. The breaking method was a physical method, including cutting or grinding.

8. The application according to claim 1, characterized in that, The cosmetics also include at least one of moisturizers, emulsifiers, thickeners, or preservatives.

9. The application according to claim 8, characterized in that, The moisturizer includes at least one of butylene glycol, glycerin, 1,3-propanediol, pentanediol, isopentanediol, diglycerol, triglyceride, polyglycerol, sorbitol, polyethylene glycol, polypropylene glycol, ethylene glycol, or diethylene glycol.

10. The application according to claim 8, characterized in that, The emulsifier includes at least one of stearic acid, cetearyl alcohol, cetearyl glucose, cetearyl alcohol ethylhexanoate, stearyl alcohol polyether-20, cetyl phosphate potassium, lanolin wax, or coconut oil PEG-10 ester.

11. The application according to claim 8, characterized in that, The thickener includes one or more of the following: C14-C22 alcohols, C12-C20 alkyl glucosides, carbomer, xanthan gum, gellan gum, gum arabic, sclerotium gum, hydroxymethyl cellulose, hydroxyethyl cellulose, acrylate / C10-30 alkanol acrylate crosspolymers, polyacrylate-13, ammonium acrylate and acrylamide copolymers, hydroxyethyl acrylates, sodium acryloyl dimethyl taurate copolymers, sodium polyacrylate, ammonium acryloyl dimethyl taurate / VP copolymers, or ammonium acryloyl dimethyl taurate / behenol polyether-25 methacrylate crosspolymers.

12. The application according to claim 8, characterized in that, The preservatives include at least one of methylparaben, ethylparaben, propylparaben, phenoxyethanol, chlorophenoxyethanol, potassium sorbate, sodium benzoate, benzoic acid, salicylic acid, imidazolidinyl urea, p-hydroxyacetophenone, capryloyl hydroxamic acid, pentylene glycol, hexanediol, or ethylhexylglycerin.

13. The application according to claim 8, characterized in that, The cosmetic product comprises, by weight, 15-25 parts of umbilical cord mesenchymal stem cell active peptide secretion, 10-20 parts of stearic acid, 1-3 parts of ethylhexyl palmitate, 0.5-2 parts of lanolin, 0.1-0.4 parts of cetearyl alcohol, 0.1-0.4 parts of cetearyl glucoside, 0.1-0.5 parts of C14-C22 alcohol, 0.1-0.5 parts of C12-C20 alkyl glucoside, 5-15 parts of butylene glycol, 1-5 parts of sorbitol, 0.5-2 parts of potassium hydroxide, 0.1-0.7 parts of phenoxyethanol, 0.1-0.5 parts of methylparaben, 0.05-0.2 parts of fragrance, and 40-50 parts of water.

14. The application according to claim 13, characterized in that, The cosmetic product comprises, by weight, 20 parts of umbilical cord mesenchymal stem cell active peptide secretion, 15 parts of stearic acid, 2 parts of ethylhexyl palmitate, 1 part of lanolin, 0.5 parts of cetearyl alcohol and cetearyl glucoside, 0.6 parts of C14-C22 alcohol and C12-C20 alkyl glucoside, 10 parts of butylene glycol, 3 parts of sorbitol, 1 part of potassium hydroxide, 0.5 parts of phenoxyethanol, 0.2 parts of methylparaben, 0.1 parts of fragrance, and 46.1 parts of water.

15. The application according to claim 8, characterized in that, The dosage forms of the cosmetics include creams, lotions, liquids, gels, oils, powders, muds, patches, films, or freeze-dried products.

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