A composition with facial repair efficacy, its preparation method and its application

Abstract

The present invention relates to a composition with facial repair efficacy. By weight percentage, the composition contains 0.01-5% of cell protein extract, 3-10% of propylene glycol, 0.1-5% of xanthan gum, 0.01-0.1% of hyaluronic acid, 0.1-5% of symex, 0.01-1% of disodium EDTA, 0.5-10% of erythritol, 0.5-10% of β-glucan, 0.1-5% of carbomer, 0.1-5% of triethanolamine, 0.01-1% of dipotassium glycyrrhizinate, 0.1-5% of Piper methysticum root extract, 0.1-5% of 1,2-hexanediol, 0.01-1% of fullerene, 0.0001-0.1% of superoxide dismutase (SOD), and the balance is water. The composition of the present invention can effectively promote cell repair and regeneration, delay skin aging, and achieve the effects of whitening, anti-aging, calming and soothing.

Description

Technical Field

[0001] The present invention relates to the field of biology, and specifically relates to a composition having a facial repair effect, a preparation method thereof, and its application. Background Art

[0002] Facial skin is sensitive and fragile, and it is necessary to do a good job in moisturizing and sun protection of the skin. Frequent exfoliation will cause damage to the skin barrier, and it is easy to have skin sensitivity, resulting in problems such as redness, itching, and desquamation of the facial skin. Commonly used facial repair active ingredients include hyaluronic acid, collagen, amino acids, ceramides, EGF epidermal growth factor, etc. However, there are defects such as slow onset and easy allergy. Therefore, it is necessary to develop safe and effective repair products. The technical contents disclosed in CN 2023100429139 and PCT / CN2023 / 073566 are essential components of the present invention. Summary of the Invention

[0003] The purpose of the present invention is to provide a composition having a facial repair effect. Calculated by weight percentage, the composition contains 0.01-5% of cell protein extract, 3-10% of propylene glycol, 0.1-5% of xanthan gum, 0.01-0.1% of hyaluronic acid, 0.1-5% of symexanthone, 0.01-1% of disodium EDTA, 0.5-10% of erythritol, 0.5-10% of β-glucan, 0.1-5% of carbomer, 0.1-5% of triethanolamine, 0.01-1% of dipotassium glycyrrhizinate, 0.1-5% of Piper methysticum rhizome extract, 0.1-5% of 1,2-hexanediol, 0.01-1% of fullerene, 0.0001-0.1% of superoxide dismutase (SOD), and the balance is water.

[0004] In a preferred technical solution of the present invention, the preparation of the cell protein extract includes the following steps:

[0005] S-1: The density is 5.0×10 6 cells / mL - 1.0×10 7Mesenchymal passaged cells at [specific number] cells / mL are placed in a medium containing 40 - 50% DMEM / F12, 40 - 50% RPMI1640, 0.1 - 2% bovine serum albumin (BSA), 1 - 15 μg / mL epidermal growth factor (EGF), 1 - 15 μg / mL fibroblast growth factor (FGF), 1 - 15 μg / mL insulin transferrin, 0.01 - 0.1% compound amino acids (18AA), and 2 - 10 μmol / L stressor. Then, they are placed under the conditions of 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 - 60 min, and then separated, washed, and the cells are collected. Among them, the stressor is selected from any one or a combination of compounds 1 - 16.

[0006]

[0007] S-2: The collected cells are dispersed in a solvent at a density of 5.0×10 6 cells / mL - 5.0×10 7 cells / mL, and then ultrasonicated under the conditions of 2°C - 8°C to obtain a cell lysate. Among them, the solvent is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution.

[0008] S-3: After separating the cell lysate prepared in step S-2, the obtained separated liquid is filtered through 0.45 μm and 0.22 μm filter membranes in sequence to obtain the product.

[0009] In a preferred technical solution of the present invention, the medium in step S-1 contains 42 - 45% DMEM / F12, 42 - 45% RPMI1640, 0.5 - 1.5% bovine serum albumin (BSA), 5 - 10 μg / mL epidermal growth factor (EGF), 5 - 10 μg / mL fibroblast growth factor (FGF), 5 - 10 μg / mL insulin transferrin, 0.02 - 0.05% compound amino acids (18AA), and 3 - 8 μmol / L stressor.

[0010] In a preferred technical solution of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 μg / mL epidermal growth factor (EGF), 10 μg / mL fibroblast growth factor (FGF), 10 μg / mL insulin transferrin, 0.05% compound amino acids (18AA), and 4 - 6 μmol / L stressor.

[0011] In a preferred technical solution of the present invention, the density of the mesenchymal passaged cells in step S-1 is 6.0×106 -2.0×10 7 cells / mL, preferably 8.0×10 6 -1.0×10 7 cells / mL.

[0012] In a preferred technical solution of the present invention, the mesenchymal passage cells in step S-1 are cultured in a medium for 15 - 50 min, preferably 20 - 40 min.

[0013] In a preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution. The number of times of washing cells is 2 - 5 times, preferably 3 - 4 times.

[0014] In a preferred technical solution of the present invention, the separation in step S-1 is selected from any one or a combination of centrifugation and filtration.

[0015] In a preferred technical solution of the present invention, the centrifugation conditions in step S-1 are 1000 - 2000 rpm * 3 - 15 min, preferably 1200 rpm - 1500 rpm * 5 - 10 min.

[0016] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are: working at 2°C - 8°C, 25 kHz, and 360 W for 3 s and then with a 1 s interval, and the ultrasonic treatment is for 1 - 5 min.

[0017] In a preferred technical solution of the present invention, the separation in step S-3 is selected from any one or a combination of centrifugation, multi-stage centrifugation, and multi-stage filtration.

[0018] In a preferred technical solution of the present invention, the centrifugation conditions in step S-3 are 2000 - 8000 rpm * 10 - 30 min, preferably 3000 - 7000 rpm * 15 - 25 min.

[0019] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is successively 3000 - 4000 rpm * 3 - 5 min, 5000 - 6000 rpm * 3 - 5 min, and 7000 rpm * 5 - 8 min.

[0020] In a preferred technical solution of the present invention, the pore size of the filter membrane for the multi-stage filtration is selected from any one of 80 um, 50 um, 30 um, 10 um, and 5 um.

[0021] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically hydrolyzed with any one of nuclease or omnipotent nuclease and then separated and purified.

[0022] In a preferred technical solution of the present invention, the nuclease is selected from any one or a combination of RNA nucleases and DNA nucleases.

[0023] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is frozen, preferably frozen at -40°C to -20°C.

[0024] In a preferred technical solution of the present invention, a lyoprotectant is added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2), and then freeze-dried to obtain a freeze-dried preparation of the cell protein extract or a freeze-dried preparation of the protein composition, wherein the lyoprotectant is selected from any one or a combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, glyceryl trioctanoate (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, starch.

[0025] In a preferred technical solution of the present invention, calculated by mass percentage, the lyoprotectant contained in the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 0.5 - 8%, preferably 1 - 5%.

[0026] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

[0027] In a preferred technical solution of the present invention, the pH of the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 6 - 8, preferably pH 7 - 7.5.

[0028] In a preferred technical solution of the present invention, before use, the composition is reconstituted with an isotonic solution or a buffer solution and then applied to the scalp, and absorbed by any one or a combination of methods such as rolling needles, microneedles, and massage, wherein the isotonic solution or the buffer solution is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, Tris (tris(hydroxymethyl)aminomethane) buffer solution, citrate buffer solution, carbonate buffer solution, acetate buffer solution, barbiturate buffer solution.

[0029] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is a culture method in the art.

[0030] In a preferred technical solution of the present invention, the culture of the mesenchymal passaged stem cells includes the following steps: The primary mesenchymal stem cells are seeded at an initial density of 5.0×10 5 -5.0×10 6Add it to the subculture medium at a concentration of [[ID=]], and then culture it under the conditions of 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 - 15 days. Every 2 - 3 days, observe the yellowing of the subculture medium, and then replace half of the subculture medium. Among them, the subculture medium is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0031] In the preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells includes the following steps:

[0032] A: After cleaning and disinfecting the umbilical cord, perform tissue dissection, take the Wharton's jelly tissue, cut it into small pieces of 3 mm 3 , centrifuge, wash, collect the tissue pieces, place them in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 μg / ml penicillin, and 100 μg / ml streptomycin, and then culture them under the conditions of 37.0°C ± 0.5°C and 5% ± 1.0% CO2. Replace half of the medium every 2 - 3 days until cells crawl out of the tissue pieces;

[0033] B: Shake, collect the cells at the bottom layer, wash them with PBS, add 0.25% trypsin and digest for 2 - 3 minutes, add an equal volume of trypsin inhibitor to stop digestion, gently pipette, centrifuge at 1200 - 1500 rpm / min for 5 - 8 minutes, and collect the cells to obtain the cells.

[0034] In the preferred technical solution of the present invention, by weight percentage, the composition contains 0.01 - 1% of cell protein extract, 8 - 10% of propylene glycol, 0.1 - 1% of xanthan gum, 0.01 - 0.1% of hyaluronic acid, 0.1 - 1% of symex, 0.01 - 0.1% of disodium EDTA, 1 - 10% of erythritol, 1 - 10% of β - glucan, 0.1 - 1% of carbomer, 0.1 - 1% of triethanolamine, 0.01 - 0.1% of dipotassium glycyrrhizinate, 0.1 - 1% of Piper methysticum root extract, 0.1 - 1% of 1,2 - hexanediol, 0.01 - 0.1% of fullerene, and 0.0001 - 0.001% of superoxide dismutase (SOD).

[0035] In a preferred technical solution of the present invention, by weight percentage, the composition contains 0.06% of cell protein extract, 8% of propylene glycol, 0.15% of xanthan gum, 0.025% of hyaluronic acid, 0.5% of symexanthone, 0.03% of disodium EDTA, 2% of erythritol, 1% of oat β-glucan, 0.18% of carbomer, 0.18% of triethanolamine, 0.06% of dipotassium glycyrrhizinate, 0.25% of kava kava rhizome extract, 0.5% of 1,2 - hexanediol, 0.03% of fullerene, 0.0002% of superoxide dismutase (SOD), and the balance is water.

[0036] In a preferred technical solution of the present invention, by weight percentage, the composition contains 0.02% of cell protein extract, 4% of propylene glycol, 0.4% of xanthan gum, 0.02% of hyaluronic acid, 0.8% of symexanthone, 0.02% of disodium EDTA, 4% of erythritol, 2% of oat β-glucan, 0.5% of carbomer, 0.6% of triethanolamine, 0.06% of dipotassium glycyrrhizinate, 0.3% of kava kava rhizome extract, 0.8% of 1,2 - hexanediol, 0.02% of fullerene, 0.01% of superoxide dismutase (SOD), and the balance is water.

[0037] In a preferred technical solution of the present invention, by weight percentage, the composition contains 0.06% of cell protein extract, 3% of propylene glycol, 0.2% of xanthan gum, 0.029% of hyaluronic acid, 0.3% of symexanthone, 0.02% of disodium EDTA, 1% of erythritol, 2% of oat β-glucan, 0.3% of carbomer, 0.4% of triethanolamine, 0.03% of dipotassium glycyrrhizinate, 0.44% of kava kava rhizome extract, 0.2% of 1,2 - hexanediol, 0.02% of fullerene, 0.0007% of superoxide dismutase (SOD), and the balance is water.

[0038] Another object of the present invention is to provide a preparation method of a composition with a repair effect. The composition contains 0.01 - 1% of cell protein extract, 8 - 10% of propylene glycol, 0.1 - 1% of xanthan gum, 0.01 - 0.1% of hyaluronic acid, 0.1 - 1% of symexanthone, 0.01 - 0.1% of disodium EDTA, 1 - 10% of erythritol, 1 - 10% of β-glucan, 0.1 - 1% of carbomer, 0.1 - 1% of triethanolamine, 0.01 - 0.1% of dipotassium glycyrrhizinate, 0.1 - 1% of kava kava rhizome extract, 0.1 - 1% of 1,2 - hexanediol, 0.01 - 0.1% of fullerene, 0.0001 - 0.001% of superoxide dismutase (SOD), and the balance is water. The preparation method of the composition includes the following steps:

[0039] (1) Weigh the required amounts of propylene glycol, xanthan gum, hyaluronic acid, thymol and disodium EDTA, dissolve them in water, heat to 80 - 90 °C, stir at 100 - 300 rpm for 1 - 4 h, and let it stand and cool down.

[0040] (2) Cool down to ≤40 °C, add the required amounts of erythritol, β - glucan, carbomer, triethanolamine and dipotassium glycyrrhizinate, stir at 100 - 300 rpm for 1 - 4 h, and let it stand and cool down.

[0041] (3) When cooled down to ≤30 °C, add the required amounts of Piper methysticum rhizome extract, 1,2 - hexanediol, fullerene, cell protein extract and superoxide dismutase (SOD), adjust the viscosity to 10,000 - 13,000, and make up the volume with water to obtain the product.

[0042] In the preferred technical solution of the present invention, the preparation of the cell protein extract includes the following steps:

[0043] S - 1: Place mesenchymal passage cells with a density of 5.0×10 6 cells / mL - 1.0×10 7 cells / mL in a medium containing 40 - 50% DMEM / F12, 40 - 50% RPMI1640, 0.1 - 2% bovine serum albumin (BSA), 1 - 15 μg / mL epidermal growth factor (EGF), 1 - 15 μg / mL fibroblast growth factor (FGF), 1 - 15 μg / mL insulin - transferrin, 0.01 - 0.1% compound amino acids (18AA) and 2 - 10 μmol / L stress agent, and then place it in an environment of 37.0 °C ± 0.5 °C and 5% ± 1.0% CO2 for 10 - 60 min, separate, wash, and collect the cells. Among them, the stress agent is selected from any one or a combination of compounds 1 - 16.

[0044]

[0045] S - 2: Disperse the collected cells at a density of 5.0×10 6 cells / mL - 5.0×10 7 cells / mL in a solvent, and then place it under ultrasonic treatment at 2 °C - 8 °C to prepare a cell lysate. Among them, the solvent is selected from any one or a combination of normal saline, 5% glucose solution, phosphate - buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer.

[0046] S - 3: After separating the cell lysate prepared in step S - 2, the obtained separation liquid is filtered through 0.45 μm and 0.22 μm filter membranes in sequence to obtain the product.

[0047] In the preferred technical solution of the present invention, the culture medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 μg / mL epidermal growth factor (EGF), 5-10 μg / mL fibroblast growth factor (FGF), 5-10 μg / mL insulin transferrin, 0.02-0.05% compound amino acids (18AA), and 3-8 μmol / L stress substance.

[0048] In the preferred technical solution of the present invention, the culture medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 μg / mL epidermal growth factor (EGF), 10 μg / mL fibroblast growth factor (FGF), 10 μg / mL insulin transferrin, 0.05% compound amino acids (18AA), and 4-6 μmol / L stress substance.

[0049] In the preferred technical solution of the present invention, the density of mesenchymal passage cells in step S-1 is 6.0×10 6 -2.0×10 7 cells / mL, preferably 8.0×10 6 -1.0×10 7 cells / mL.

[0050] In the preferred technical solution of the present invention, the mesenchymal passage cells in step S-1 are cultured in the culture medium for 15-50 min, preferably 20-40 min.

[0051] In the preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one or a combination of physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution. The number of times of washing cells is 2-5 times, preferably 3-4 times.

[0052] In the preferred technical solution of the present invention, the separation in step S-1 is selected from any one or a combination of centrifugation and filtration.

[0053] In the preferred technical solution of the present invention, the centrifugation conditions in step S-1 are 1000-2000 rpm * 3-15 min, preferably 1200 rpm - 1500 rpm * 5-10 min.

[0054] In the preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are: working at 2℃-8℃, 25 kHz, and 360 W for 3 s and then having a 1 s interval, and the ultrasonic treatment is 1-5 min.

[0055] In a preferred technical solution of the present invention, the separation in step S-3 is selected from any one or a combination of centrifugation, multi-stage centrifugation, and multi-stage filtration.

[0056] In a preferred technical solution of the present invention, the centrifugation conditions in step S-3 are 2000 - 8000 rpm * 10 - 30 min, preferably 3000 - 7000 rpm * 15 - 25 min.

[0057] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is successively 3000 - 4000 rpm * 3 - 5 min, 5000 - 6000 rpm * 3 - 5 min, and 7000 rpm * 5 - 8 min.

[0058] In a preferred technical solution of the present invention, the pore size of the filter membrane for the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

[0059] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is enzymatically hydrolyzed with either a nuclease or a universal nuclease and then separated and purified.

[0060] In a preferred technical solution of the present invention, the nuclease is selected from any one or a combination of RNA nuclease and DNA nuclease.

[0061] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 or the protein composition obtained in step (2) is frozen, preferably frozen at -40°C to -20°C.

[0062] In a preferred technical solution of the present invention, a lyoprotectant is added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), and then freeze-dried to obtain a freeze-dried preparation of the cell protein extract or a freeze-dried preparation of the protein composition. Among them, the lyoprotectant is selected from any one or a combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, triglyceride (HES), polyethylene glycol, ethylene vinylidene, phosphate, acetate, citrate, sorbitol, and starch.

[0063] In a preferred technical solution of the present invention, calculated by mass percentage, the lyoprotectant contained in the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 0.5 - 8%, preferably 1 - 5%.

[0064] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), and the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

[0065] In a preferred technical solution of the present invention, the freeze-dried preparation of the cellular protein extract or the freeze-dried preparation of the protein composition has a pH of 6-8, preferably 7-7.5.

[0066] In a preferred technical solution of the present invention, before use, the composition is reconstituted with an isotonic solution or a buffer solution and then applied to the scalp, and absorbed by any one or a combination of rolling needles, microneedles, and massage. Among them, the isotonic solution or buffer solution is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, Tris (tris(hydroxymethyl)aminomethane) buffer solution, citrate buffer solution, carbonate buffer solution, acetate buffer solution, and barbiturate buffer solution.

[0067] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells is a culture method in the art.

[0068] In a preferred technical solution of the present invention, the culture of the mesenchymal passaged stem cells includes the following steps: The primary mesenchymal stem cells are added to the passage medium at an initial density of 5.0×10 5 -5.0×10 6 cells / ml, and then cultured at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10-15 days. Every 2-3 days, after observing that the passage medium turns yellow, half of the passage medium is replaced. Among them, the passage medium is a DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0069] In a preferred technical solution of the present invention, the culture of the primary mesenchymal stem cells includes the following steps:

[0070] A: After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, cut it into small pieces of 3 mm 3 , centrifuge, wash, collect the tissue blocks, place them in a DMEM / F12 medium containing 10% fetal bovine serum FBS, 100 μg / ml penicillin, and 100 μg / ml streptomycin, and then culture them at 37.0°C ± 0.5°C and 5% ± 1.0% CO2. Half of the medium is replaced every 2-3 days until cells crawl out of the tissue blocks;

[0071] B: Shake, collect the low-layer cells, wash them with PBS, add 0.25% trypsin and digest for 2-3 minutes, add an equal volume of trypsin inhibitor to stop digestion, gently pipette, centrifuge at 1200-1500 rpm / min for 5-8 minutes, and collect the cells to obtain the cells.

[0072] Another object of the present invention is to provide the use of a composition with facial repair efficacy in the preparation of cell repair products.

[0073] In a preferred technical solution of the present invention, the cell repair is any one of moisturizing, facial dullness, melanin repair, alleviating allergies, redness and swelling symptoms, freckle removal, acne treatment, and acne therapy.

[0074] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between a liquid and a solid, the percentage is volume / weight percentage; when the present invention relates to the percentage between a solid and a liquid, the percentage is weight / volume percentage; the rest is weight / weight percentage.

[0075] Compared with the prior art, the present invention has the following beneficial technical effects:

[0076] 1. The present invention scientifically combines cell protein extracts with cell repair efficacy and other components to prepare proteins and growth factors required for effectively activating and repairing cells. The composition has obvious advantages in cell repair, can effectively promote cell protection and repair regeneration, accelerate injury recovery, delay skin aging, repair melanocytes, increase the hyaluronidase inhibition rate, improve skin gloss, reduce the transdermal water loss rate of the skin and the area of the red skin area, and the skin lactate sting value score, achieving the effects of whitening, anti-aging, calming, soothing, alleviating allergies, redness and swelling symptoms, freckle removal, acne treatment, and acne therapy.

[0077] 2. The preparation method of the composition of the present invention has the advantages of simple operation, low cost, and being suitable for industrial-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Research on the repair effect of the cell protein extract of the present invention on oxidatively damaged skin;

[0079] Figure 2 Research on the repair effect of the cell protein extract of the present invention on skin damage caused by UVB irradiation;

[0080] Figure 3 Influence of the cell protein extract of the present invention on the generation of oxides (IOD) in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * p < 0.05 compared with the NC (negative control) group, ** indicates p < 0.01 compared with the NC (negative control) group;

[0081] Figure 4Effect of the cell protein extract of the present invention on the generation of superoxide dismutase (SOD) in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * indicates p < 0.05 compared with the NC (negative control) group, and ** indicates p < 0.01 compared with the NC (negative control) group;

[0082] Figure 5 Effect of the cell protein extract of the present invention on the level of MDA in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * indicates p < 0.05 compared with the NC (negative control) group, and ** indicates p < 0.01 compared with the NC (negative control) group;

[0083] Figure 6 Study on the effect of the composition of the present invention on facial repair, ***p < 0.001, ***p < 0.01, *p < 0.05. Detailed implementation manners

[0084] The following further explains and describes the detailed content of the present invention with reference to specific embodiments, but does not limit the protection scope of the present invention thereby.

[0085] Except for the cell protein extract, the rest of the raw materials in the examples are commercially available.

[0086] 1. Culture of primary mesenchymal stem cells

[0087] The culture of primary mesenchymal stem cells includes the following steps:

[0088] 1) After cleaning and disinfecting the umbilical cord, anatomize the tissue, take the Wharton's jelly tissue, cut it into small pieces of 3 mm 3 , centrifuge, wash, collect the tissue pieces, place them in a culture flask, add DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 μg / ml penicillin, and 100 μg / ml streptomycin, and then culture it at 37 °C and 5% CO2 to promote its adhesion. Every 2 - 3 days, observe the yellowing of the medium and then replace half of the medium. Culture for 10 - 12 days until cells can be seen crawling out from the edge of the tissue pieces;

[0089] 2) Gently shake to make the tissue pieces fall off, and collect the tissue pieces and low - layer cells respectively. Among them, the collected tissue pieces are cultured by re - adhesion;

[0090] 3) After washing the collected low - layer cells with PBS, add an appropriate amount of 0.25% trypsin to digest for 2 - 3 min, add an equal volume of trypsin inhibitor to stop digestion, gently pipette the bottom of the flask, centrifuge at 1500 rpm / min for 5 min, and collect the cells to obtain the cells.

[0091] 2. Subculture of primary mesenchymal stem cells

[0092] Subculture of primary mesenchymal stem cells: The primary mesenchymal stem cells were added to DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin and 100 μg / ml streptomycin at an initial density of 1.0×10 5 -6.0×10 5 cells / ml, and then cultured at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 - 15 days. Every 2 - 3 days, after observing that the medium turned yellow, half of the medium was replaced.

[0093] 3. The preparation of compounds 1 - 16 refers to Literature 1 (New limonophyllines A - C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431–437).

[0094] Reference Example 1 Preparation of the cell protein extract of the present invention

[0095] The preparation method of the cell protein extract of the present invention includes the following steps:

[0096] (1) The mesenchymal subculture cells were added to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10 μg / mL epidermal growth factor (EGF), 10 μg / mL fibroblast growth factor (FGF), 10 μg / mL insulin transferrin, 0.05% compound amino acid (18AA) and 5 μmol / L of compound 16 at a density of 5.0×10 6 cells / mL, and then cultured at 37°C and 5% CO2 for 30 min. After that, they were centrifuged at 1200 rpm for 5 min, washed 3 times with PBS, and the cells were collected;

[0097] (2) The cells collected in step (1) were dispersed in physiological saline at a density of 8.0×10 6 cells / mL, and sonicated at 2 - 8°C, 25 kHz, and 360 W for 3 s with a 1 s interval, and sonicated for 2 min to obtain a cell lysate;

[0098] (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate successively through 0.45 μm and 0.22 μm filter membranes to obtain the product.

[0099] Reference Example 2 Preparation of the freeze-dried preparation of the cell protein extract of the present invention

[0100] Add the required amount of mannitol to the cell protein extract prepared in Example 1, stir, mix evenly, and then lyophilize. The obtained freeze-dried preparation contains 2% mannitol (m / m).

[0101] Example 1 Preparation of the pharmaceutical composition of the present invention

[0102] The pharmaceutical composition of the present invention is composed of:

[0103] Composition Dosage (g) Cell Protein Extract (Reference Example 2) 0.06 Propylene Glycol 8.00 Xanthan Gum 0.15 Hyaluronic Acid 0.025 Fresh Protect 0.50 Disodium EDTA 0.03 Erythritol 2.00 Oat β-Glucan 1.00 Carbomer 940 0.18 Triethanolamine 0.18 Dipotassium Glycyrrhizinate 0.06 Kava Rhizome Extract 0.25 1,2-Hexanediol 0.50 Fullerene 0.03 Superoxide Dismutase SOD 0.0002

[0104] The preparation of the pharmaceutical composition of the present invention includes the following steps:

[0105] (1) Dissolve the required amounts of propylene glycol, xanthan gum, hyaluronic acid, symex, and disodium EDTA in 10 ml of water, heat to 80 - 90 °C, and slowly stir at 150 rpm for 1 h, then let it stand and cool down.

[0106] (2) When the temperature drops below 40 °C, add the required amounts of erythritol, oat β-glucan, carbomer, triethanolamine, and dipotassium glycyrrhizinate, and slowly stir at 150 rpm for 1 h, then let it stand and cool down.

[0107] (3) When the temperature drops below 30 °C, add the required amounts of kava rhizome extract, 1,2 - hexanediol, fullerene, cell protein extract, and superoxide dismutase SOD, adjust the viscosity to 10000 - 13000, and make up the volume to 100 ml with water to obtain the product.

[0108] Example 2 Preparation of the pharmaceutical composition of the present invention

[0109] The pharmaceutical composition of the present invention is composed of:

[0110] Composition Dosage (g) Cell Protein Extract (Reference Example 2) 0.02 Propylene Glycol 4.00 Xanthan Gum 0.40 Hyaluronic Acid 0.02 Fresh Protect 0.80 Disodium EDTA 0.02 Erythritol 4.00 Oat β-Glucan 2.00 Carbomer 940 0.50 Triethanolamine 0.60 Dipotassium Glycyrrhizinate 0.06 Kava Rhizome Extract 0.30 1,2-Hexanediol 0.80 Fullerene 0.02 Superoxide Dismutase SOD 0.01

[0111] The preparation of the pharmaceutical composition of the present invention includes the following steps:

[0112] (1) Dissolve the required amounts of propylene glycol, xanthan gum, hyaluronic acid, symex, and disodium EDTA in 10 ml of water, heat to 80 - 90 °C, and slowly stir at 150 rpm for 1 h, then let it stand and cool down.

[0113] (2) When the temperature drops below 40 °C, add erythritol, oat β-glucan, carbomer 940, triethanolamine, dipotassium glycyrrhizinate, and stir slowly at 150 rpm for 1 h, then let it stand and cool down;

[0114] (3) When the temperature drops below 30 °C, add Piper methysticum rhizome extract, 1,2-hexanediol, fullerene, freeze-dried preparation of the cell protein extract of Reference Example 2, superoxide dismutase (SOD), adjust the viscosity to 10,000 - 13,000, and make up the volume to 100 ml with water to obtain the product.

[0115] Example 3 Preparation of the pharmaceutical composition of the present invention

[0116] The pharmaceutical composition of the present invention consists of:

[0117] Composition Dosage (g) Cell Protein Extract (Reference Example 2) 0.06 Propylene Glycol 3.00 Xanthan Gum 0.20 Hyaluronic Acid 0.029 Fresh Protect 0.30 Disodium EDTA 0.02 Erythritol 1.00 Oat β-Glucan 2.00 Carbomer 940 0.30 Triethanolamine 0.40 Dipotassium Glycyrrhizinate 0.03 Kava Rhizome Extract 0.44 1,2-Hexanediol 0.20 Fullerene 0.02 Superoxide Dismutase SOD 0.0007

[0118] The preparation of the pharmaceutical composition of the present invention includes the following steps:

[0119] (1) Dissolve propylene glycol, xanthan gum, hyaluronic acid, symex, disodium EDTA in 10 ml of water, heat up to 80 - 90 °C, stir slowly at 150 rpm for 1 h, then let it stand and cool down;

[0120] (2) When the temperature drops below 40 °C, add erythritol, β-glucan, carbomer, triethanolamine, dipotassium glycyrrhizinate, and stir slowly at 150 rpm for 1 h, then let it stand and cool down;

[0121] (3) When the temperature drops below 30 °C, add Piper methysticum rhizome extract, 1,2-hexanediol, fullerene, freeze-dried preparation of the cell protein extract of Reference Example 2, SOD, adjust the viscosity to 10,000 - 13,000, and make up the volume to 100 ml with water to obtain the product.

[0122] Test Example 1 Study on the repair effect of the cell protein extract of the present invention on oxidatively damaged skin

[0123] 3D skin model: , commercially available from EpiKutis.

[0124] SLS working solution: Weigh 0.0080 g of SLS, dissolve it in 2 ml of PBS solution, filter it through 0.22 µm, and prepare a 0.4% SLS mother solution. Pipette 0.5 ml of the 0.4% SLS mother solution, add 0.5 ml of PBS, and prepare a 0.2% SLS working solution.

[0125] WY14643 working solution: Weigh 10 mg of WY14643 (PPARα agonist), dissolve it in 1 mL of DMSO, and prepare a 30 mM WY14643 mother solution. Then add 10 μL of the WY14643 mother solution (30 mM) to 6 mL of the model culture medium to prepare a 50 μM WY14643 working solution.

[0126] Model culture medium: DMEM basal medium.

[0127] Add 0.9 mL of the model culture medium to a 6-well plate, transfer the 3D skin model to the 6-well plate, and label the test number.

[0128] Blank control (BC): Do not perform any treatment on the skin model, incubate it in a CO2 incubator (37 °C, 5% CO2) for 48 h;

[0129] Negative control (NC): Add 25 μL of 0.2% SLS working solution to the surface of the skin model, and incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h;

[0130] Positive control (PC): Add 25 μL of 0.2% SLS working solution to the surface of the skin model, and incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h; after adding 25 μL of 50 μM WY14643 working solution, then incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h;

[0131] Test group: Add 25 μL of 0.2% SLS working solution to the surface of the skin model, and incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h; after adding 25 μL of 1% cell protein extract solution (the freeze-dried preparation of the cell protein extract in Reference Example 2 is made into a 1% solution with physiological saline), then incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h.

[0132] After incubating for 48 h, wash and remove the residual liquid inside and outside the skin model with PBS solution. After fixing with 4% paraformaldehyde for 24 h, cut the model and observe it after H&E staining. The results are shown in Figure 1 . The protein extract of the present invention has an obvious repair effect on oxidatively damaged skin.

[0133] Test Example 2 Study on the repair effect of the cell protein extract of the present invention on UVB-irradiated damaged skin

[0134] 3D skin model: , commercially purchased from EpiKutis.

[0135] Model culture medium: DMEM basal medium.

[0136] VE working solution: Pipette 0.5 g of VE stock solution, dissolve it in 10 mL of absolute ethanol to prepare a 5% mother solution; Pipette 100 μL of the 5% mother solution, dissolve it in 10 mL of the model culture medium to prepare a 0.05% VE working solution.

[0137] Add 0.9 mL of the model culture medium to a 6-well plate, transfer the 3D skin model to the 6-well plate, and label the test number.

[0138] Blank control (BC): Do not perform any treatment on the skin model, and incubate it in a CO2 incubator (37 °C, 5% CO2) for 48 h;

[0139] Negative control (NC): After irradiating the skin model with a UVB dose of 600 mJ / cm 2 incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h; Add 25 μL of 0.2% SLS working solution, and incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h;

[0140] Positive control (PC): After irradiating the skin model with a UVB dose of 600 mJ / cm 2 incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h; Add 25 μL of 0.05% VE working solution, and then incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h;

[0141] Test group: After irradiating the skin model with a UVB dose of 600 mJ / cm 2 incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h; Add 25 μL of 1% cell protein extract solution (prepare a 1% solution by dissolving the freeze-dried preparation of the cell protein extract obtained in Reference Example 2 of the present invention with physiological saline), and then incubate it in a CO2 incubator (37 °C, 5% CO2) for 24 h.

[0142] After incubating for 48 h, wash and remove the residual liquid inside and outside the skin model with PBS solution. After fixing with 4% paraformaldehyde for 24 h, cut the model and observe it after H&E staining. The results are shown in Figure 2 . Detect the levels of reactive oxygen species (IOD), superoxide dismutase, and malondialdehyde (MDA) in the skin damaged by UVB radiation. The results are shown in Figures 3 - 5 . The cell protein extract of the present invention has an obvious repair effect on the skin damaged by UVB radiation.

[0143] Test Example 3Research on the Effect of the Composition of the Invention on Facial Repair

[0144] Thirty-one volunteers were selected as subjects, aged 31 - 59 years old, with an average age of 44.94 + 1.61 years old.

[0145] Inclusion criteria: Healthy people, people with poor facial skin barrier, people with obvious facial telangiectasia, who can cooperate with the experiment, maintain regular life during the experiment, can complete the experiment as required and sign the informed consent form, and agree not to use any cosmetics, drugs and health products that affect the results during the experiment; The lactic acid stinging score is greater than 3 points, and other corresponding inclusion criteria;

[0146] Exclusion criteria: Those who have used antihistamines in the past week or immunosuppressants in the past month; Those who have applied any anti-inflammatory drugs to the test site in the past two months; Subjects with inflammatory skin diseases that have not healed clinically; Insulin-dependent diabetes patients; Patients with asthma or other chronic respiratory diseases undergoing treatment; Those who have received anti-cancer chemotherapy in the past six months; Immunodeficiency or autoimmune disease patients; Lactating or pregnant women; Those who have had bilateral mastectomy and bilateral axillary lymph node dissection; Those who have scars, pigmentation, atrophy, nevus flammeus or other defects at the skin test site that affect the determination of the test results; Those who participate in other clinical trials; Highly sensitive individuals; Non-volunteers or those who cannot complete the specified content according to the test requirements.

[0147] The subjects used the composition of Example 1 to prepare facial masks (each facial mask contains 30 g of the composition of Example 1). After cleansing and toning the face, take out the facial mask, apply it on the face for 20 - 30 minutes, then remove the facial mask, gently pat the facial skin until absorbed, and then wipe it with clean water. Twice a week, visit and follow up at D0, D14, and D29 respectively.

[0148] Lactic acid stinging value score: The subjects cleaned their faces and dried them with lint-free paper. After sitting still for 30 min in a compliant environment, 50 μL of 10% lactic acid aqueous solution was applied to either side of the nasolabial groove on the cheek. The subjects evaluated the discomfort degrees of itching, stinging, and burning pain at the test site at 30 s, 2.5 min, and 5 min respectively. Scoring was performed according to the 4-point method (0 point means no sensation, 1 point means mild, 2 points means moderate, and 3 points means severe). If the cumulative stinging sensation at 30 s, 2.5 min, and 5 min is greater than or equal to 3 points, it is positive for lactic acid stinging.

[0149] Transepidermal water loss rate TEWL: An AS-TEWL transcutaneous water loss tester was used to measure the transepidermal water loss rate TEWL value to observe the skin barrier function.

[0150] Skin red area: Using a VISIA 7 skin detector, the measured area of the face was selected, and mainly the skin red area was measured.

[0151] Skin glossiness: Use a Ci6x portable spectrocolorimeter to observe the glossiness of the skin, and measure three times for each test area.

[0152] Safety assessment: Adverse reactions are judged according to the following three-level criteria:

[0153] - Mild - Easy to tolerate, causing very little discomfort and not affecting normal life.

[0154] - Moderate - Enough to affect normal life.

[0155] - Severe - Hindering normal life.

[0156] If the test data is normally distributed, the t-test method is used for statistical analysis; if the test data is non-normally distributed, the rank sum test method is used for statistical analysis. The statistical method uses a two-tailed test, and the significance level α = 0.05.

[0157] Compared with the initial value, after 14 days of use, the TEWL value of the transdermal water loss rate of the facial skin of the subjects decreased significantly, the skin glossiness of the face increased significantly, and the proportion of the area of the red area of the facial skin decreased significantly (see Figure 6 A - C). Compared with the initial value, after 29 days of use, the lactic acid stinging value score of the facial skin of the subjects decreased significantly (see Figure 6 D), and there were no adverse reactions.

[0158] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or deformations according to the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A composition with facial repair efficacy. By weight percentage, the composition contains 0.01 - 5% of cell protein extract, 3 - 10% of propylene glycol, 0.1 - 5% of xanthan gum, 0.01 - 0.1% of hyaluronic acid, 0.1 - 5% of symex, 0.01 - 1% of disodium EDTA, 0.5 - 10% of erythritol, 0.5 - 10% of β - glucan, 0.1 - 5% of carbomer, 0.1 - 5% of triethanolamine, 0.01 - 1% of dipotassium glycyrrhizinate, 0.1 - 5% of Piper methysticum rhizome extract, 0.1 - 5% of 1,2 - hexanediol, 0.01 - 1% of fullerene, 0.0001 - 0.1% of superoxide dismutase (SOD), and the balance is water; The preparation of the cell protein extract includes the following steps: S-1: Place mesenchymal passage cells with a density of 5.0×10 6 cells / mL - 1.0×10 7 cells / mL in a medium containing 40 - 50% DMEM / F12, 40 - 50% RPMI1640, 0.1 - 2% bovine serum albumin (BSA), 1 - 15 μg / mL epidermal growth factor (EGF), 1 - 15 μg / mL fibroblast growth factor (FGF), 1 - 15 μg / mL insulin transferrin, 0.01 - 0.1% compound amino acids (18AA), and 2 - 10 μmol / L stressor. Then place it in an incubator at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 - 60 minutes, followed by separation, washing, and collection of the cells. Among them, The stressor is selected from Compound 16, ; S-2: Disperse the collected cells at a density of 5.0×10 6 cells / mL - 5.0×10 7 cells / mL in a solvent, and then place it under ultrasonic treatment at 2°C - 8°C to obtain a cell lysate, wherein the solvent is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution; S - 3: After separating the cell lysate obtained in step S - 2, the obtained separation liquid is filtered through 0.45um and 0.22um filter membranes in sequence to obtain it.

2. The composition according to claim 1, wherein the culture medium in step S - 1 contains 42 - 45% of DMEM / F12, 42 - 45% of RPMI1640, 0.5 - 1.5% of bovine serum albumin (BSA), 5 - 10ug / mL of epidermal growth factor (EGF), 5 - 10ug / mL of fibroblast growth factor (FGF), 5 - 10ug / mL of insulin transferrin, 0.02 - 0.05% of compound amino acids (18AA), and 3 - 8μmol / L of stress substance.

3. The composition according to claim 2, wherein the culture medium in step S - 1 contains 45% of DMEM / F12, 45% of RPMI1640, 0.5% of bovine serum albumin (BSA), 10ug / mL of epidermal growth factor (EGF), 10ug / mL of fibroblast growth factor (FGF), 10ug / mL of insulin transferrin, 0.05% of compound amino acids (18AA), and 4 - 6μmol / L of stress substance.

4. The composition according to claim 3, wherein the density of the mesenchymal passage cells in step S-1 is 6.0×10 6 -2.0×10 7 cells / mL.

5. The composition according to claim 4, wherein the mesenchymal passage cell density in step S-1 is 8.0×10 6 -1.0×10 7 cells / mL.

6. The composition according to claim 4, wherein the mesenchymal passaged cells in step S - 1 are cultured in the culture medium for 15 - 50min.

7. The composition according to claim 6, wherein the mesenchymal passaged cells in step S - 1 are cultured in the culture medium for 20 - 40min.

8. The composition according to claim 4, wherein the solvent for washing cells in step S - 1 is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, and Tris buffer solution, and the number of times of washing cells is 2 - 5 times.

9. The composition according to claim 8, wherein the number of times of washing cells in step S - 1 is 3 - 4 times.

10. The composition according to claim 4, wherein the separation in step S - 1 is selected from any one or a combination of centrifugation and filtration.

11. The composition according to claim 10, wherein the centrifugation conditions in step S - 1 are 1000 - 2000rpm, 3 - 15min.

12. The composition according to claim 11, wherein the centrifugation conditions in step S-1 are 1200 rpm - 1500 rpm for 5 - 10 min.

13. The composition according to claim 4, wherein the ultrasonic conditions in step S-2 are: working at 2°C - 8°C, 25 kHz, and 360 W for 3 s and then with a 1 s interval, and the ultrasonic treatment is carried out for 1 - 5 min.

14. The composition according to claim 4, wherein the separation in step S-3 is selected from any one or a combination of centrifugation, multi-stage centrifugation, and multi-stage filtration.

15. The composition according to claim 14, wherein the centrifugation conditions in step S-3 are 2000 - 8000 rpm for 10 - 30 min.

16. The composition according to claim 15, wherein the centrifugation conditions in step S-3 are 3000 - 7000 rpm for 15 - 25 min.

17. The composition according to claim 4, wherein the multi-stage centrifugation in step S-3 is successively 3000 - 4000 rpm for 3 - 5 min; 5000 - 6000 rpm for 3 - 5 min; 7000 rpm for 5 - 8 min.

18. The composition according to claim 4, wherein the pore size of the filter membrane for the multi-stage filtration is selected from any one of 80 um, 50 um, 30 um, 10 um, and 5 um.

19. The composition according to claim 4, wherein the cell protein extract prepared in step S-3 is enzymatically hydrolyzed with either a nuclease or a universal nuclease and then separated and purified.

20. The composition according to claim 19, wherein the nuclease is selected from any one or a combination of RNA nuclease and DNA nuclease.

21. The composition according to claim 4, wherein the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is cryopreserved.

22. The composition according to claim 21, wherein the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is cryopreserved at -40°C to -20°C.

23. The composition according to claim 4, wherein a lyoprotectant is added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2), and then freeze-dried to obtain a freeze-dried preparation of the cell protein extract or a freeze-dried preparation of the protein composition, wherein, The lyoprotectant is selected from any one or a combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, glyceryl trioctanoate (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, and starch.

24. The composition according to claim 23, wherein, by mass percentage, the lyoprotectant contained in the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 1 - 5%.

25. The composition according to claim 23, wherein, by mass percentage, the lyoprotectant contained in the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 0.5 - 8%.

26. The composition according to claim 4, wherein a protein stabilizer is optionally added to the cellular protein extract prepared in step S-3 or the protein composition prepared in step (2), wherein, The protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

27. The composition according to claim 23, wherein the pH of the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 6 - 8.

28. The composition according to claim 27, wherein the pH of the freeze-dried preparation of the cell protein extract or the freeze-dried preparation of the protein composition is 7 - 7.

5.

29. The composition according to claim 4, wherein the composition is reconstituted with an isotonic solution or a buffer solution before use and then applied to the scalp, and absorbed by any one or a combination of a roller needle, a microneedle, and massage, wherein, The isotonic solution or buffer solution described above is selected from any one or a combination of normal saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer solution, TBST buffer solution, Tris (tris(hydroxymethyl)aminomethane) buffer solution, citrate buffer solution, carbonate buffer solution, acetate buffer solution, and barbiturate buffer solution.

30. The composition according to claim 1, wherein the culture of the mesenchymal passage cells comprises the following steps: adding primary mesenchymal stem cells at an initial density of 5.0×10 5 -5.0×10 6 cells / ml to a passage medium, and then culturing the cells at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 - 15 days. Every 2 - 3 days, after observing that the passage medium turns yellow, half of the passage medium is replaced. Among them, The subculture medium is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin.

31. The composition according to claim 30, wherein the culture of the primary mesenchymal stem cells comprises the following steps: A: After cleaning and disinfecting the umbilical cord, anatomize the tissue, take the Wharton's jelly tissue, cut it into small pieces of 3 mm 3 , centrifuge, wash, collect the tissue pieces, place them in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100 μg / ml penicillin, and 100 μg / ml streptomycin, and then culture them under the conditions of 37.0°C ± 0.5°C and 5% ± 1.0% CO2. Replace half of the medium every 2 - 3 days until cells crawl out of the tissue pieces; B: Shake, collect the lower-layer cells, wash them with PBS, add 0.25% trypsin and digest for 2-3 minutes, add an equal volume of trypsin inhibitor to stop digestion, gently pipette, centrifuge at 1200-1500 rpm for 5-8 minutes, and collect the cells to obtain the product.

32. The composition according to any one of claims 1-31, by weight percentage, the cell protein extract contained in the composition is 0.01-1%, propylene glycol is 8-10%, xanthan gum is 0.1-1%, hyaluronic acid is 0.01-0.1%, symexone is 0.1-1%, disodium EDTA is 0.01-0.1%, erythritol is 1-10%, β-glucan is 1-10%, carbomer is 0.1-1%, triethanolamine is 0.1-1%, dipotassium glycyrrhizinate is 0.01-0.1%, Piper methysticum rhizome extract is 0.1-1%, 1,2-hexanediol is 0.1-1%, fullerene is 0.01-0.1%, superoxide dismutase (SOD) is 0.0001-0.001%, and the balance is water.

33. The composition according to claim 32, by weight percentage, the cell protein extract contained in the composition is 0.06%, propylene glycol is 8%, xanthan gum is 0.15%, hyaluronic acid is 0.025%, symexone is 0.5%, disodium EDTA is 0.03%, erythritol is 2%, oat β-glucan is 1%, carbomer is 0.18%, triethanolamine is 0.18%, dipotassium glycyrrhizinate is 0.06%, Piper methysticum rhizome extract is 0.25%, 1,2-hexanediol is 0.5%, fullerene is 0.03%, superoxide dismutase SOD is 0.0002%, and the balance is water.

34. The composition according to claim 32, by weight percentage, the cell protein extract contained in the composition is 0.02%, propylene glycol is 4%, xanthan gum is 0.4%, hyaluronic acid is 0.02%, symexone is 0.8%, disodium EDTA is 0.02%, erythritol is 4%, oat β-glucan is 2%, carbomer is 0.5%, triethanolamine is 0.6%, dipotassium glycyrrhizinate is 0.06%, Piper methysticum rhizome extract is 0.3%, 1,2-hexanediol is 0.8%, fullerene is 0.02%, superoxide dismutase SOD is 0.01%, and the balance is water.

35. The composition according to claim 32, wherein, by weight percentage, the composition contains 0.06% of cell protein extract, 3% of propylene glycol, 0.2% of xanthan gum, 0.029% of hyaluronic acid, 0.3% of fresh-keeping ketone, 0.02% of disodium EDTA, 1% of erythritol, 2% of oat β-glucan, 0.3% of carbomer, 0.4% of triethanolamine, 0.03% of dipotassium glycyrrhizinate, 0.44% of kava kava rhizome extract, 0.2% of 1,2 - hexanediol, 0.02% of fullerene, 0.0007% of superoxide dismutase (SOD), and the balance is water.

36. A method for preparing a composition with repair efficacy according to any one of claims 1 - 35, wherein the composition contains 0.01 - 1% of cell protein extract, 8 - 10% of propylene glycol, 0.1 - 1% of xanthan gum, 0.01 - 0.1% of hyaluronic acid, 0.1 - 1% of fresh-keeping ketone, 0.01 - 0.1% of disodium EDTA, 1 - 10% of erythritol, 1 - 10% of β-glucan, 0.1 - 1% of carbomer, 0.1 - 1% of triethanolamine, 0.01 - 0.1% of dipotassium glycyrrhizinate, 0.1 - 1% of kava kava rhizome extract, 0.1 - 1% of 1,2 - hexanediol, 0.01 - 0.1% of fullerene, 0.0001 - 0.001% of superoxide dismutase (SOD), and the balance is water. The method for preparing the composition comprises the following steps: (1) Weigh the required amounts of propylene glycol, xanthan gum, hyaluronic acid, fresh-keeping ketone and disodium EDTA, dissolve them in water, heat up to 80 - 90 °C, stir at 100 - 300 rpm for 1 - 4 h, and let it stand and cool down. (2) When the temperature drops to ≤40 °C, add the required amounts of erythritol, β-glucan, carbomer, triethanolamine and dipotassium glycyrrhizinate, stir at 100 - 300 rpm for 1 - 4 h, and let it stand and cool down. (3) When the temperature drops to ≤30 °C, add the required amounts of kava kava rhizome extract, 1,2 - hexanediol, fullerene, cell protein extract and superoxide dismutase (SOD), adjust the viscosity to 10000 - 13000, and make up the volume with water to obtain the product.

37. Use of a composition with facial repair efficacy according to any one of claims 1 - 35 for preparing cell repair products.

38. The use according to claim 37, wherein the cell repair is any one of moisturizing, facial dullness, melanin repair, relieving allergy, redness and swelling symptoms, freckle removal, acne treatment, and acne cure.

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