CN110934815A - Anti-saccharification mask containing mesenchymal stem cell culture supernatant and preparation method thereof - Google Patents
Anti-saccharification mask containing mesenchymal stem cell culture supernatant and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant, which comprises the following steps: preparing a mesenchymal stem cell culture supernatant, preparing a base solution, adding the mesenchymal stem cell culture supernatant obtained in the step (1) into the base solution obtained in the step (2) after a high-speed mixer cools to room temperature, and carrying out high-speed mixing and stirring to obtain the anti-saccharification mask containing the mesenchymal stem cell culture supernatant, wherein the mask solution comprises: consists of 0.1 to 5 percent of mesenchymal stem cell culture supernatant and 95 to 99.9 percent of base solution by mass ratio. According to the invention, the mesenchymal stem cell culture supernatant is combined with the anti-glycation functional components, so that the skin improvement effect is better than that of the anti-glycation functional components such as the mesenchymal stem cell culture supernatant or resveratrol, carnosine, lipoic acid and the like, the skin elasticity is increased, the color and the glossiness of the skin are improved, the barrier function of the skin is improved, the water is locked and the moisture is preserved, the wrinkles and the skin texture are improved, and the skin problem is solved from the internal cell level.
Description
Technical Field
The invention relates to the technical field of cosmetics, in particular to an anti-saccharification mask containing mesenchymal stem cell culture supernatant and a preparation method thereof.
Background
Along with the social development and the improvement of living standard of people, people pay more attention to the self care and skin care, especially women, but the cosmetics in the market at present have various varieties, great effect difference and difficult discrimination of consumers. Some fine chemical skin care products are added with substances harmful to human skin, such as lead, mercury and the like, in order to pursue whitening effect, so that the problems of skin inflammation and infection are caused. On the other hand, the existing cosmetic products contain less bioactive substances, cannot fundamentally improve the growth state and metabolism of epidermal cells, and have limited repair effect on skin.
With the increase of human age, the activity of cells is weakened, and the metabolism function in vivo is deteriorated, thereby causing various phenomena such as aging and saccharification. Glycation is mainly due to the fact that excessive sugar ingested by a human body can be combined with protein structures in the body,product produced by birthThe aging substances, namely saccharification end products (AGEs), can intervene in human metabolism, cross-link protein structures, make skin difficult to repair, cause the skin color to be yellow and dark, loose, increase wrinkles and generate aging signs. Saccharification can also result in large oil output, large pores, acne, and rough and unsmooth skin. Anti-glycation cosmetic mainly containing resveratrol, carnosine, thioctic acid, etcHas antioxidant and whitening effects, and can improve skin saccharification.
The mesenchymal stem cells exist in various tissues and organs of an organism, are a group of pluripotent stem cells with multidirectional differentiation potential, comprise mesenchymal stem cells from bone marrow, umbilical cord, fat and the like, and have the characteristics of convenient material acquisition, easy amplification, low immunogenicity and the like. The existing research shows that the mesenchymal stem cells have the functions of tissue repair, immune function regulation and the like, and can secrete various growth factors, including epidermal growth factor EGF, basic fibroblast growth factor bFGF and the like. The mesenchymal stem cell culture supernatant contains the growth factor, can remarkably promote the proliferation of dermal fibroblasts and the generation of collagen and elastin, and has anti-aging potential in the field of cosmetics. At present, the method for culturing the mesenchymal stem cells and obtaining the culture supernatant of the mesenchymal stem cells still needs to be improved so as to meet the requirement of mass stable production.
The inventor finds that the anti-saccharification product provided by the prior art does not have a high-efficiency anti-saccharification facial mask containing mesenchymal stem cell culture supernatant. Based on this, the invention provides an anti-glycation mask which is superior to the existing anti-glycation components, has a good moisturizing effect, increases the skin elasticity, improves the skin color and glossiness, can obviously reduce fine lines, enables the skin to be fine and smooth, improves the skin inflammation and the like, and has an effect superior to a mask which is only added with anti-glycation components such as adipose mesenchymal stem cell culture supernatant or resveratrol, carnosine, lipoic acid and the like, thereby completing the invention.
Disclosure of Invention
The invention aims to provide an anti-saccharification mask containing mesenchymal stem cell culture supernatant and a preparation method thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant comprises the following steps:
(1) preparing a mesenchymal stem cell culture supernatant: pretreating a cell culture container by using fibronectin, sucking away the fibronectin after pretreatment, then paving the domesticated P1-P30 generation mesenchymal stem cells in the culture container, changing the liquid on the next day, collecting culture supernatant on the third day, and filtering the culture supernatant with a 0.22 mu m filter membrane for later use;
(2) preparing a base solution: heating a component A comprising 1-10% of glycerol, 1-4% of butanediol, 1-5% of 1, 2-pentanediol, 0.1-5% of dipropylene glycol, 0.5-5% of diglycerol, 0.5-4% of sucrose, 0.5-3% of sodium PCA, 0.01-1% of PEG-60 hydrogenated castor oil, 0.1-0.3% of xanthan gum and the balance of water to 80-85 ℃, carrying out high-speed mixing stirring, adding a component B comprising 0.1-0.5% of citric acid, 0.1-0.5% of sodium citrate and the balance of water when the high-speed mixer is cooled to below 40 ℃, carrying out high-speed mixing stirring, and then adding 0.1-0.5% of phenoxyethanol, 0.1-1% of betaine, 0.1-1% of hydroxyethyl cellulose, 0.0001-1% of carnosine, 0.0001-0.1% of lipoic acid, 0.0001-1% of resveratrol, 0.001-1% of grape seed extract, 0.001-1% of wild soybean extract, 0.001-1% of centella asiatica extract, 0.01-1% of hydrolyzed silk, 0.0001-1% of oligopeptide-5, 0.001-1% of hydrolyzed yeast extract, 0.001-0.3% of daily essence and the balance of water;
(3) and (3) after the high-speed mixer is cooled to room temperature, adding the mesenchymal stem cell culture supernatant in the step (1) into the base solution in the step (2), and carrying out high-speed mixing and stirring.
Preferably, the domestication method of the mesenchymal stem cells is serum-free culture medium culture.
Preferably, the tissue containing mesenchymal stem cells is derived from umbilical cord, placenta, fat, bone marrow or dental pulp of a healthy subject.
Preferably, the mesenchymal stem cells are extracted by washing the blood cells with phosphate buffer solution and cutting into pieces of about 1mm3Adding 2mL of 1mg/mL collagenase I digestive juice into the fine tissue blocks, transferring the mixture into a 15mL centrifuge tube, carrying out shaking digestion on a constant temperature shaking table at 37 ℃ for 30min, and adding the equal volume mesenchymal stem cell complete culture medium after no large tissue blocks existFinal (a Chinese character of 'gan')Stopping digestion, centrifuging at 1000rpm for 8min to settle cells, discardingRemoving supernatant and suspended adipose tissue, adding complete culture medium of adipose mesenchymal stem cells, inoculating in cell culture dish, and placing in CO at 37 deg.C2Culturing in an incubator, changing the culture solution for the first time after 48 hours, culturing for 4-7 days, then changing a new complete culture medium, culturing for 2-3 days again, discarding the culture solution when 80-90% fusion is achieved, washing the cells for three times by using a phosphate buffer solution, then adding a serum-free culture solution, continuously culturing for 48 hours, and carrying out subculture to obtain the mesenchymal stem cells cultured by the serum-free culture medium.
Preferably, the pH value of the phosphate buffer is 7.0-7.2.
Preferably, the density of the mesenchymal stem cells plated in the culture container is more than 1.8 x 104/cm2。
Preferably, the pretreatment using fibronectin is carried out at 37 ℃ for 1 hour.
Preferably, the fibronectin is iMatrix-511 fibronectin.
An anti-saccharification facial mask containing mesenchymal stem cell culture supernatant is characterized in that the facial mask liquid comprises: consists of 0.1 to 5 percent of mesenchymal stem cell culture supernatant and 95 to 99.9 percent of base solution by mass ratio.
Preferably, the base solution comprises a component A, a component B and a component C:
the component A comprises 1-10% of glycerol, 1-4% of butanediol, 1-5% of 1, 2-pentanediol, 0.1-5% of dipropylene glycol, 0.5-5% of diglycerol, 0.5-4% of cane sugar, 0.5-3% of PCA sodium, 0.01-1% of PEG-60 hydrogenated castor oil, 0.1-0.3% of xanthan gum and the balance of water in percentage by mass;
the component B comprises 0.1-0.5% of citric acid, 0.1-0.5% of sodium citrate and the balance of water in percentage by mass;
the component C comprises 0.1-0.5% of phenoxyethanol, 0.1-1% of betaine, 0.1-1% of hydroxyethyl cellulose, 0.0001-1% of carnosine, 0.0001-0.1% of lipoic acid, 0.0001-1% of resveratrol, 0.001-1% of grape seed extract, 0.001-1% of wild soybean extract, 0.001-1% of centella asiatica extract, 0.01-1% of hydrolyzed silk, 0.0001-1% of oligopeptide-5, 0.001-1% of hydrolyzed yeast extract, 0.001-0.3% of daily essence and the balance of water in percentage by mass.
Compared with the prior art, the invention has the beneficial effects that:
1. the facial mask provided by the invention comprises a mesenchymal stem cell culture supernatant and a base solution for promoting and maintaining biological activity. Through reasonable preparation process and formula, the components in the mask liquid interact, coordinate and synergize, good moisturizing effect is realized, skin color is improved, fine lines can be obviously reduced, the skin is fine and smooth, and the skin inflammation is improved. The active substances with various growth factors are produced by utilizing the culture supernatant of the mesenchymal stem cells, and the mask which has the effects of increasing skin elasticity, moisturizing, locking water, resisting inflammation and brightening the skin, is safe and has no toxic or side effect and the preparation method are provided by a special formula.
2. The invention combines the mesenchymal stem cell culture supernatant with the anti-glycation functional components, obtains better skin improvement effect than the anti-glycation functional components such as the mesenchymal stem cell culture supernatant or resveratrol, carnosine, lipoic acid and the like, brightens skin color, improves skin barrier function, locks water and preserves moisture, improves wrinkles, skin textures and the like, and solves the skin problem from the internal cell level.
3. The invention improves the existing method for culturing and collecting the stem cell culture solution, and improves the original method for culturing the stem cell by using the stem cell complete cell growth solution until 80-90% of cells are fused, abandoning the culture solution, washing the cells three times by using a phosphate buffer solution, then adding a serum-free culture solution for continuous culture for 72 hours, and collecting the serum-free culture solution into the method for acclimatizing the cells into the cells which normally grow under the serum-free culture medium, and under the condition of certain number of plates, continuously collecting the culture supernatant of the stem cells after the solution is changed for the second day of subculture, wherein the culture supernatant has good proliferation effect on adult dermal fibroblasts without influencing the activity of the adult dermal fibroblasts. In the process of culturing the mesenchymal stem cells, culture bottles pretreated by the adhesive protein are used for culturing, so that 1:7-1:10 times of cell amplification can be realized, and the requirement of large-scale stable production can be met.
Drawings
FIG. 1 is a graph showing the effect of continuously collecting culture supernatant of human adipose mesenchymal stem cells on proliferation of human dermal fibroblasts (HDF-a) according to the present invention;
FIG. 2 is a graph of skin moisture results according to the present invention;
FIG. 3 is a graph showing the results of the transepidermal water loss rate of the present invention;
FIG. 4 is a graph showing the results of skin wrinkles in accordance with the present invention;
FIG. 5 is a graph of skin complexion results of the present invention;
FIG. 6 is a process flow diagram.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Referring to fig. 1-6, the present invention provides a technical solution: a preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant comprises the following steps:
(1) preparing a mesenchymal stem cell culture supernatant: preparing a mesenchymal stem cell culture supernatant: pretreating a cell culture container by using fibronectin, wherein the cell culture container can adopt a cell culture dish, a cell culture bottle and other containers, sucking away the fibronectin after pretreatment, then paving the domesticated P1-generation mesenchymal stem cells in the culture container, changing the liquid on the next day, collecting culture supernatant on the third day, and filtering the culture supernatant with a 0.22-micron filter membrane for later use;
(2) preparing a base solution: heating a component A high-speed mixer comprising 8% of glycerol, 2% of butanediol, 1% of 1, 2-pentanediol, 0.2% of dipropylene glycol, 0.5% of diglycerol, 1% of sucrose, 0.2% of sodium PCA, 0.2% of PEG-60 hydrogenated castor oil, 0.2% of xanthan gum and the balance of water to 80 ℃ for high-speed mixing and stirring, adding a component B comprising 0.2% of citric acid, 0.2% of sodium citrate and the balance of water by mass when the high-speed mixer is cooled to 35 ℃ for high-speed mixing and stirring, then adding C components comprising 0.5% of phenoxyethanol, 0.3% of betaine, 0.15% of hydroxyethyl cellulose, 0.025% of carnosine, 0.03% of lipoic acid, 0.0005% of resveratrol, 0.2% of grape seed extract, 0.15% of wild soybean extract, 0.01% of centella asiatica extract, 0.25% of hydrolyzed silk, 0.01% of oligopeptide-5, 0.015% of hydrolyzed yeast extract, 0.14% of daily essence and the balance of water by mass ratio, and carrying out high-speed mixing and stirring;
(3) and (3) after the high-speed mixer is cooled to room temperature, adding the mesenchymal stem cell culture supernatant in the step (1) into the base solution in the step (2), and carrying out high-speed mixing and stirring.
Specifically, the domestication method of the mesenchymal stem cells is culture in a serum-free culture medium.
Specifically, the tissue containing the mesenchymal stem cells is derived from fat of a healthy person.
Specifically, the extraction method of the mesenchymal stem cells comprises washing blood cells with phosphate buffer solution, and cutting into pieces of about 1mm3Adding 2mL of 1mg/mL collagenase I digestive juice into the fine tissue blocks, transferring the mixture into a 15mL centrifuge tube, carrying out shaking digestion on a constant temperature shaking table at 37 ℃ for 30min, and adding the equal volume mesenchymal stem cell complete culture medium after no large tissue blocks existFinal (a Chinese character of 'gan')Stopping digestion, centrifuging at 1000rpm for 8min to settle cells, removing supernatant and suspended adipose tissue, adding complete culture medium of adipose-derived mesenchymal stem cells, inoculating into cell culture dish, and placing in CO at 37 deg.C2Culturing in an incubator, firstly changing the culture solution after 48 hours, after culturing for 4 days, changing a new complete culture medium, culturing for 2 days again, discarding the culture solution when 80% fusion is achieved, firstly washing the cells for three times by using a phosphate buffer solution, then adding a serum-free culture solution, continuously culturing for 48 hours, and carrying out subculture to obtain the mesenchymal stem cells cultured by the serum-free culture medium.
Specifically, the pH value of the phosphate buffer solution is 7.0.
Specifically, the density of the mesenchymal stem cells plated in the culture container is more than 1.9 multiplied by 104/cm2。
Specifically, the pretreatment using fibronectin was carried out at 37 ℃ for 1 hour.
Specifically, the fibronectin is iMatrix-511 fibronectin.
An anti-glycation mask containing mesenchymal stem cell culture supernatant, which comprises: consists of 0.1 percent of mesenchymal stem cell culture supernatant and 99.9 percent of base solution by mass ratio.
Specifically, the base solution comprises a component A, a component B and a component C:
the component A comprises 8% of glycerol, 2% of butanediol, 1% of 1, 2-pentanediol, 0.2% of dipropylene glycol, 0.5% of diglycerol, 1% of cane sugar, 0.2% of PCA sodium, 0.2% of PEG-60 hydrogenated castor oil, 0.2% of xanthan gum and the balance of water in percentage by mass;
the component B comprises 0.2 percent of citric acid, 0.2 percent of sodium citrate and the balance of water in percentage by mass;
the component C comprises 0.5% of phenoxyethanol, 0.3% of betaine, 0.15% of hydroxyethyl cellulose, 0.025% of carnosine, 0.03% of lipoic acid, 0.0005% of resveratrol, 0.2% of grape seed extract, 0.15% of wild soybean extract, 0.01% of centella asiatica extract, 0.25% of hydrolyzed silk, 0.01% of oligopeptide-5, 0.015% of hydrolyzed yeast extract, 0.14% of daily essence and the balance of water in percentage by mass.
Example 2
A preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant comprises the following steps:
(1) preparing a mesenchymal stem cell culture supernatant: pretreating a cell culture container by using fibronectin, sucking away the fibronectin after pretreatment, then paving the domesticated P15 generation mesenchymal stem cells in the culture container, changing the liquid on the next day, collecting culture supernatant on the third day, and filtering the culture supernatant by using a 0.22 mu m filter membrane for later use;
(2) preparing a base solution: heating a component A high-speed mixer comprising 10% of glycerol, 2% of butanediol, 1% of 1, 2-pentanediol, 0.2% of dipropylene glycol, 0.5% of diglycerol, 1% of sucrose, 0.5% of sodium PCA, 0.2% of PEG-60 hydrogenated castor oil, 0.2% of xanthan gum and the balance of water to 80 ℃ for high-speed mixing and stirring, adding a component B comprising 0.1% of citric acid, 0.1% of sodium citrate and the balance of water by mass when the high-speed mixer is cooled to 35 ℃ for high-speed mixing and stirring, then adding C components comprising 0.5% of phenoxyethanol, 0.3% of betaine, 0.15% of hydroxyethyl cellulose, 0.05% of carnosine, 0.03% of lipoic acid, 0.0005% of resveratrol, 0.25% of grape seed extract, 0.15% of wild soybean extract, 0.01% of centella asiatica extract, 0.25% of hydrolyzed silk, 0.01% of oligopeptide-5, 0.015% of hydrolyzed yeast extract, 0.14% of daily essence and the balance of water by mass ratio, and carrying out high-speed mixing and stirring;
(3) and (3) after the high-speed mixer is cooled to room temperature, adding the mesenchymal stem cell culture supernatant in the step (1) into the base solution in the step (2), and carrying out high-speed mixing and stirring.
Specifically, the domestication method of the mesenchymal stem cells is culture in a serum-free culture medium.
Specifically, the tissue containing the mesenchymal stem cells is derived from the umbilical cord of a healthy person.
Specifically, the extraction method of the mesenchymal stem cells comprises washing blood cells with phosphate buffer solution, and cutting into pieces of about 1mm3Adding 2mL of 1mg/mL collagenase I digestive juice into the fine tissue blocks, transferring the mixture into a 15mL centrifuge tube, carrying out shaking digestion on a constant temperature shaking table at 37 ℃ for 30min, and adding the equal volume mesenchymal stem cell complete culture medium after no large tissue blocks existFinal (a Chinese character of 'gan')Stopping digestion, centrifuging at 1000rpm for 8min to settle cells, removing supernatant and suspended adipose tissue, adding complete culture medium of adipose-derived mesenchymal stem cells, inoculating into cell culture dish, and placing in CO at 37 deg.C2Culturing in an incubator, firstly changing the culture solution after 48 hours, after culturing for 5 days, changing a new complete culture medium, culturing for 3 days again, when the fusion reaches 85%, discarding the culture solution, washing the cells for three times by using a phosphate buffer solution, then adding a serum-free culture solution, continuously culturing for 48 hours, and carrying out subculture to obtain the mesenchymal stem cells which can be cultured by the serum-free culture medium.
Specifically, the pH value of the phosphate buffer solution is 7.1.
Specifically, the density of the mesenchymal stem cells plated in the culture container is 3.6 multiplied by 104/cm2。
Specifically, the pretreatment using fibronectin was carried out at 37 ℃ for 1 hour.
Specifically, the fibronectin is iMatrix-511 fibronectin.
An anti-glycation mask containing mesenchymal stem cell culture supernatant, which comprises: consists of 0.25 percent of mesenchymal stem cell culture supernatant and 99.75 percent of base solution by mass ratio.
Specifically, the base solution comprises a component A, a component B and a component C:
the component A comprises 10% of glycerol, 2% of butanediol, 1% of 1, 2-pentanediol, 0.2% of dipropylene glycol, 0.5% of diglycerol, 1% of sucrose, 2% of PCA sodium, 0.2% of PEG-60 hydrogenated castor oil, 0.2% of xanthan gum and the balance of water in percentage by mass;
the component B comprises 0.1 percent of citric acid, 0.1 percent of sodium citrate and the balance of water in percentage by mass;
the component C comprises 0.5% of phenoxyethanol, 0.3% of betaine, 0.15% of hydroxyethyl cellulose, 0.05% of carnosine, 0.03% of lipoic acid, 0.0005% of resveratrol, 0.25% of grape seed extract, 0.15% of wild soybean extract, 0.01% of centella asiatica extract, 0.25% of hydrolyzed silk, 0.01% of oligopeptide-5, 0.015% of hydrolyzed yeast extract, 0.14% of daily essence and the balance of water in percentage by mass.
Example 3
A preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant comprises the following steps:
(1) preparing a mesenchymal stem cell culture supernatant: pretreating a cell culture container by using fibronectin, sucking away the fibronectin after pretreatment, then paving the domesticated P30 generation mesenchymal stem cells in the culture container, changing the liquid on the next day, collecting culture supernatant on the third day, and filtering the culture supernatant by using a 0.22 mu m filter membrane for later use;
(2) preparing a base solution: heating a component A high-speed mixer comprising 5% of glycerol, 4% of butanediol, 5% of 1, 2-pentanediol, 5% of dipropylene glycol, 5% of diglycerol, 4% of sucrose, 3% of sodium PCA, 1% of PEG-60 hydrogenated castor oil, 0.3% of xanthan gum and the balance of water to 80 ℃ for high-speed mixing and stirring, adding a component B comprising 0.5% of citric acid, 0.5% of sodium citrate and the balance of water by mass when the high-speed mixer is cooled to 35 ℃ for high-speed mixing and stirring, then adding C components comprising 0.25% of phenoxyethanol, 1% of betaine, 1% of hydroxyethyl cellulose, 1% of carnosine, 0.1% of lipoic acid, 1% of resveratrol, 1% of grape seed extract, 1% of wild soybean extract, 1% of centella asiatica extract, 1% of hydrolyzed silk, 1% of oligopeptide-5, 1% of hydrolyzed yeast extract, 0.3% of daily essence and the balance of water by mass ratio, and mixing and stirring at a high speed;
(3) and (3) after the high-speed mixer is cooled to room temperature, adding the mesenchymal stem cell culture supernatant in the step (1) into the base solution in the step (2), and carrying out high-speed mixing and stirring.
Specifically, the domestication method of the mesenchymal stem cells is culture in a serum-free culture medium.
Specifically, the tissue containing the mesenchymal stem cells is derived from the placenta of a healthy person.
Specifically, the extraction method of the mesenchymal stem cells comprises washing blood cells with phosphate buffer solution, and cutting into pieces of about 1mm3Adding 2mL of 1mg/mL collagenase I digestive juice into the fine tissue blocks, transferring the mixture into a 15mL centrifuge tube, carrying out shaking digestion on a constant temperature shaking table at 37 ℃ for 30min, and adding the equal volume mesenchymal stem cell complete culture medium after no large tissue blocks existFinal (a Chinese character of 'gan')Stopping digestion, centrifuging at 1000rpm for 8min to settle cells, removing supernatant and suspended adipose tissue, adding complete culture medium of adipose-derived mesenchymal stem cells, inoculating into cell culture dish, and placing in CO at 37 deg.C2Culturing in incubator, changing culture medium for the first time after 48 hr, culturing for 7 days, changing new complete culture medium, culturing for 3 days, discarding culture medium when 90% fusion is achieved, washing cells with phosphate buffer solution for three times, adding serum-free culture medium, culturing for 48 hr, and subculturing to obtain the final productCultured mesenchymal stem cells.
Specifically, the pH value of the phosphate buffer solution is 7.2.
Specifically, the density of the mesenchymal stem cells plated in the culture container is more than 2.1 x 104/cm2。
Specifically, the pretreatment using fibronectin was carried out at 37 ℃ for 1 hour.
An anti-saccharification facial mask containing mesenchymal stem cell culture supernatant is characterized in that the facial mask liquid comprises: the culture medium consists of 5% of mesenchymal stem cell culture supernatant and 95% of base solution by mass ratio.
Specifically, the base solution comprises a component A, a component B and a component C:
the component A comprises 5% of glycerol, 4% of butanediol, 5% of 1, 2-pentanediol, 5% of dipropylene glycol, 5% of diglycerol, 4% of sucrose, 3% of PCA sodium, 1% of PEG-60 hydrogenated castor oil, 0.3% of xanthan gum and the balance of water in percentage by mass;
the component B comprises 0.5 percent of citric acid, 0.5 percent of sodium citrate and the balance of water in percentage by mass;
the component C comprises 0.25% of phenoxyethanol, 1% of betaine, 1% of hydroxyethyl cellulose, 1% of carnosine, 0.1% of lipoic acid, 1% of resveratrol, 1% of grape seed extract, 1% of wild soybean extract, 1% of centella asiatica extract, 1% of hydrolyzed silk, 1% of oligopeptide-5, 1% of hydrolyzed yeast extract, 0.3% of daily essence and the balance of water in percentage by mass.
And (3) effect testing:
1. and evaluating the water replenishing, moisturizing and locking effects.
1.1 test population: gender and age were randomized, 24 in total, into 3 groups of 8 people each, each group tested one mask each.
1.2 test period: and 28 days.
1.3 test samples: example 1, comparative example 1 (facial mask consisting of base solution of example one without mesenchymal stem cell culture supernatant), and comparative example 2 (facial mask without resveratrol, carnosine and lipoic acid anti-glycation component), all samples were stored at low temperature of 4-8 ℃.
1.4 test conditions: the test environment is a constant temperature and humidity test room, the indoor temperature is kept at 20-22 ℃, and the humidity is kept at 40-60%.
1.5 test instruments: skin tester MP 580 (CK, Germany) and skin moisture test probe Corneometer CM 825 (CK, Germany), skin transepidermal moisture loss rate test probe Tewameter TM300 (CK, Germany).
1.6 test methods: the subjects did not use a functional analogue skin care and cosmetic on the face during and within one month before the test, and the testers used a test mask every night during the test period, which had to be kept cold in a home refrigerator (4-8 ℃). Before each test, the test subjects clean their faces with facial cleanser and makeup remover, sit still in the test room for 30 minutes until the skin is in accordance with the environment. The moisture content of the skin surface of the subject was measured using Corneometer CM 825 at the forehead and left and right cheeks, 3 measurements per area being averaged. The test for transepidermal water loss rate was performed on the skin surface of the subject using a Tewameter TM300, the test areas being the forehead and the left and right cheeks, and the average was taken 3 times. The test period is the first week, the second week and the fourth week before and after the mask is used. Comparing the results of the skin moisture and the trans-epidermal water loss rate (TEWL), wherein the increase of the skin moisture value represents that the skin moisturizing effect is good, and the smaller the TEWL value is, the less the moisture loss is, the better the skin barrier function is, and the stronger the water locking capacity is; conversely, the skin barrier function is impaired, the weaker the water-holding capacity.
1.7 test results: the test results are shown in fig. 2 and 3. According to the detection result, the skin moisture of the testee is obviously improved after the product of the example 1 is used, and the transepidermal water loss rate of the skin of the testee is obviously reduced after the product is used for 28 days, which means that the mask disclosed by the invention has good water replenishing, moisturizing and water locking effects, has the functions of repairing the skin barrier and preventing the skin epidermal water from being lost. Compared with the results of the comparative example 1 and the comparative example 2, the facial mask added with the mesenchymal stem cell culture supernatant component and the three anti-saccharification components of resveratrol, carnosine and lipoic acid has better moisturizing and skin barrier repair effects.
2. Evaluation of anti-wrinkle efficacy
2.1 test population: gender and age were randomized, 24 in total, into 3 groups of 8 people each, each group tested one mask each.
2.2 test period: 28 days
2.3 test samples: example 1, comparative example 1 (facial mask consisting of base solution of example one without mesenchymal stem cell culture supernatant), and comparative example 2 (facial mask without resveratrol, carnosine and lipoic acid anti-glycation component), all samples were stored at low temperature of 4-8 ℃.
2.4 test conditions: the test environment is a constant temperature and humidity test room, the indoor temperature is kept at 20-22 ℃, and the humidity is kept at 40-60%.
2.5 test instruments: VISIA seventh generation skin monitor (Canfield, USA).
2.6 test methods: the subjects did not use a functional analogue skin care and cosmetic on the face during and within one month before the test, and the testers used a test mask every night during the test period, which had to be kept cold in a home refrigerator (4-8 ℃). Before each test, the test subjects clean their faces with facial cleanser and makeup remover, sit still in the test room for 30 minutes until the skin is in accordance with the environment. VISIA was used to photograph the front, left, and right sides of the face of the subject, and the wrinkle detection data was analyzed by VISIA, and 3 times of data were averaged. The results compare, the lower the absolute number of skin wrinkles, representing a lower number of skin wrinkles.
2.7 test results: the test results are shown in fig. 4. As can be seen from the test results, the skin wrinkles of the subjects were significantly improved after the products of example 1 were used, and wrinkles of the corners of the eyes of the subjects were significantly reduced after 28 days of use, indicating that the anti-glycation mask of the present invention has anti-wrinkle and repairing effects. Compared with the results of the comparative example 1 and the comparative example 2, the mesenchymal stem cell culture supernatant component has better anti-wrinkle and repairing effects when being matched with resveratrol, carnosine and lipoic acid anti-saccharification components.
3. Skin complexion efficacy evaluation:
3.1 test population: gender and age were randomized, 24 in total, into 3 groups of 8 people each, each group tested one mask each.
3.2 test period: 28 days
3.3 test samples: example 1, comparative example 1 (facial mask consisting of base solution of example one without mesenchymal stem cell culture supernatant), and comparative example 2 (facial mask without resveratrol, carnosine and lipoic acid anti-glycation component), all samples were stored at low temperature of 4-8 ℃.
3.4 test conditions: the test environment is a constant temperature and humidity test room, the indoor temperature is kept at 20-22 ℃, and the humidity is kept at 40-60%.
3.5 test instruments: skin tester MP 580 (CK, germany) and skin color test probe colorimeter cl400 (CK, germany).
3.6 test methods: the subjects did not use a functional analogue skin care and cosmetic on the face during and within one month before the test, and the testers used a test mask every night during the test period, which had to be kept cold in a home refrigerator (4-8 ℃). Before each test, the test subjects clean their faces with facial cleanser and makeup remover, sit still in the test room for 30 minutes until the skin is in accordance with the environment. Subjects were tested for skin complexion using a Colorimeter CL400, with the test areas being the forehead and the left and right cheeks, and 3 measurements were averaged for each test area. The test period is the first week, the second week and the fourth week before and after the mask is used. The skin complexion results were compared to the mask before use, with an increase in the complexion value indicating a whitening of the skin.
3.7 test results: the test results are shown in fig. 5. According to the detection results, the skin complexion of the testee is obviously improved after the product of the example 1 is used, the skin complexion of the testee is obviously improved after the product is used for 28 days, and the anti-saccharification mask has the effect of improving the skin complexion. Compared with the results of comparative example 1 and comparative example 2, it can be seen that the mesenchymal stem cell culture supernatant component has a better skin color improvement effect when being combined with resveratrol, carnosine and lipoic acid anti-glycation components.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (10)
1. A preparation method of an anti-saccharification mask containing mesenchymal stem cell culture supernatant is characterized by comprising the following steps:
(1) preparing a mesenchymal stem cell culture supernatant: pretreating a cell culture container by using fibronectin, sucking away the fibronectin after pretreatment, then paving the domesticated P1-P30 generation mesenchymal stem cells in the culture container, changing the liquid on the next day, collecting culture supernatant on the third day, and filtering the culture supernatant with a 0.22 mu m filter membrane for later use;
(2) preparing a base solution: heating the A component comprising 1-10% of glycerol, 1-4% of butanediol, 1-5% of 1, 2-pentanediol, 0.1-5% of dipropylene glycol, 0.5-5% of diglycerol, 0.5-4% of sucrose, 0.5-3% of sodium PCA, 0.01-1% of PEG-60 hydrogenated castor oil, 0.1-0.3% of xanthan gum and the balance of water to 80-85 ℃ by a high-speed mixer, carrying out high-speed mixing stirring, adding the B component comprising 0.1-0.5% of citric acid, 0.1-0.5% of sodium citrate and the balance of water when the temperature of the high-speed mixer is cooled to below 40 ℃, carrying out high-speed mixing stirring, and then adding the B component comprising 0.1-0.5% of phenoxyethanol, 0.1-1% of betaine, 0.1-1% of hydroxyethyl cellulose, 0.0001-1% of carnosine, 0.lipoic acid-0.1% of, 0.0001-1% of resveratrol, 0.001-1% of grape seed extract, 0.001-1% of wild soybean extract, 0.001-1% of centella asiatica extract, 0.01-1% of hydrolyzed silk, 0.0001-1% of oligopeptide-5, 0.001-1% of hydrolyzed yeast extract, 0.001-0.3% of daily essence and the balance of water;
(3) and (3) after the high-speed mixer is cooled to room temperature, adding the mesenchymal stem cell culture supernatant in the step (1) into the base solution in the step (2), and carrying out high-speed mixing and stirring.
2. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the domestication method of the mesenchymal stem cells is culture in a serum-free culture medium.
3. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the tissue containing mesenchymal stem cells is derived from umbilical cord, placenta, fat, bone marrow or dental pulp of a healthy subject.
4. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the extraction method of the mesenchymal stem cells comprises washing blood cells with phosphate buffer solution, and cutting into pieces of about 1mm3Adding 2mL of 1mg/mL collagenase I digestive juice into the fine tissue blocks, transferring the mixture into a 15mL centrifuge tube, carrying out shaking digestion on a constant temperature shaking table at 37 ℃ for 30min, and adding the equal volume mesenchymal stem cell complete culture medium after no large tissue blocks existFinal (a Chinese character of 'gan')Stopping digestion, centrifuging at 1000rpm for 8min to settle cells, removing supernatant and suspended adipose tissue, adding complete culture medium of adipose-derived mesenchymal stem cells, inoculating into cell culture dish, and placing in CO at 37 deg.C2Culturing in an incubator, changing the culture solution for the first time after 48 hours, culturing for 4-7 days, then changing a new complete culture medium, culturing for 2-3 days again, discarding the culture solution when 80-90% fusion is achieved, washing the cells for three times by using a phosphate buffer solution, then adding a serum-free culture solution, continuously culturing for 48 hours, and carrying out subculture to obtain the mesenchymal stem cells cultured by the serum-free culture medium.
5. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the pH value of the phosphate buffer solution is 7.0-7.2.
6. Mesenchymal stem-containing composition according to claim 1The preparation method of the anti-saccharification mask of the cell culture supernatant is characterized by comprising the following steps: the density of the mesenchymal stem cells plated in the culture container is more than 1.8 multiplied by 104/cm2。
7. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the pretreatment conditions using fibronectin were 1 hour at 37 ℃.
8. The method for preparing an anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 1, wherein the method comprises the following steps: the fibronectin is iMatrix-511 fibronectin.
9. An anti-saccharification facial mask containing mesenchymal stem cell culture supernatant is characterized in that the facial mask liquid comprises: consists of 0.1 to 5 percent of mesenchymal stem cell culture supernatant and 95 to 99.9 percent of base solution by mass ratio.
10. The anti-glycation mask containing mesenchymal stem cell culture supernatant according to claim 7, wherein: the base solution comprises a component A, a component B and a component C:
the component A comprises 1-10% of glycerol, 1-4% of butanediol, 1-5% of 1, 2-pentanediol, 0.1-5% of dipropylene glycol, 0.5-5% of diglycerol, 0.5-4% of cane sugar, 0.5-3% of PCA sodium, 0.01-1% of PEG-60 hydrogenated castor oil, 0.1-0.3% of xanthan gum and the balance of water in percentage by mass;
the component B comprises 0.1-0.5% of citric acid, 0.1-0.5% of sodium citrate and the balance of water in percentage by mass;
the component C comprises 0.1-0.5% of phenoxyethanol, 0.1-1% of betaine, 0.1-1% of hydroxyethyl cellulose, 0.0001-1% of carnosine, 0.0001-0.1% of lipoic acid, 0.0001-1% of resveratrol, 0.001-1% of grape seed extract, 0.001-1% of wild soybean extract, 0.001-1% of centella asiatica extract, 0.01-1% of hydrolyzed silk, 0.0001-1% of oligopeptide-5, 0.001-1% of hydrolyzed yeast extract, 0.001-0.3% of daily essence and the balance of water in percentage by mass.
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