CN113559047A - Preparation method of beauty skin care product and skin care essence containing active factor extract secreted by mesenchymal stem cells - Google Patents
Preparation method of beauty skin care product and skin care essence containing active factor extract secreted by mesenchymal stem cells Download PDFInfo
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- CN113559047A CN113559047A CN202010349250.1A CN202010349250A CN113559047A CN 113559047 A CN113559047 A CN 113559047A CN 202010349250 A CN202010349250 A CN 202010349250A CN 113559047 A CN113559047 A CN 113559047A
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Images
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/60—Sugars; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/65—Collagen; Gelatin; Keratin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/67—Vitamins
- A61K8/678—Tocopherol, i.e. vitamin E
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
Abstract
The invention discloses a preparation method of a beauty skin care product and a beauty essence containing an active factor extract secreted by mesenchymal stem cells, wherein the beauty skin care product contains the active factor extract secreted by mesenchymal stem cells, bFGF basic fibroblast growth factor, water-soluble collagen, hyaluronic acid, VE vitamin E and trehalose; the weight ratio of active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE and trehalose is 0.02ug-20 ug: 10ng-5000 ng: 0.01g-20 g: 0.01g-3 g: 0.01g-10 g: 0.01g-20 g; the skin care product containing the extract of the active factors secreted by the mesenchymal stem cells can improve the defects of skin elasticity, aging resistance and the like, and meanwhile, the preparation method has the advantages of simple process and convenient operation.
Description
Technical Field
The invention relates to a beauty skin care product, in particular to a beauty skin care product containing an active factor extract secreted by mesenchymal stem cells and a preparation method of a skin care essence.
Background
The skin of modern people is affected by a large number of harmful factors such as ultraviolet rays, harmful microorganisms, various harmful chemical and physical factors and the like every day, and when the defense system and the repair system of the skin are damaged, the skin is damaged, so that various skin problems such as color spots, wrinkles, skin dryness, elasticity loss, sensitivity and the like are generated.
At present, the cosmetics are various in types, and are classified according to skin care types, and the cosmetics can be cleansing cream, facial cleanser, bath agent, hair washing and care agent, shaving cream and the like; although these cosmetics have a certain effect of improving the skin, it is difficult to improve the skin elasticity and anti-aging.
Disclosure of Invention
The invention aims to provide a preparation method of a beauty skin care product containing an active factor extract secreted by mesenchymal stem cells and a skin care essence.
In order to achieve the above object, the present invention provides a skin care product containing an active factor extract secreted by mesenchymal stem cells, comprising an active factor extract secreted by mesenchymal stem cells, bFGF basic fibroblast growth factor, water-soluble collagen, hyaluronic acid, VE vitamin E, trehalose;
wherein the weight ratio of the active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE and trehalose is 0.02ug-20 ug: 10ng-5000 ng: 0.01g-20 g: 0.01g-3 g: 0.01g-10 g: 0.01g to 20 g.
The invention also provides a preparation method of the skin care essence, wherein the preparation method comprises the following steps:
1) mixing bFGF, water-soluble collagen, VE and trehalose in water to obtain a mixture W1;
2) filtering and sterilizing the mixture W1 to obtain a mixture W2, and mixing the extract of the active factors secreted by the mesenchymal stem cells with the mixture W2 to obtain a mixture W3;
3) and mixing the sterile hyaluronic acid with the mixture W3 under sterile conditions to obtain the skin care essence.
In the technical scheme, the applicant finds that the traditional cosmetic ingredients cannot permeate into dermis to act on fibroblasts through creative work, and only can protect the skin from being damaged by the outside, and particularly the traditional cosmetic is difficult to ensure that the skin has a long-term moisturizing effect, so that the defects of skin elasticity, aging resistance and the like are difficult to improve.
A stem cell refers to a cell that is capable of self-renewal, division, and differentiation into other cell populations. From the stage of development of stem cells, stem cells can be classified into embryonic stem cells (ES cells) and adult stem cells (adult stem cells). Mesenchymal Stem Cells (MSCs) are a subgroup of adult stem cells, and can be derived from multiple tissues, such as bone marrow, fat, umbilical cord, etc., with all the commonalities of stem cells, i.e., self-renewal and multipotential differentiation capacity. MSCs have potential immunomodulatory functions, anti-inflammatory capabilities, and repair of tissue and cell damage. The repair of MSCs is mainly dependent on their differentiation and paracrine actions. In both of these modes, paracrine action is primarily used, rather than differentiation. The mesenchymal stem cells have unique cytokine secretion function, and the secreted active factors play an important role in repairing tissues and cells. The mesenchymal stem cells secrete at least chemokines (RANTES, SDF-1 alpha, fractalkine, MIP-1 alpha, MCP-1, MCP-2), immunoregulatory factors (HGF, LIF), nutritional factors supporting progenitor cells (IL-6, FGF-2, PDGF-AA, PDGF-BB), wound healing-related factors (IL-6, IL-8, TGF-beta 1, MCP-1, VEGF, GM-CSF, TIMP-1), scar-inhibiting factors (HGF, FGF-2), anti-apoptotic factors (VEGF165, FGF-2, HGF), vascular regeneration factors (VEGF165, FGF-2, PDGF-AA, PDGF-BB, EGF), collagen types (I, II, III, IV protein), lysozyme and the like. These cytokines can effectively regulate and control the cell signal transmission of the organism and activate the stem cells of the human body, thereby physiologically repairing or replacing the damaged, pathological and aged cells of the organism. For example, Vascular Endothelial Growth Factor (VEGF) promotes skin regeneration, tissue recovery and wound healing, regulates nutrients and epidermal cell proliferation; fibronectin (Fibronectin) has wound healing efficacy as a skin-forming component; transforming growth factor (TGF- β) restores damaged tissues and regulates the proliferation of immune cells. Hepatocyte Growth Factor (HGF) activates cells, and has wound treatment effect; fibroblast growth factor (bFGF) promotes skin regeneration, recovers aged tissues, treats wounds, regulates nutrients and epidermal cell proliferation, moves, regulates differentiation and the like.
The active factors can still keep high activity under the conditions of hypoxia and low nutrition, and the defect that stem cells are easy to die in a short period is overcome; meanwhile, the active factors are very easy to fuse with cell membranes of epidermal cells due to small diameters, so that the active factors enter receptor cells, provide substances and signal sources for cell regeneration and repair, further awaken and activate epidermal stem cells of which the skin basal layers are in a dormant state, strengthen cell regeneration, promote metabolism, enhance activity of epidermal cells, activate fibroblasts, repair collagen cells, enhance skin elasticity, remove wrinkles, resist aging and enhance water retention of skin.
bFGF can promote the growth of tissues and nerve cells and repair wound surfaces which are difficult to heal and damaged nerves; can induce a series of cell reactions for tissue repair, maintain the integrity of the skin, and keep the normal color and luster and fineness of the skin. BFGF can penetrate into skin cells, starts from the regulation mechanism of the cells, promotes the growth and differentiation of the cells, promotes the development of elastic fiber cells, enhances the function of the elastic fiber cells, induces the formation, development and differentiation of subcutaneous microvessels, thereby improving the microcirculation of the skin, and comprehensively improves the nutritional status of the skin in multiple layers to ensure that the skin reaches the optimal physiological state.
The collagen has skin whitening, moisturizing, wrinkle preventing, repairing, nourishing, weight reducing, and skin caring effects. Collagen is a water-insoluble fibrous glycoprotein in the connective tissue of the spinal animals, accounts for about 25 to 35% of the protein in the human body, is the main structural protein of the skin, accounts for more than 50%, has very important function in the skin of the human body, has good supporting force, is like a steel bar framework for supporting the skin tissue, and can ensure that the skin looks very rich. The collagen is similar to the structure of human skin collagen, contains a large amount of amino acids and hydrophilic groups in molecules, has certain surface activity and good compatibility, and when the collagen is used as an active substance in cosmetics, the active substance can be diffused to the deep layer of the skin to supplement the amino acids required by the human skin, so that the activity of the collagen in the skin is enhanced, the integrity of stratum corneum moisture and a fiber structure is maintained, the metabolism of skin tissues is promoted, and good moistening, moisturizing, wrinkle-removing and beautifying effects are generated on the skin.
VE, also known as tocopherol, is a fat-soluble vitamin that is liked by scientists as the "spring of youth" in myth. It is one of the most prominent natural antioxidants. VE can protect cell membranes from oxidative damage of free radicals, help repair and consolidate natural protective barriers of skin, and lock moisture in skin. Wrinkles often occur due to loss of subcutaneous fat, rupture of elastic fibers and degradation of connective collagen tissue. VE can inhibit lipid peroxidation, maintain tissue connection, and make skin smooth and elastic.
Trehalose is a stable non-reducing disaccharide, has stable performance, can form a special protective film on the surface of cells under severe conditions of high temperature, high cold, drying and dehydration and the like, and effectively protects the biomolecular structure from being damaged. The trehalose has small molecular weight, is easy to be absorbed by the skin, enters cells to play the unique water substitution stress factor effect and the function of protecting cell membranes, and improves the anti-drying and anti-freezing capacity of the cells, thereby improving the capacity of the skin adapting to the environment. In addition, trehalose can prevent oil and fat separation and protein denaturation; has radiation resistance and oxidation resistance; also has excellent water retentivity, hygroscopicity, etc.
Hyaluronic acid is one of the main components constituting extracellular matrix and intercellular substance, is an intercellular filler, and plays an important role in the form, structure and function of skin; has effects in keeping moisture, repairing, and nourishing.
The beauty skin care product provided by the invention can be skin-friendly and transdermal through the combined action of the active factor extract secreted by the mesenchymal stem cells and other components, so that effective substances can easily enter a skin basal layer, the epidermal stem cells of the skin basal layer in a dormant state are awakened and activated, metabolism is promoted, the activity of the epidermal cells is enhanced, fibroblasts are activated, collagen cells are repaired, the skin elasticity is enhanced, wrinkles are removed, aging is resisted, and the water-retaining property of the skin is enhanced; the product does not contain any preservative, essence, hormone and the like.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a morphological feature diagram of umbilical cord mesenchymal stem cells
FIG. 2 shows the result of flow-through assay of umbilical cord mesenchymal stem cells for P5 generation;
FIG. 3 is a detection report chart in detection example 1;
FIG. 4 is a graph showing the results of measuring the transdermal water loss value in test example 2;
FIG. 5 is a graph showing the results of measuring the skin hydration degree in test example 3.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a beauty skin care product containing an active factor extract secreted by mesenchymal stem cells, which contains the active factor extract secreted by the mesenchymal stem cells, bFGF basic fibroblast growth factor, water-soluble collagen, hyaluronic acid, VE vitamin E and trehalose;
wherein the weight ratio of the active factor extract secreted by the mesenchymal stem cells, EGF, bFGF, water-soluble collagen, hyaluronic acid, VE and trehalose is 0.02ug-20 ug: 10ng-5000 ng: 0.01g-20 g: 0.01g-3 g: 0.01g-10 g: 0.01g to 20 g.
In the present invention, the content of each component may be selected within a wide range, but in order to further improve the moisture retention of the prepared cosmetic skin care product to improve the elasticity and aging resistance of the skin, it is preferable that the weight ratio of the active factor extract secreted from the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose is 0.05ug to 5 ug: 500ng-2000 ng: 0.05g-8 g: 0.05g-1 g: 0.05g-1.2 g: 0.1g to 8 g.
In the present invention, the source of the extract of active factors secreted from the mesenchymal stem cells can be selected in a wide range, but in order to further improve the moisture retention of the prepared cosmetic skin care product to improve the skin's elasticity and aging resistance, it is preferable that the extract of active factors secreted from the mesenchymal stem cells is obtained by the following method:
1) continuously culturing the mesenchymal stem cell primary cells to obtain cells of P1 generation, P2 generation and P5 generation;
2) collecting culture solution supernatants from generation P3 to generation P5 to obtain crude active factor solution secreted by the mesenchymal stem cells;
3) centrifuging the crude active factor solution, collecting supernatant, and filtering to obtain an active factor extract secreted by the mesenchymal stem cells;
wherein the mesenchymal stem cell primary cell is a human umbilical cord-derived mesenchymal stem cell primary cell.
In the above method for obtaining an extract of an active factor secreted from mesenchymal stem cells, the density of the cells at the beginning of continuous culture may be selected within a wide range, but in order to improve the efficiency and effect of culture, it is preferable that, in step 1), the density of the cells at the beginning of continuous culture is 0.7 × 104-1.3×104Individual cell/cm2。
In the above method for obtaining an active factor extract secreted from mesenchymal stem cells, the medium used for continuous culture may be selected from a wide range, but in order to further improve the moisture retention of the obtained cosmetic skin care product to improve the skin elasticity and aging resistance, it is preferable that the medium used for continuous culture is a serum-free medium.
In the above method for obtaining the extract of active factors secreted from mesenchymal stem cells, the temperature of the centrifugation may be selected within a wide range, but in order to secure the stability of the extract of active factors secreted from mesenchymal stem cells, it is preferable that, in step 3), the temperature of the centrifugation is 2 to 8 ℃.
In the above method for obtaining an active factor extract secreted from mesenchymal stem cells, the step of centrifugation may be selected within a wide range, but in order to further remove residual cells and cell debris, it is preferable that the step of centrifugation is: first treated under the centrifugal force of 280-320g for 8-12min (mainly for removing residual cells), and then treated under the centrifugal force of 1900-2100g for 18-22min (mainly for removing cell debris).
In the above method for obtaining an active factor extract secreted from mesenchymal stem cells, the step of filtration may be selected within a wide range, but in order to further remove impurities, it is preferable that, in step 3), the step of filtration is: the supernatant was filtered through a 0.20-0.25 μm pore size filter, followed by a first ultrafiltration of the filtrate under a nitrogen blanket at 500 and 518kPa, and then repeated ultrafiltration 2-4 times with PBS solution.
In the ultrafiltration process, in order to further improve the ultrafiltration effect, the vortex height is preferably 0.3-0.4 of the liquid height (based on the liquid height) in each ultrafiltration process.
In the present invention, the type of the beauty skin care product can be selected in a wide range, but in order to further increase the range of use of the beauty skin care product, it is preferable that the beauty skin care product is divided into a solution, a gel, a suspension or an emulsion according to a dispersion system; preferably, the beauty skin care product is divided into cream, paste, milk, dry powder, wet powder or spray according to the shape. More preferably, the beauty and skin care product is skin care essence, and the dispersing agent of the skin care essence is water; the weight ratio of the active factor extract secreted by the mesenchymal stem cells to the water is 0.05ug-5 ug: 50g-150 g.
The invention also provides a preparation method of the skin care essence, wherein the preparation method comprises the following steps:
1) mixing bFGF, water-soluble collagen, VE and trehalose in water to obtain a mixture W1;
2) filtering and sterilizing the mixture W1 to obtain a mixture W2, and mixing the extract of the active factors secreted by the mesenchymal stem cells with the mixture W2 to obtain a mixture W3;
3) and mixing the sterile hyaluronic acid with the mixture W3 under sterile conditions to obtain the skin care essence.
In the above production method, the pore size of the filtration membrane used for filtration may be used in a wide range, but in order to further improve the filtration effect, it is preferable that in step 2), the pore size of the filtration membrane used for filtration is 0.1 to 0.3 μm;
in the above production method, the mixing conditions may be used in a wide range, but for further mixing effect, it is preferable that the mixing satisfies the following conditions: the mixing temperature is 0-4 ℃, and the mixing time is 4-8 h.
In the above preparation method, the supply manner of the extract of the active factor secreted from the mesenchymal stem cell may be used in a wide range, but for further mixing effect, it is preferable that the extract of the active factor secreted from the mesenchymal stem cell is supplied in the form of a solution, and the solvent is a PBS buffer solution.
The present invention will be described in detail below by way of examples. In the following examples, primary human umbilical cord-derived mesenchymal stem cells were obtained by the following method:
and (3) completely soaking and rinsing collected newborn umbilical cords qualified in health examination by using physiological saline containing double antibodies for 3 times, and completely soaking and rinsing by using physiological saline without double antibodies for 2 times. The rinsed umbilical cord was cut into 2cm pieces and rinsed three times with physiological saline without antibiotics. And (3) putting the cleaned umbilical cord segment into a new sterile culture dish, replacing two new forceps, cutting along the umbilical vein, and stripping the inner vein, the inner artery and the outer skin by using the two forceps to obtain the Wharton jelly. Cutting the peeled Wharton jelly into pieces of 1-4mm3Placing the tissue blocks with the sizes into a new 10cm culture dish, adding 15mL of complete culture medium, uniformly distributing the tissue blocks in the culture dish by using forceps, and then placing the culture dish into a carbon dioxide incubator for culture; after 5 days, the culture medium was replaced with a new one. And (3) climbing out the human umbilical cord mesenchymal stem cells from the tissue block after 7 to 10 days, and digesting the human umbilical cord mesenchymal stem cells by using pancreatin to obtain the human umbilical cord source mesenchymal stem cell primary cells when the cell density reaches 70 to 80 percent.
The morphological characteristic diagram of the umbilical cord mesenchymal stem cells is shown in figure 1, the P5 flow-induced detection result of the umbilical cord mesenchymal stem cells is shown in figure 2, the positive rates of CD14, CD19, CD34, CD45 and HLA-DR in figure 2 are all less than 2%, the positive rates of CD73 and CD90 are all more than 95%, and the purity is satisfactory. As can be seen from FIGS. 1-2, the umbilical cord mesenchymal stem cells with satisfactory morphology and phenotype can be obtained by the above-described operations according to the examples.
Preparation example 1
Obtaining an active factor extract secreted by human umbilical cord-derived mesenchymal stem cells:
the primary mesenchymal stem cell of human umbilical cord is treated according to the method of 104Individual cell/cm2The cells were inoculated into a culture flask and cultured continuously using a serum-free medium to obtain cells of P1 passage, P2 passage to P5 passage. Collecting culture solution from P3 generation to P5 generationAnd (4) clearing to obtain a crude solution of the active factors secreted by the human umbilical cord-derived mesenchymal stem cells. The crude liquid of the active factors secreted by the human umbilical cord-derived mesenchymal stem cells is subpackaged into 50ml centrifuge tubes, centrifuged at 4 ℃ for 10min under the centrifugal force of 300g to remove residual cells, centrifuged at 2000g for 20min to remove cell debris, the supernatant is carefully collected and filtered by a 0.22 mu m pore size filter to obtain a filtrate A1. A1 was placed in a Model8050 rotary ultrafilter equipped with a 100000NW-CO ultrafiltration membrane, nitrogen was switched on and the maximum inlet pressure was controlled below 517.125kPa, and the magnetic stirrer was switched on to make the vortex height 1/3 the liquid height. After completion of the A1 ultrafiltration, 50ml of PBS buffer was added and the ultrafiltration was repeated 3 times. After completion of ultrafiltration, the active factor on the ultrafiltration membrane was suspended in 0.5mL of PBS buffer solution and transferred to a 1.5mL Eppendorf tube for storage in a refrigerator at-80 ℃.
Example 1
1) Dissolving and uniformly mixing bFGF, water-soluble collagen, VE and trehalose by using ultrapure water to prepare a mixture W1;
2) filtering and sterilizing W1 through a filter membrane with 0.2 μm diameter micropore to obtain W2; adding a sterile solution of an active factor extract secreted by the human umbilical cord mesenchymal stem cells (prepared in preparation example 1) into the mixture W2 under aseptic operation, and uniformly mixing to obtain a mixture W3;
3) adding sterile sodium hyaluronate powder into the mixture W3 under aseptic operation, stirring at 0-4 ℃ for 6h, and carrying out aseptic subpackaging to obtain skin care essence;
wherein the weight ratio of the active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose and water is 1.5 ug: 1 ug: 3 g: 0.4 g: 0.5 g: 3 g: the weight ratio of the active factor extract secreted by the human umbilical cord mesenchymal stem cells to the PBS buffer solution in 100g of the active factor extract solution secreted by the human umbilical cord mesenchymal stem cells is 20 ug: 500 ug.
Example 2
A skin care essence was prepared in the manner of example 1, except that:
the weight ratio of active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose and water is 20 ug: 5 ug: 20 g: 3 g: 10 g: 20 g: the weight ratio of the active factor extract secreted by the human umbilical cord mesenchymal stem cells to the PBS buffer solution in 100g of the active factor extract solution secreted by the human umbilical cord mesenchymal stem cells is 20 ug: 500 ug.
Example 3
A skin care essence was prepared in the manner of example 1, except that:
the weight ratio of active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose and water is 0.02 ug: 10 ng: 0.01 g: 0.01 g: 0.01 g: 0.01 g: the weight ratio of the active factor extract secreted by the human umbilical cord mesenchymal stem cells to the PBS buffer solution in 100g of the active factor extract solution secreted by the human umbilical cord mesenchymal stem cells is 20 ug: 500 ug.
Example 4
A skin care essence was prepared in the manner of example 1, except that:
the weight ratio of active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose and water is 0.05 ug: 500 ng: 0.05 g: 0.05 g: 0.05 g: 0.1 g: the weight ratio of the active factor extract secreted by the human umbilical cord mesenchymal stem cells to the PBS buffer solution in 100g of the active factor extract solution secreted by the human umbilical cord mesenchymal stem cells is 20 ug: 500 ug.
Example 5
A skin care essence was prepared in the manner of example 1, except that:
the weight ratio of active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE, trehalose and water is 5 ug: 2000 ng: 8 g: 1 g: 1.2 g: 8 g: 100g of the total weight of the mixture; the weight ratio of the active factor extract secreted by the human umbilical cord mesenchymal stem cells to the PBS buffer solution in the active factor extract solution secreted by the human umbilical cord mesenchymal stem cells is 20 ug: 500 ug.
Comparative example
A skin care essence was prepared in the manner of example 1, except that: active factor extract and hyaluronic acid secreted by mesenchymal stem cells are not added in the formula.
Detection example 1
The products prepared in the examples 1 to 5 are sent to the Ningbo entry-exit inspection and quarantine bureau inspection and quarantine technical center, and the health safety test is carried out according to the technical specification of cosmetic safety (2015 edition); the detection results of example 1 are shown in FIG. 3, and the products prepared in examples 2-5 also meet the requirements of sanitary safety.
Detection example 2
TEWL determination: transdermal water loss (TEWL) measures the change in water vapor pressure adjacent to the skin surface. The measurement principle of the test instrument derives from fick's diffusion law. The moisture content of the epidermis is directly measured by measuring the near epidermis (within about 1 cm) through 2 groups of temperature and humidity sensors and the water vapor pressure gradient formed by the water loss of the cuticle at different two points. TEWL values represent the loss of water from the stratum corneum and are an important criterion for evaluating the barrier function of the stratum corneum. A lower TEWL value of the skin indicates a better barrier function of the skin, whereas the opposite is the worse.
And (3) testing environmental conditions: the test environment temperature is 22 ℃ plus or minus 1 ℃ and the humidity is 50 percent plus or minus 5 percent, and real-time dynamic monitoring is carried out.
Testing the population: 30 persons; age between 16 and 40 years; no serious systemic disease, no immunodeficiency or autoimmune disease; those with inactive allergic disease; those who have not had a history of severe allergy to skin care cosmetics. Any product (cosmetic or external medicine) cannot be used 2 days before the test site is measured, and water cannot be contacted within 2 hours.
Testing an instrument: tester for testing moisture loss of skin under Tewameter TM300 trademark
Test samples: examples 1 to 5, comparative example 1
Comparison: blank control (blank track)
A measurement step: before formal measurement, the patient should sit still in a room meeting the standard for 30min, and cannot drink water, and the forearm is exposed and placed in a measurement state to keep relaxed. In the experiment, the inner sides of the left and right arms are marked with 3cm multiplied by 3cm test areas, and the same arm can be simultaneously marked with a plurality of areasThe domains are spaced 1cm apart. Both the test product and the placebo were randomly distributed on the left and right arms. Blank values were measured for each test area and then measured as 2.0 mg. + -. 0.1mg sample/cm2The test product is evenly coated in the test area by using the latex finger sleeve. Each area was assayed in 5 replicates and averaged. TEWL values were measured before application (noted 0h) and at 1h, 2h, 3h and 4h before application, respectively. The test results are shown in FIG. 4.
As shown in FIG. 4, the values of the water loss through skin of the sample (marked as 0h) before smearing (sequentially comprising the products of examples 1-5 and comparative example 1) and the blank control are as follows: 46.2, 45.7, 45.5, 45.6, 45.9, 45.8, 45.6. When the samples (products of examples 1-5 and comparative example 1 in sequence) are used for 4 hours, the values of the skin permeation water loss of the essence (products of examples 1-5 and comparative example 1 in sequence) are respectively as follows: 13.4, 22.5, 25.1, 18.6, 16.5, 26.6, all decreased to different extents as compared to the initial blank; the specific results are shown in FIG. 5.
As can be seen from FIG. 4, the products of examples 1, 4 and 5 have lower decrease in the transdermal water loss value compared to the products of examples 2 to 3, which shows that the essence of the present invention can effectively reduce the transdermal water evaporation of the skin, can improve the barrier function of the stratum corneum of the skin, and has a direct or indirect moisturizing effect on the skin.
Detection example 3
Skin stratum corneum hydration degree measurement:
the test environmental conditions, test population, test sample, control and test example 2 were the same.
Testing an instrument: corneometer CM 825 skin moisture tester
A measurement step: before formal measurement, the patient should sit still in a room meeting the standard for 30min, and cannot drink water, and the forearm is exposed and placed in a measurement state to keep relaxed. In the experiment, the inner sides of the left and right arms are marked with 3cm multiplied by 3cm test areas, and the same arm can be simultaneously marked with a plurality of areas at intervals of 1 cm. Exposing the detected area 10min before the test, and measuring the blank value of each test area as blank control. Both test products and controls were then randomly distributed on the left and right arms. Then 2.0 mg. + -. 0.1mg of sample/cm2Using latex finger-cotThe test product is uniformly coated in the test area. Each area was assayed in 5 replicates and averaged. After application, skin hydration values were measured at 0h, 0.5h, 1h, 1.5h, 2h and 3h, respectively. The skin hydration change rate was calculated. Skin hydration change rate (degree of skin hydration increase) ═ skin hydration (test value) -skin hydration (control))/skin hydration (control) × 100% test results: as shown in fig. 5.
Even though the skin hydration degree is the greatest after the essence is used, the skin hydration degree is rapidly reduced as the moisture in the essence is rapidly evaporated from the skin surface. Thereafter, the moisturizing component acts and the hydration degree gradually decreases to be stable. After the essence (the products of examples 1-5 and comparative example 1 in sequence) is used for 3 hours, the skin hydration degree is respectively improved compared with the original skin hydration degree: 47.1%, 34.0%, 26.1%, 15.0%, 13.9%, 12.1%; the specific results are shown in FIG. 5. The results show that the essence of the invention can effectively moisturize the skin, while the moisturizing effect of the product of example 1 is most obvious, and the moisturizing effects of the products of examples 2-3 are general.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A skin care product containing the extract of active factors secreted by mesenchymal stem cells is characterized by comprising the extract of active factors secreted by mesenchymal stem cells, bFGF basic fibroblast growth factor, water-soluble collagen, hyaluronic acid, VE vitamin E and trehalose;
wherein the weight ratio of the active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE and trehalose is 0.02ug-20 ug: 10ng-5000 ng: 0.01g-20 g: 0.01g-3 g: 0.01g-10 g: 0.01g to 20 g.
2. The cosmetic skin care product according to claim 1, wherein the weight ratio of the active factor extract secreted by the mesenchymal stem cells, bFGF, water-soluble collagen, hyaluronic acid, VE and trehalose is 0.05ug-5 ug: 500ng-2000 ng: 0.05g-8 g: 0.05g-1 g: 0.05g-1.2 g: 0.1g to 8 g.
3. The cosmetic skin care product according to claim 1, wherein the extract of active factors secreted from the mesenchymal stem cells is obtained by the following method:
1) continuously culturing the mesenchymal stem cell primary cells to obtain cells of P1 generation, P2 generation and P5 generation;
2) collecting culture solution supernatants from generation P3 to generation P5 to obtain crude active factor solution secreted by the mesenchymal stem cells;
3) centrifuging the crude active factor solution, collecting supernatant, and filtering to obtain an active factor extract secreted by the mesenchymal stem cells;
wherein the mesenchymal stem cell primary cell is a human umbilical cord-derived mesenchymal stem cell primary cell.
4. The cosmetic skin care preparation according to claim 3, wherein, in step 1), the density of cells at the beginning of the continuous culture is 0.7 x 104-1.3×104Individual cell/cm2；
Preferably, the medium used for continuous culture is a serum-free medium.
5. The cosmetic skin care product according to claim 3, wherein, in step 3), the temperature of the centrifugation treatment is 2-8 ℃;
preferably, the centrifugation step is: first treated under the centrifugal force of 280-320g for 8-12min, and then treated under the centrifugal force of 1900-2100g for 18-22 min.
6. The cosmetic skin care product according to claim 3, wherein in step 3), the step of filtering is: filtering the supernatant through a filter with the aperture of 0.20-0.25 mu m, performing primary ultrafiltration on the filtrate under the protection of 500-518kPa and nitrogen, and adding PBS solution to perform repeated ultrafiltration for 2-4 times;
preferably, the vortex height is 0.3-0.4 of the liquid height per ultrafiltration.
7. The cosmetic skin care product according to claim 1, wherein the cosmetic skin care product is divided into a solution, a gel, a suspension or an emulsion according to a dispersion system;
preferably, the beauty skin care product is divided into cream, paste, milk, dry powder, wet powder or spray according to the shape.
8. The beauty skin care product according to claim 1, wherein the beauty skin care product is a skin care essence, and a dispersant of the skin care essence is water; the weight ratio of the active factor extract secreted by the mesenchymal stem cells to the water is 0.05ug-5 ug: 50g-150 g.
9. A method of preparing the skin care essence according to claim 8, wherein the method comprises:
1) mixing bFGF, water-soluble collagen, VE and trehalose in water to obtain a mixture W1;
2) filtering and sterilizing the mixture W1 to obtain a mixture W2, and mixing the extract of the active factors secreted by the mesenchymal stem cells with the mixture W2 to obtain a mixture W3;
3) and mixing the sterile hyaluronic acid with the mixture W3 under sterile conditions to prepare the skin care essence.
10. The production method according to claim 9, wherein, in step 2), the filtration membrane used for the filtration has a pore size of 0.1 to 0.3 μm;
preferably, the mixing satisfies the following condition: the mixing temperature is 0-4 ℃, and the mixing time is 4-8 h;
preferably, the extract of the active factors secreted by the mesenchymal stem cells is provided by a solution, and the solvent is PBS buffer solution.
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| CN114681387A (en) * | 2022-03-16 | 2022-07-01 | 和携科技有限公司 | Animal-derived exosome composition and application thereof in skin wound repair |
| CN116392575A (en) * | 2023-03-22 | 2023-07-07 | 广东唯泰生物科技有限公司 | Preparation for treating acne by combining stem cell active ingredients with hyaluronic acid |
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| CN110840758A (en) * | 2019-10-10 | 2020-02-28 | 湖南源品细胞生物科技有限公司 | Essence containing umbilical cord mesenchymal stem cell factor and preparation method thereof |
| CN110898078A (en) * | 2019-12-09 | 2020-03-24 | 上海市东方医院（同济大学附属东方医院） | Preparation and application of mesenchymal stem cell secretory factor |
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| CN106074356A (en) * | 2016-08-04 | 2016-11-09 | 深圳市合康生物科技股份有限公司 | A kind of preparation method and applications of living cells essence |
| CN110840758A (en) * | 2019-10-10 | 2020-02-28 | 湖南源品细胞生物科技有限公司 | Essence containing umbilical cord mesenchymal stem cell factor and preparation method thereof |
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| CN116392575A (en) * | 2023-03-22 | 2023-07-07 | 广东唯泰生物科技有限公司 | Preparation for treating acne by combining stem cell active ingredients with hyaluronic acid |
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