A cosmetic composition containing stem cell extract and its preparation method and application

Abstract

The present invention proposes a cosmetic composition containing a stem cell extract, a preparation method and an application thereof, and belongs to the field of cosmetic technology. Stem cells are separated from umbilical cord tissue, and then cultured with the culture medium to separate stem cells, and exosomes are extracted from the supernatant. The stem cells and exosomes are mixed and embedded to prepare embedded microspheres, which are stirred and mixed with sodium hyaluronate and collagen peptides to obtain a cosmetic composition containing a stem cell extract. The cosmetic composition containing a stem cell extract prepared by the present invention can significantly improve the storage stability of stem cells and exosomes, extend the storage period, and the stem cells and exosomes are widely available, and the preparation method is simple. They have good anti-wrinkle, skin elasticity promotion, whitening, moisturizing and other effects on the skin. Together with sodium hyaluronate and collagen peptides, they have a good improvement effect on the skin.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a cosmetic composition containing a stem cell extract, and a preparation method and application thereof. Background Art

[0002] With the development of society and modern science, people's material living standards have greatly improved, and people's pursuit of beauty and youth has become higher and higher. How to effectively improve the skin and keep the skin relatively young is the goal that the beauty and cosmetics industry has been pursuing. At present, most of the cosmetics on the market only replenish the nutrition of the skin surface or cover the aging skin, but cannot really penetrate into the dermis of the cells effectively, cannot deeply improve the aging and dullness of the skin, and cannot inhibit the aging of skin cells and promote the production of new cells.

[0003] Mesenchymal stem cells (MSCs) are a type of multipotent stem cells derived from the mesoderm, with strong proliferation ability and multidirectional differentiation potential. Under a suitable environment, they have the ability to differentiate into multiple cells such as muscle cells, hepatocytes, osteoblasts, adipocytes, etc. MSCs can be isolated and prepared from bone marrow, fat, and umbilical cord. Studies have shown that compared with MSCs from other sources, umbilical cord-derived MSCs have many advantages such as rich sources, convenient collection, strong proliferation and differentiation ability, low immunogenicity, and no ethical issues. They play an extremely important role in the field of stem cell research and application. Recent research reports have shown that the repair function of MSCs on tissues and organs depends not only on its ability to self-replicate and differentiate, but also on the large amount of cytokines it secretes to regulate the microenvironment in the body and activate dormant stem cells in the patient's body, thereby achieving the effect of improving the body's pathological and physiological conditions. Mesenchymal stem cells cultured in vitro can also release a large number of cytokines, such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), stem cell growth factor (SCF), fibroblast growth factor (bFGF), epidermal growth factor (EGF), transforming growth factor β1 (TGF-β1), insulin-like growth factor (IGF), tissue matrix metalloproteinase inhibitor 1 (TIMP-1), etc. These factors have been proven to have the functions of scavenging free radicals, anti-oxidation, wound repair, enhancing skin's anti-allergic ability, delaying skin aging, synthesizing collagen, and maintaining sufficient collagen fibers and elastic fibers. Therefore, the application of stem cell active factors in the field of medical cosmetology will subvert the traditional cosmetics industry.

[0004] Although stem cells have a very significant effect on the skin, due to the limitations of existing technologies, it is difficult for cell preparations to act on the skin during the optimal period of action. In addition, as in vitro active preparations, cell preparations are greatly affected by timeliness and viability. How to further enhance the effect of stem cells, extend their shelf life, and reduce the allergenicity of animal-derived ingredients will be an urgent problem to be solved in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a cosmetic composition containing a stem cell extract, a preparation method and an application thereof, which significantly improves the storage stability of stem cells and exosomes and prolongs the storage period. The stem cells and exosomes have a wide range of sources and a simple preparation method. They have good anti-wrinkle, skin elasticity, whitening, moisturizing and other effects on the skin. Together with sodium hyaluronate and collagen peptides, they have a good improvement effect on the skin.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a method for preparing a cosmetic composition containing a stem cell extract. The method comprises adding fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 into a DMEM culture medium to prepare a culture medium, separating stem cells from umbilical cord tissue, culturing with the culture medium to separate stem cells, extracting exosomes from the supernatant, embedding the stem cells and the exosomes to prepare embedded microspheres, and stirring and mixing the microspheres with sodium hyaluronate and collagen peptide to obtain a cosmetic composition containing a stem cell extract.

[0008] As a further improvement of the present invention, the following steps are included:

[0009] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium;

[0010] S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium for culture. The tissue clumps are removed. After the cells adhere to the wall and grow to 80-90% fusion, the cells are centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in the culture medium, and frozen to obtain a stem cell solution.

[0011] S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes;

[0012] S4. Embedding: After the stem cell solution in step S2 is revived, the exosomes prepared in step S3 are added, sodium alginate is added, stirred and mixed evenly, emulsified, calcium chloride solution is added dropwise, solidified at room temperature, centrifuged, washed, and dried to obtain embedded microspheres;

[0013] S5. Preparation of a cosmetic composition containing a stem cell extract: Sodium hyaluronate, collagen peptide and embedded microspheres are stirred and mixed uniformly to prepare a cosmetic composition containing a stem cell extract.

[0014] As a further improvement of the present invention, the concentration of fetal bovine serum in the culture medium in step S1 is 10-20wt% of fetal bovine serum, 5-15nmol / L of dexamethasone, 100-120U / mL of penicillin, 100-120μg / mL of streptomycin, 50-70μg / mL of melatonin, 5-10μg / mL of metformin, and 10-20μg / mL of interleukin-6.

[0015] As a further improvement of the present invention, the digestion solution in step S2 is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, a CO2 concentration of 3-6v / v%, and the culture is carried out for 2-3 weeks.

[0016] As a further improvement of the present invention, the ultra-high speed centrifugation method combined with the ultrafiltration tube method in step S3 is as follows: the cell supernatant is centrifuged at 4°C, 500-1000 r / min for 10-20 min to remove cells, and then centrifuged at 3-5°C, 3000-4000 r / min for 15-25 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C, 12000-15000 r / min for 20-30 min to remove large vesicles and proteins, and the supernatant is concentrated by ultrafiltration tube. Filter with a 0.22 μm filter membrane, centrifuge at 4°C, 30000-35000 r / min for 50-70 min, resuspend the precipitate in PBS with pH = 6.7-7, centrifuge again at 4°C, 30000-35000 r / min for 50-70 min, and the precipitate obtained is exosomes, which is resuspended in PBS with pH = 6.7-7. Perform protein quantification according to the BCA kit steps to control the protein concentration to 80-100 mg / L and store in a -80°C refrigerator.

[0017] As a further improvement of the present invention, the mass ratio of the stem cell solution, exosomes, and sodium alginate in step S4 is 10-20:1-2:5-7.

[0018] As a further improvement of the present invention, the mass ratio of sodium hyaluronate, collagen peptide and embedded microspheres in step S5 is 2-3:1-2:4-6.

[0019] The present invention further protects a cosmetic composition containing stem cell extract obtained by the above preparation method.

[0020] The present invention further protects the use of the above-mentioned cosmetic composition containing stem cell extract in the preparation of cosmetics.

[0021] As a further improvement of the present invention, the cosmetic has anti-wrinkle, whitening and moisturizing effects.

[0022] The present invention has the following beneficial effects:

[0023] Autophagy is a cell protection process that degrades misfolded proteins and damaged organelles under starvation or hypoxia conditions to maintain cell homeostasis. The present invention prepares a culture medium by adding melatonin, metformin and interleukin-6, wherein melatonin can scavenge free radicals, reduce oxidative stress damage to cells, inhibit apoptosis-related signaling pathways, reduce stem cell apoptosis, and promote the growth of umbilical cord stem cells by affecting intracellular signal transduction pathways, such as MAPK, PI3K / Akt, etc.; metformin can promote endothelial cell angiogenesis, increase the level of stem cell autophagy to promote vascularization, thereby improving the ability of stem cells to adapt in vitro. IL-6 inhibits cell apoptosis, binds to its receptor IL-6R to form a complex, and then binds to the membrane protein gp130 to initiate intracellular signal transduction, improve the proliferation, survival and differentiation ability of stem cells, and the three have a synergistic effect.

[0024] The prepared stem cells and exosomes are embedded in sodium alginate. In the sodium alginate-embedded microspheres, sodium alginate can replace water molecules to form hydration bonds with hydrophilic groups on cell membranes and proteins, thereby avoiding structural changes of cell membranes and protein molecules and maintaining the stability of their structures and functions, so that stem cells can be preserved for a long time, thereby improving their storage stability, and also improving the storage stability of exosomes.

[0025] The cosmetic composition containing stem cell extract prepared by the present invention can significantly improve the storage stability of stem cells and exosomes and prolong the storage period. The stem cells and exosomes have a wide range of sources and a simple preparation method. They have good anti-wrinkle, skin elasticity promotion, whitening, moisturizing and other effects on the skin. Together with sodium hyaluronate and collagen peptide, they have a good improvement effect on the skin. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Trypsin, 200,000 U / g, and alkaline protease, 200,000 U / g, were purchased from Xiasheng (Beijing) Enzyme Biotechnology Development Co., Ltd.

[0028] Sodium hyaluronate, glucuronic acid content 46-48%, content greater than 97%, purchased from Zhejiang Yicun Biotechnology Co., Ltd.

[0029] The collagen peptides were fish skin collagen peptides with a content of >99%, purchased from Shandong Guoyao Peptide Valley Health Technology Co., Ltd. Example 1

[0030] This embodiment provides a method for preparing a cosmetic composition containing a stem cell extract, comprising the following steps:

[0031] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium;

[0032] The concentration of fetal bovine serum in the culture medium is 10 wt %, the content of dexamethasone is 5 nmol / L, the content of penicillin is 100 U / mL, the content of streptomycin is 100 μg / mL, the content of melatonin is 50 μg / mL, the content of metformin is 5 μg / mL, and the content of interleukin-6 is 10 μg / mL;

[0033] S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution for 30 minutes, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium in step S1. The culture is carried out at 36°C and a CO2 concentration of 3v / v% for 2 weeks. The tissue paste is removed, and the cells are grown to 80-90% confluence after attachment, centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested with 0.25wt% trypsin for 30 minutes, resuspended in the culture medium, and frozen to obtain a stem cell solution;

[0034] The digestion solution is a PBS buffer with a pH of 7.5 containing 1wt% trypsin and 0.5wt% alkaline protease;

[0035] S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes;

[0036] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 500 r / min for 10 min to remove cells, and then centrifuged at 4°C and 3000 r / min for 15 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 12000 r / min for 20 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 30000 r / min for 50 min, and the precipitate is resuspended in PBS with pH=6.7, and centrifuged again at 4°C and 30000 r / min for 50 min. The obtained precipitate is exosomes, which is resuspended in PBS with pH=6.7, and the protein concentration is controlled to be 80 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator;

[0037] S4. Encapsulation: After resuscitating 10 g of the stem cell solution in step S2, add 1 g of the exosomes prepared in step S3, add 5 g of sodium alginate, 0.5 g of lecithin and 1 L of water, stir and mix for 20 min, drop into fish oil, emulsify at 5000 r / min for 20 min, add 10 mL of 1 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain embedded microspheres;

[0038] S5. Preparation of a cosmetic composition containing a stem cell extract: 2 g of sodium hyaluronate, 1 g of collagen peptide and 4 g of embedded microspheres were stirred and mixed for 20 min to prepare a cosmetic composition containing a stem cell extract. Example 2

[0039] This embodiment provides a method for preparing a cosmetic composition containing a stem cell extract, comprising the following steps:

[0040] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium;

[0041] The concentration of fetal bovine serum in the culture medium is 20wt%, the content of dexamethasone is 15nmol / L, the content of penicillin is 120U / mL, the content of streptomycin is 120μg / mL, the content of melatonin is 70μg / mL, the content of metformin is 10μg / mL, and the content of interleukin-6 is 20μg / mL;

[0042] S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution for 30 minutes, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium in step S1. The culture is carried out at 38°C and a CO2 concentration of 6v / v% for 3 weeks. The tissue blocks are removed, and the cells are grown to 80-90% confluence after attachment. The cells are centrifuged and the supernatant is collected. The solid is washed with D-Hank's solution, digested with 0.25wt% trypsin for 30 minutes, resuspended in the culture medium, and frozen to obtain a stem cell solution.

[0043] The digestion solution is a PBS buffer solution with a pH of 7.7 containing 2wt% trypsin and 1wt% alkaline protease;

[0044] S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes;

[0045] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 20 min to remove cells, and then centrifuged at 4°C and 4000 r / min for 25 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 15000 r / min for 30 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 35000 r / min for 70 min, the precipitate is resuspended in PBS with pH=7, and centrifuged again at 4°C and 35000 r / min for 70 min. The precipitate obtained is exosomes, which is resuspended in PBS with pH=7, and the protein concentration is controlled to be 100 mg / L according to the steps of the BCA kit, and the protein is frozen in a refrigerator at -80°C;

[0046] S4. Encapsulation: After resuscitating 20 g of the stem cell solution in step S2, add 2 g of the exosomes prepared in step S3, add 7 g of sodium alginate, 0.5 g of lecithin and 1 L of water, stir and mix for 20 min, drop into fish oil, emulsify at 5000 r / min for 20 min, add 10 mL of 1 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain embedded microspheres;

[0047] S5. Preparation of a cosmetic composition containing a stem cell extract: 3 g of sodium hyaluronate, 2 g of collagen peptide and 6 g of embedded microspheres were stirred and mixed for 20 min to prepare a cosmetic composition containing a stem cell extract. Example 3

[0048] This embodiment provides a method for preparing a cosmetic composition containing a stem cell extract, comprising the following steps:

[0049] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium;

[0050] The concentration of fetal bovine serum in the culture medium is 10wt% fetal bovine serum, 10nmol / L dexamethasone, 110U / mL penicillin, 110μg / mL streptomycin, 60μg / mL melatonin, 7μg / mL metformin, and 15μg / mL interleukin-6;

[0051] S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution for 30 minutes, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium in step S1. The culture is carried out at 37°C and a CO2 concentration of 5v / v% for 3 weeks. The tissue blocks are removed, and the cells are grown to 80-90% confluence after attachment. The cells are centrifuged and the supernatant is collected. The solid is washed with D-Hank's solution, and digested with 0.25wt% trypsin for 30 minutes, resuspended in the culture medium, and frozen to obtain a stem cell solution.

[0052] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0053] S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes;

[0054] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13500 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The precipitate obtained is exosomes, which is resuspended in PBS with pH=6.7, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator;

[0055] S4. Embedding: After resuscitating 15 g of the stem cell solution in step S2, add 1.5 g of the exosomes prepared in step S3, add 6 g of sodium alginate, 0.5 g of lecithin and 1 L of water, stir and mix for 20 min, drop into fish oil, emulsify at 5000 r / min for 20 min, add 10 mL of 1 wt% calcium chloride solution, solidify at room temperature for 30 min, centrifuge, wash, and dry to obtain embedded microspheres;

[0056] S5. Preparation of a cosmetic composition containing a stem cell extract: 2.5 g of sodium hyaluronate, 1.5 g of collagen peptide and 5 g of embedded microspheres were stirred and mixed for 20 min to prepare a cosmetic composition containing a stem cell extract.

[0057] Comparative Example 1

[0058] Compared with Example 3, the difference is that melatonin is not added in step S1.

[0059] The details are as follows:

[0060] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, metformin and interleukin-6 were added to DMEM medium to prepare a culture medium;

[0061] The concentration of fetal bovine serum in the culture medium is 10wt% fetal bovine serum, 10nmol / L dexamethasone, 110U / mL penicillin, 110μg / mL streptomycin, 7μg / mL metformin, and 15μg / mL interleukin-6.

[0062] Comparative Example 2

[0063] Compared with Example 3, the difference is that metformin is not added in step S1.

[0064] The details are as follows:

[0065] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin and interleukin-6 were added to DMEM medium to prepare a culture medium;

[0066] The concentration of fetal bovine serum in the culture medium is 10wt% fetal bovine serum, 10nmol / L dexamethasone, 110U / mL penicillin, 110μg / mL streptomycin, 60μg / mL melatonin, and 15μg / mL interleukin-6.

[0067] Comparative Example 3

[0068] Compared with Example 3, the difference is that interleukin-6 is not added in step S1.

[0069] The details are as follows:

[0070] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, and metformin were added to DMEM culture medium to prepare a culture medium;

[0071] The concentration of fetal bovine serum in the culture medium is 10wt% fetal bovine serum, 10nmol / L dexamethasone, 110U / mL penicillin, 110μg / mL streptomycin, 60μg / mL melatonin, and 7μg / mL metformin.

[0072] Comparative Example 4

[0073] Compared with Example 3, the difference is that step S4 is not performed.

[0074] The details are as follows:

[0075] S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium;

[0076] The concentration of fetal bovine serum in the culture medium is 10wt% fetal bovine serum, 10nmol / L dexamethasone, 110U / mL penicillin, 110μg / mL streptomycin, 60μg / mL melatonin, 7μg / mL metformin, and 15μg / mL interleukin-6;

[0077] S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution for 30 minutes, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium in step S1. The culture is carried out at 37°C and a CO2 concentration of 5v / v% for 3 weeks. The tissue blocks are removed, and the cells are grown to 80-90% confluence after attachment. The cells are centrifuged and the supernatant is collected. The solid is washed with D-Hank's solution, and digested with 0.25wt% trypsin for 30 minutes, resuspended in the culture medium, and frozen to obtain a stem cell solution.

[0078] The digestion solution is a PBS buffer with a pH of 7.6 containing 1.5wt% trypsin and 0.7wt% alkaline protease;

[0079] S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes;

[0080] The ultra-high speed centrifugation method combined with the ultrafiltration tube method is as follows: the cell supernatant is centrifuged at 4°C and 1000 r / min for 15 min to remove cells, and then centrifuged at 4°C and 3500 r / min for 20 min to remove cell debris, etc., the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 13500 r / min for 25 min to remove large vesicles and proteins, the supernatant is filtered with a 0.22 μm filter membrane, and finally centrifuged at 4°C and 32000 r / min for 60 min, the precipitate is resuspended in PBS with pH=6.8, and centrifuged again at 4°C and 32000 r / min for 60 min. The precipitate obtained is exosomes, which is resuspended in PBS with pH=6.7, and the protein concentration is controlled to be 90 mg / L according to the steps of the BCA kit, and the protein is frozen in a -80°C refrigerator;

[0081] S4. Mixing: 15 g of the resuscitated stem cell solution in step S2 and 1.5 g of the exosomes obtained in step S3 were mixed to obtain a mixture;

[0082] S5. Preparation of a cosmetic composition containing a stem cell extract: 2.5 g of sodium hyaluronate, 1.5 g of collagen peptide and 5 g of the mixture were stirred and mixed for 20 min to prepare a cosmetic composition containing a stem cell extract.

[0083] Comparative Example 5

[0084] Compared with Example 3, the difference is that no stem cell solution is added in step S4.

[0085] The details are as follows:

[0086] S4. Encapsulation: 1.5 g of the exosomes prepared in step S3, 6 g of sodium alginate, 0.5 g of lecithin and 1 L of water were mixed evenly, stirred for 20 min, emulsified at 5000 r / min for 20 min, 10 mL of 1 wt% calcium chloride solution was added dropwise, cured at room temperature for 30 min, centrifuged, washed and dried to obtain encapsulated microspheres.

[0087] Comparative Example 6

[0088] Compared with Example 3, the difference is that no exosomes are added in step S4.

[0089] The details are as follows:

[0090] S4. Encapsulation: After resuscitating 15 g of the stem cell solution in step S2, add 6 g of sodium alginate, 0.5 g of lecithin and 1 L of water, stir and mix for 20 min, emulsify at 5000 r / min for 20 min, add 10 mL of 1 wt% calcium chloride solution dropwise, cure at room temperature for 30 min, centrifuge, wash, and dry to obtain encapsulated microspheres.

[0091] Test Example 1 Human skin patch test and allergy test

[0092] Test samples: cosmetic compositions containing stem cell extracts prepared in Examples 1-3 and Comparative Examples 1-6 of the present invention, prepared into 1 mg / mL sample solutions.

[0093] Experimental method: Subjects aged 24-57 were selected and randomly divided into groups, with 6 people in each group. Among them, the subject's right forearm flexor side was marked with a 5cm×5cm area and medical disinfection was used to create an injury in the marked area. The method was: 36-needle microneedle introduced the sample solution for 30s, and the residual liquid was gently wiped with a cotton ball soaked in physiological saline to keep the marked area dry; the test sample solution was placed in the 1-9 spot tester wells, with a dosage of 10-20μL. Bathing was prohibited during the test, and the normal reaction was negative. The test sample was distributed to the subject and acted on the subject's face, neck and back of the ear. The skin condition of the subject was observed after 10 minutes.

[0094] The experimental results are shown in Table 1.

[0095] Table 1

[0096]

[0097] As can be seen from the above table, the patch reactions of the cosmetic compositions containing stem cell extracts prepared in Examples 1-3 of the present invention and Comparative Examples 1-6 are negative reactions, and they are safe to use without any allergic reactions.

[0098] Test Example 2

[0099] Female subjects aged 22-49 were selected on a voluntary basis and randomly divided into 9 groups, 6 people in each group, respectively, Examples 1-3 and Comparative Examples 1-6. No drugs or cosmetics irrelevant to the test were applied during the test, and the subjects used samples on both sides of their faces. The sample group used the corresponding cosmetic composition solution containing stem cell extract (1 mg / mL). Once a day for a total of 1 week. On the 8th day, before the test, the subjects washed the test area with clean water, wiped it dry, and sat quietly for 30 minutes in a constant environment of 25±0.5℃ and relative humidity of 50±5%. Each group was tested 5 times and the average value was taken.

[0100] The results are shown in Table 2.

[0101] 1. Skin moisturizing test

[0102] The stratum corneum moisture content was measured before the start of the experiment, and the stratum corneum moisture content of the subjects was tested after using the samples on the 8th day, and the change rate (%) of skin moisture content was calculated.

[0103] Skin moisture content change rate (%) = (moisture content after use - moisture content before use) / moisture content before use × 100%

[0104] 2. Skin elasticity test

[0105] Skin elasticity and roughness parameters were tested before the start of the experiment. One hour after using the samples on the seventh day, the stratum corneum water content of the subjects was tested, and the change rate (%) of skin water content was calculated.

[0106] Skin elasticity change rate (%) = (skin elasticity after use - skin elasticity before use) / skin elasticity before use × 100%

[0107] Roughness change rate (%) = (roughness after use - roughness before use) / roughness before use × 100%

[0108] Table 2

[0109]

[0110] As can be seen from the above table, the cosmetic compositions containing stem cell extracts prepared in Examples 1-3 of the present invention have good moisturizing, improving skin elasticity, reducing skin roughness, and anti-wrinkle effects.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a cosmetic composition containing a stem cell extract, characterized in that: The following steps are involved: S1. Preparation of culture medium: fetal bovine serum, dexamethasone, penicillin, streptomycin, melatonin, metformin and interleukin-6 were added to DMEM culture medium to prepare a culture medium; S2. In vitro culture of stem cells: The excised umbilical cord tissue is washed with tissue protection solution to remove residual fat, connective tissue and blood, and then disinfected with medical alcohol. The tissue is then digested with digestive solution, centrifuged, and the supernatant is discarded. The digested tissue is collected, washed, and added to the culture medium for culture. The tissue clumps are removed. After the cells adhere to the wall and grow to 80-90% fusion, the cells are centrifuged, and the supernatant is collected. The solid is washed with D-Hank's solution, digested, resuspended in the culture medium, and frozen to obtain a stem cell solution. S3. Extraction of exosomes: The supernatant in step S2 is extracted with ultracentrifugation combined with ultrafiltration tube method to extract exosomes; S4. Embedding: After the stem cell solution in step S2 is revived, the exosomes prepared in step S3 are added, sodium alginate and an emulsifier are added, the mixture is stirred and evenly mixed, the mixture is added to fish oil, emulsified, a calcium chloride solution is added dropwise, the mixture is solidified at room temperature, centrifuged, washed, and dried to prepare embedded microspheres; the mass ratio of the stem cell solution, exosomes, and sodium alginate is 10-20:1-2:5-7; S5. Preparation of a cosmetic composition containing a stem cell extract: Sodium hyaluronate, collagen peptide and embedded microspheres are stirred and mixed uniformly to prepare a cosmetic composition containing a stem cell extract.

2. The preparation method according to claim 1, characterized in that: The concentration of fetal bovine serum in the culture medium in step S1 is 10-20wt% fetal bovine serum, 5-15nmol / L dexamethasone, 100-120U / mL penicillin, 100-120μg / mL streptomycin, 50-70μg / mL melatonin, 5-10μg / mL metformin, and 10-20μg / mL interleukin-6.

3. The preparation method according to claim 1, characterized in that: The digestion solution in step S2 is a PBS buffer with a pH of 7.5-7.7 containing 1-2wt% trypsin and 0.5-1wt% alkaline protease; the culture conditions are 36-38°C, a CO2 concentration of 3-6v / v%, and the culture is carried out for 2-3 weeks.

4. The preparation method according to claim 1, characterized in that: The ultra-high speed centrifugation method combined with the ultrafiltration tube method in step S3 is as follows: the cell supernatant is centrifuged at 4°C and 500-1000r / min for 10-20min to remove cells, and then centrifuged at 3-5°C and 3000-4000r / min for 15-25min to remove cell debris, the supernatant is concentrated by ultrafiltration tube, and centrifuged at 4°C and 12000-15000r / min for 20-30min to remove large vesicles and proteins, and the supernatant is filtered with a 0.22μm filter membrane, and finally centrifuged at 4°C and 30000-35000r / min for 50-70min, and the precipitate is resuspended in PBS with pH=6.7-7, and centrifuged again at 4°C and 30000-35000r / min for 50-70min. The precipitate obtained is exosomes, which is resuspended in PBS with pH=6.7-7, and the protein concentration is controlled to be 80-100mg / L according to the steps of the BCA kit, and stored in a refrigerator at -80°C.

5. The preparation method according to claim 1, characterized in that: The mass ratio of sodium hyaluronate, collagen peptide and embedded microspheres in step S5 is 2-3:1-2:4-6.

6. A cosmetic composition containing a stem cell extract obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the cosmetic composition containing stem cell extract according to claim 6 in the preparation of cosmetics.

8. The use according to claim 7, characterized in that: The cosmetic has the effects of anti-wrinkle, whitening and moisturizing.

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