CN110548001A - Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes - Google Patents
Repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes Download PDFInfo
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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
- A61K8/55—Phosphorus compounds
- A61K8/553—Phospholipids, e.g. lecithin
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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
- A61K8/67—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
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/68—Sphingolipids, e.g. ceramides, cerebrosides, gangliosides
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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
- A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
- A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
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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
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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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
- 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
- A61K2800/20—Chemical, physico-chemical or functional or structural properties of the composition as a whole
- A61K2800/30—Characterized by the absence of a particular group of ingredients
Abstract
the invention discloses a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes, which comprises the following components: mesenchymal stem cell exosomes, mannitol, EDTA-2Na, xanthan gum, butylene glycol, glycerol, water-soluble jojoba oil, olive oil, polydimethylsiloxane, hydroxyethylcellulose, hydrogenated lecithin, bacitracin sodium, PCA Na, urea, hyaluronic acid, β -glucan, stem cell secretin, serum albumin, ceramide-2 and water, further comprising: jojoba oil, squalane, shea butter, caprylic/capric triglyceride, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract, and vitamin E acetate. The repair anti-aging skin care product containing the umbilical cord mesenchymal stem cell exosome can effectively ensure the activity of the stem cell exosome, furthest ensure the activity of factors under the condition of no preservative and emulsifier, and can better repair skin barriers by matching with various skin maintenance components.
Description
Technical Field
the invention relates to the technical field of cosmetics, in particular to a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes.
Background
exosome (Exosome) is a vesicle-like corpuscle secreted by cells to the outside of cells, has a diameter of 30-150 nanometers, has a typical lipid bilayer membrane structure, and can exist in biological fluids such as cell culture supernatant, plasma, serum, saliva, urine, amniotic fluid and the like; it carries important information of various proteins, lipids, DNA and RNA of the mother cell, and plays an important role in substance and information transmission between cells. The existing research proves that the exosome secreted by the mesenchymal stem cell plays an important role in the aspects of promoting skin injury repair, relieving inflammatory reaction, promoting blood vessel regeneration and the like. Research shows that the mesenchymal stem cells construct a steady-state tissue repair microenvironment by releasing a large amount of exosomes, so that inflammatory reaction is eliminated, and wound healing is promoted. When the mesenchymal stem cell exosome is applied to skin repair and anti-aging, the problem that effective preservation of active ingredients before application is difficult to solve is solved, and the emulsifier and the preservative of the product also have certain influence on the efficacy of the active ingredients.
In order to better preserve the active ingredients of the mesenchymal stem cell exosomes and better exert the skin repairing and anti-aging effects of the mesenchymal stem cell exosomes, the invention provides a repairing and anti-aging skin care product containing the umbilical cord mesenchymal stem cell exosomes and a preparation method thereof.
disclosure of Invention
Based on the technical problems in the background art, the invention provides a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes, which has the advantages that active ingredients can be well stored, the effects of promoting skin hyperplasia capability, improving skin elasticity, improving skin quality and the like are achieved, the skin repair anti-aging effect of the umbilical cord mesenchymal stem cell exosomes can be better exerted, and anaphylactic reaction can not be caused.
The technical scheme of the invention is as follows:
A repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following components: mesenchymal stem cell exosomes, mannitol, EDTA-2Na, xanthan gum, butylene glycol, glycerol, water-soluble jojoba oil, olive oil, polydimethylsiloxane, hydroxyethylcellulose, hydrogenated lecithin, bacitracin sodium, PCA Na (sodium pyrrolidone carboxylate), urea, hyaluronic acid, β -glucan, stem cell secretin, serum albumin, ceramide-2, and water.
The skin care product for repairing and resisting senility of the umbilical cord mesenchymal stem cell exosome comprises the following components in percentage by weight: 0.1-1% of mesenchymal stem cell exosome, 5-7% of mannitol, 0.05-0.25% of EDTA-2Na, 0.10-0.25% of xanthan gum, 3-5% of butanediol, 1-3% of glycerol, 0.3-0.6% of water-soluble jojoba oil, 2-10% of olive oil, 1.5-2.5% of polydimethylsiloxane, 0.4-0.6% of hydroxyethyl cellulose, 1.0-2.0% of hydrogenated lecithin, 0.4-0.8% of bacillus subtilis lipopeptide sodium, 0.4-0.6% of PCA Na, 0.2-0.5% of urea, 0.4-0.6% of hyaluronic acid, 0.2-0.5% of beta-glucan, 2-5% of dry cell secretin, 0.05-0.15% of serum albumin, 20.05-0.15% of ceramide and the balance of water.
Preferably, the skin care product for repairing and resisting aging containing the umbilical cord mesenchymal stem cell exosome further comprises the following components: jojoba oil (which is not a substance other than water-soluble jojoba oil, and is an oily raw material), squalane, shea butter, caprylic/capric triglyceride, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract, and vitamin E acetate.
Preferably, the repair anti-aging skin care product containing the umbilical cord mesenchymal stem cell exosome comprises the following components in percentage by weight: 0.1-1% of mesenchymal stem cell exosome, 5-7% of mannitol, 0.05-0.25% of EDTA-2Na, 0.10-0.25% of xanthan gum, 3-5% of butanediol, 1-3% of glycerol, 0.3-0.6% of water-soluble jojoba oil, 2-10% of olive oil, 1.5-2.5% of polydimethylsiloxane, 0.4-0.6% of hydroxyethyl cellulose, 1.0-2.0% of hydrogenated lecithin, 0.4-0.8% of bacillus subtilis lipopeptide sodium, 0.4-0.6% of PCA Na, 0.2-0.5% of urea, 0.4-0.6% of hyaluronic acid, 0.2-0.5% of beta-glucan, 2-5% of dry cell secretin, 0.05-0.15% of serum albumin, 20.05-0.15% of ceramide, 1.5-2.5% of jojoba oil, 0.3-0.6% of squalane, 0.8% of shea butter, 0.1.2-0.5% of caprylic acid potassium bikolate, 0.02-0.03% of retinol palmitate, 0.4-0.6% of centella asiatica extract, 0.4-0.6% of vitamin E acetate and the balance of water.
Preferably, the mesenchymal stem cell exosome is a mixture of multiple active factors secreted by human umbilical cord mesenchymal stem cells.
further preferably, the preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. Taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. Inoculating cells into a DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the liquid once at intervals and removing non-adherent cells, digesting the cells for subculture when the cell fusion rate reaches 80% -90%, centrifuging to remove cells, debris and the like when the cell fusion rate reaches the required number, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), mixing uniformly, incubating for 8-14 hours at 4 ℃, centrifuging, discarding the supernatant, resuspending the precipitate with a PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, resuspending the precipitate with the PBS buffer solution to obtain the mesenchymal stem cell exosomes, and subpackaging at-80 ℃.
Preferably, in the step B, a 100-mesh filter screen is used.
preferably, the polyethylene glycol is polyethylene glycol 8000.
The preparation method of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) adding EDTA-2Na into a proper amount of water, adding xanthan gum, heating to 75-80 ℃, adding butanediol, glycerol and water-soluble jojoba oil after the xanthan gum is completely hydrated, and uniformly mixing to obtain a phase A for later use;
(3) Mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 60-65 ℃ for later use;
(5) dissolving hydroxyethyl cellulose in appropriate amount of water at 40 deg.C to obtain transparent gel (C phase); adding the transparent gel (phase C) into the material obtained in the step (4), cooling to 35-40 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, an asiatic pennywort herb extract and vitamin E acetate and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the compound.
preferably, in the step (1), the vacuum freeze-drying is performed in a specific operation mode: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
The invention has the advantages that: the repair anti-aging skin care product containing the umbilical cord mesenchymal stem cell exosome can effectively ensure the activity of the stem cell exosome, furthest ensure the activity of factors under the condition of no preservative and emulsifier, and can better repair skin barriers by matching with various skin maintenance components.
Detailed Description
Example 1
A preparation method of a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) Adding EDTA-2Na into appropriate amount of water, adding xanthan gum, heating to 78 deg.C, adding butanediol, glycerol and water soluble jojoba oil after xanthan gum is completely hydrated, and mixing to obtain phase A;
(3) mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 62 ℃ for later use;
(5) dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 36 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract and vitamin E acetate and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the composition.
in the step (1), the specific operation mode of the vacuum freeze drying is as follows: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
The mesenchymal stem cell exosome is a mixture of various active factors secreted by human umbilical cord mesenchymal stem cells.
The preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. Taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. inoculating cells into a DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing nonadherent cells, digesting the cells for subculture when the cell fusion rate reaches 82%, centrifuging to remove cells, debris and the like when the cell fusion rate reaches the required number, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), mixing uniformly, incubating for 12 hours at 4 ℃, centrifuging, discarding the supernatant, re-suspending and precipitating by using a PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, re-suspending and precipitating by using the PBS buffer solution to obtain the mesenchymal stem cell exosomes, subpackaging and storing at-80 ℃.
And in the step B, filtering the mixture by a 100-mesh filter screen.
The polyethylene glycol is polyethylene glycol 8000.
The components of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging are shown in the table 1.
Table 1: the component composition of the restorative anti-aging skin care product of example 1;
Example 2
a preparation method of a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) Adding EDTA-2Na into appropriate amount of water, adding xanthan gum, heating to 80 deg.C, adding butanediol, glycerol and water soluble jojoba oil after xanthan gum is completely hydrated, and mixing to obtain phase A;
(3) mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) Adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 60 ℃ for later use;
(5) dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 40 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract and vitamin E acetate and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the composition.
In the step (1), the specific operation mode of the vacuum freeze drying is as follows: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
the mesenchymal stem cell exosome is a mixture of various active factors secreted by human umbilical cord mesenchymal stem cells.
The preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. Inoculating cells into DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing nonadherent cells, performing subculture on the digested cells when the cell fusion rate reaches 80%, centrifuging to remove cells, fragments and the like when the cell fusion rate reaches the required amount, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), uniformly mixing, incubating for 14 hours at 4 ℃, centrifuging, discarding the supernatant, re-suspending and precipitating by using PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, re-suspending and precipitating by using PBS buffer solution to obtain the mesenchymal stem cell exosomes, subpackaging and storing at-80 ℃.
and in the step B, filtering the mixture by a 100-mesh filter screen.
The polyethylene glycol is polyethylene glycol 8000.
the components of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging are shown in the table 2.
Table 2: the composition of the components of the restorative anti-aging skin care product of example 2;
Example 3
a preparation method of a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) adding EDTA-2Na into appropriate amount of water, adding xanthan gum, heating to 75 deg.C, adding butanediol, glycerol and water soluble jojoba oil after xanthan gum is completely hydrated, and mixing to obtain phase A;
(3) mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) Adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 65 ℃ for later use;
(5) Dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 35 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract and vitamin E acetate and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the composition.
In the step (1), the specific operation mode of the vacuum freeze drying is as follows: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
The mesenchymal stem cell exosome is a mixture of various active factors secreted by human umbilical cord mesenchymal stem cells.
The preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. Taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. Inoculating cells into DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing nonadherent cells, performing subculture on the digested cells when the cell fusion rate reaches 90%, centrifuging to remove cells, fragments and the like when the cell fusion rate reaches the required amount, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), mixing uniformly, incubating for 8 hours at 4 ℃, centrifuging, discarding the supernatant, resuspending the precipitate with PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, resuspending the precipitate with PBS buffer solution to obtain the mesenchymal stem cell exosome, subpackaging and storing at-80 ℃.
and in the step B, filtering the mixture by a 100-mesh filter screen.
The polyethylene glycol is polyethylene glycol 8000.
The components of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging are shown in the table 3.
table 3: the component composition of the restorative anti-aging skin care product of example 3;
example 4
A preparation method of a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) Adding EDTA-2Na into appropriate amount of water, adding xanthan gum, heating to 78 deg.C, adding butanediol, glycerol and water soluble jojoba oil after xanthan gum is completely hydrated, and mixing to obtain phase A;
(3) mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) Adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 62 ℃ for later use;
(5) Dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 36 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin and ceramide-2 and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the composition.
In the step (1), the specific operation mode of the vacuum freeze drying is as follows: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
the mesenchymal stem cell exosome is a mixture of various active factors secreted by human umbilical cord mesenchymal stem cells.
the preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. Inoculating cells into a DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing nonadherent cells, digesting the cells for subculture when the cell fusion rate reaches 82%, centrifuging to remove cells, debris and the like when the cell fusion rate reaches the required number, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), mixing uniformly, incubating for 12 hours at 4 ℃, centrifuging, discarding the supernatant, re-suspending and precipitating by using a PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, re-suspending and precipitating by using the PBS buffer solution to obtain the mesenchymal stem cell exosomes, subpackaging and storing at-80 ℃.
and in the step B, filtering the mixture by a 100-mesh filter screen.
The polyethylene glycol is polyethylene glycol 8000.
The components of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging are shown in the table 4.
table 4: the component composition of the restorative anti-aging skin care product of example 4;
Example 5
a preparation method of a repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes comprises the following steps:
(1) Mixing the mesenchymal stem cell exosome, mannitol (excipient) and a proper amount of water, and freeze-drying in vacuum to obtain freeze-dried powder (F phase) for later use;
(2) adding EDTA-2Na into appropriate amount of water, adding xanthan gum, heating to 78 deg.C, adding butanediol, glycerol and water soluble jojoba oil after xanthan gum is completely hydrated, and mixing to obtain phase A;
(3) Mixing oleum Olivarum, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C to obtain BD phase; the olive oil and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacitracin sodium are phase D;
(4) adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 62 ℃ for later use;
(5) dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 36 ℃, adding a mixture (phase E) of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract and vitamin E acetate and the freeze-dried powder (phase F) obtained in the step (1), and uniformly mixing to obtain the composition.
in the step (1), the specific operation mode of the vacuum freeze drying is as follows: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
the mesenchymal stem cell exosome is a mixture of various active factors secreted by human umbilical cord mesenchymal stem cells.
the preparation method of the mesenchymal stem cell exosome comprises the following steps:
A. Taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. inoculating cells into a DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing nonadherent cells, digesting the cells for subculture when the cell fusion rate reaches 82%, centrifuging to remove cells, debris and the like when the cell fusion rate reaches the required number, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10% (volume percentage), mixing uniformly, incubating for 12 hours at 4 ℃, centrifuging, discarding the supernatant, re-suspending and precipitating by using a PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, re-suspending and precipitating by using the PBS buffer solution to obtain the mesenchymal stem cell exosomes, subpackaging and storing at-80 ℃.
And in the step B, filtering the mixture by a 100-mesh filter screen.
The polyethylene glycol is polyethylene glycol 8000.
the components of the skin care product containing the umbilical cord mesenchymal stem cell exosome for repairing and resisting aging are shown in the table 5.
table 5: the component composition of the restorative anti-aging skin care product of example 5;
comparative example 1
the composition of the skin care product for repairing and resisting aging containing the umbilical cord mesenchymal stem cell exosome in example 1 is shown in table 6.
table 6: the components of the repair anti-aging skin care product of comparative example 1;
experimental example clinical observation of anti-aging skin care product
The test method comprises the following steps: 400 female test volunteers with aged skin were screened, with the age of 28-50 years and the average age of 35 years.
and (3) inclusion standard: the skin is dry, the elasticity is reduced, and the skin aging phenomena such as wrinkles, pigmentation, dull complexion and the like appear; during the whole test period, people are willing to avoid the sun or face use the sun-proof product with the SPF more than or equal to 30 when contacting sunlight; no other anti-aging products were used throughout the test period.
400 female volunteers were randomly divided into 5 groups of 80 control, examples 1, 4, 5 and control. Wherein, the control group subjects used the anti-skin aging essence containing the umbilical cord mesenchymal stem cell exosome sold by Nanjing Winbo Biotechnology Limited, and the essence was applied to the face after cleaning the face in the morning and at night. Examples 1, 4, 5 and comparative subjects used the anti-aging skin care products prepared in examples 1 and 4 of the present invention and example 5 and comparative example 1, respectively, and applied the skin care products on the face after cleansing the face in the morning and evening. The total test time of each group was 90d, adverse reactions occurring during the test period were recorded, and the subjects were evaluated for efficacy.
Skin aging evaluation: evaluation was performed by observing wrinkles (glabellar lines, forehead wrinkles, crow's tail lines, nasolabial folds, perilabial wrinkles) in 5 test areas of the subject.
And (4) judging the standard:
No wrinkles;
Mild: 2-3 shallow wrinkles are formed, and the length is less than 1.5 cm;
Medium: 2-6 shallow wrinkles are formed, and the length is less than 3 cm;
and (3) severe degree: there were several major wrinkles up to 4cm in length accompanied by shallow wrinkles.
And (3) safety evaluation: the occurrence of adverse reactions such as slight discomfort, erythema, dryness, pruritus and stabbing pain of a subject after using the product is mainly evaluated. And (5) detecting the water content and the oil content of the tested part of the tested subject by adopting a non-invasive instrument at the 90 th day, and carrying out statistical analysis.
And (3) test results: as shown in tables 7, 8 and 9.
Table 7: evaluating the skin aging of each group of testees after the test is finished;
| group of | no wrinkles (number of cases) | Slight (number of cases) | Moderate (number of cases) | Severe (number of cases) |
| control group | 58 | 16 | 4 | 2 |
| Practice ofExample 1 | 68 | 10 | 2 | 0 |
| example 4 | 61 | 16 | 3 | 0 |
| Example 5 | 64 | 11 | 3 | 0 |
| Comparative example | 42 | 24 | 10 | 4 |
Table 8: adverse reaction evaluation of each group of subjects during the test period;
Table 9: stratum corneum moisture and oil content evaluation during the test period for each group of subjects;
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (10)
1. A repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes is characterized by comprising the following components: mesenchymal stem cell exosomes, mannitol, EDTA-2Na, xanthan gum, butylene glycol, glycerol, water-soluble jojoba oil, olive oil, polydimethylsiloxane, hydroxyethylcellulose, hydrogenated lecithin, bacitracin sodium, PCA Na, urea, hyaluronic acid, β -glucan, stem cell secretin, serum albumin, ceramide-2, and water.
2. The repair anti-aging skin care product for umbilical cord mesenchymal stem cell exosomes according to claim 1, which is characterized by comprising the following components in percentage by weight: 0.1-1% of mesenchymal stem cell exosome, 5-7% of mannitol, 0.05-0.25% of EDTA-2 Na0.10-0.25%, 0.10-0.25% of xanthan gum, 3-5% of butanediol, 1-3% of glycerol, 0.3-0.6% of water-soluble jojoba oil, 2-10% of olive oil, 1.5-2.5% of polydimethylsiloxane, 0.4-0.6% of hydroxyethyl cellulose, 1.0-2.0% of hydrogenated lecithin, 0.4-0.8% of bacillus subtilis lipopeptide sodium, 0.4-0.6% of PCA Na, 0.2-0.5% of urea, 0.4-0.6% of hyaluronic acid, 0.2-0.5% of beta-glucan, 2-5% of dry cell secretin, 0.05-0.15% of serum albumin, 20.05-0.15% of ceramide and the balance of water.
3. The skin care product for repairing and resisting aging of umbilical cord mesenchymal stem cell exosomes according to claim 1 or 2, further comprising the following components: jojoba oil, squalane, shea butter, caprylic/capric triglyceride, dipotassium glycyrrhizinate, retinol palmitate, centella asiatica extract, and vitamin E acetate.
4. the repair anti-aging skin care product for umbilical cord mesenchymal stem cell exosomes according to claim 3, which is characterized by comprising the following components in percentage by weight: 0.1-1% of mesenchymal stem cell exosome, 5-7% of mannitol, 0.05-0.25% of EDTA-2Na0.05, 0.10-0.25% of xanthan gum, 3-5% of butanediol, 1-3% of glycerol, 0.3-0.6% of water-soluble jojoba oil, 2-10% of olive oil, 1.5-2.5% of polydimethylsiloxane, 0.4-0.6% of hydroxyethyl cellulose, 1.0-2.0% of hydrogenated lecithin, 0.4-0.8% of sodium bacillus subtilis lipopeptide, 0.4-0.6% of PCA Na, 0.2-0.5% of urea, 0.4-0.6% of hyaluronic acid, 0.2-0.5% of beta-glucan, 2-5% of dry cell secretin, 0.05-0.15% of serum albumin, 20.05-0.15% of ceramide, 1.5-2.5% of jojoba oil, 0.3-0.6% of squalane, 0.8% of shea butter, 0.1.2-0.5% of caprylic acid triglyceride, 0.08-0.08% of palmitic acid and 0.02-0.12% of potassium palmitate, centella extract 0.4-0.6%, vitamin E acetate 0.4-0.6%, and water in balance.
5. the skin care product for repairing and resisting aging of umbilical cord mesenchymal stem cell exosomes according to any one of claims 1 to 4, wherein the mesenchymal stem cell exosomes are a mixture of a plurality of active factors secreted by human umbilical cord mesenchymal stem cells.
6. The skin care product for repairing and resisting aging of umbilical cord mesenchymal stem cell exosomes according to any one of claims 1 to 5, wherein the preparation method of the mesenchymal stem cell exosomes comprises the following steps:
A. taking placenta, separating chorion, washing with PBS buffer solution to remove erythrocytes, shearing chorion into tissue blocks with the size of 10-20 mm 3, and adding collagenase digestive juice for digestion;
B. Collecting digested tissue mass, filtering to obtain filtrate 1, adding trypsin into the rest tissue, digesting, and filtering to obtain filtrate 2; mixing the filtrate 1 and the filtrate 2, adding serum to terminate digestion, centrifuging, discarding supernatant, and collecting centrifuged cells;
C. inoculating cells into a DMEM medium containing 10% FBS at a density of 2 x 10 6/cm 2, culturing at 37 ℃, 5% CO 2 and saturated humidity, changing the solution once at intervals and removing non-adherent cells, when the cell fusion rate reaches 80% -90%, digesting the cells for subculture, centrifuging to remove cells, debris and the like when the required number of the cells is expanded, collecting supernatant, passing through a sterile filter membrane of 0.22 mu m, adding polyethylene glycol with the final concentration of 10%, uniformly mixing, incubating for 8-14 hours at 4 ℃, centrifuging, discarding the supernatant, re-suspending the precipitate with PBS buffer solution, adding polyethylene glycol with the final concentration of 8%, incubating for 1 hour again, centrifuging, re-suspending the precipitate with the PBS buffer solution to obtain the mesenchymal stem cell exosomes, subpackaging and storing at-80 ℃.
7. the skin care product for repairing and resisting aging of umbilical cord mesenchymal stem cell exosome according to claim 6, wherein in the step B, the skin care product is filtered through a 100-mesh filter screen.
8. the skin care product for repairing and anti-aging umbilical cord mesenchymal stem cell exosomes according to claim 6, wherein the polyethylene glycol is polyethylene glycol 8000.
9. The skin care product for repairing and resisting aging containing umbilical cord mesenchymal stem cell exosomes according to any one of claims 3 to 8, wherein the preparation method comprises the following steps:
(1) mixing the mesenchymal stem cell exosome, mannitol and a proper amount of water, uniformly mixing, and performing vacuum freeze drying to obtain a freeze-dried powder F phase for later use;
(2) Adding EDTA-2Na into a proper amount of water, adding xanthan gum, heating to 75-80 ℃, adding butanediol, glycerol and water-soluble jojoba oil after the xanthan gum is completely hydrated, and uniformly mixing to obtain a phase A for later use;
(3) Mixing oleum Olivarum, jojoba oil, squalane, Shea butter, caprylic capric triglyceride, polydimethylsiloxane, hydrogenated lecithin and Bacillus subtilis lipopeptide sodium at 80 deg.C, and mixing to obtain BD phase; the olive oil, the jojoba oil, the squalane, the shea butter, the caprylic-capric triglyceride and the polydimethylsiloxane are phase B, and the hydrogenated lecithin and the bacillus subtilis lipopeptide sodium are phase D;
(4) adding the BD phase into the phase A, quickly stirring and uniformly mixing at 80 ℃, and cooling to 60-65 ℃ for later use;
(5) dissolving hydroxyethyl cellulose in a proper amount of water at 40 ℃ to obtain a transparent gel C phase; adding the transparent gel into the material obtained in the step (4), cooling to 35-40 ℃, adding a mixture E phase of PCA Na, urea, hyaluronic acid, beta-glucan, stem cell secretin, serum albumin, ceramide-2, dipotassium glycyrrhizinate, retinol palmitate, a centella asiatica extract and vitamin E acetate and the freeze-dried powder F phase obtained in the step (1), and uniformly mixing to obtain the composition.
10. the repair anti-aging skin care product containing umbilical cord mesenchymal stem cell exosomes according to claim 9, wherein in the step (1), the vacuum freeze-drying is performed in a specific operation mode: placing the materials in a freeze dryer at-70 deg.C, cooling to below-50 deg.C, pre-freezing for 6h, vacuumizing for 8h, slowly heating to-20 deg.C, heating to 25 deg.C at 5 deg.C/h, and drying for 2 h.
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Application publication date: 20191210 |
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| RJ01 | Rejection of invention patent application after publication |