Beautifying, whitening and repairing liquid based on skin adipose-derived stem cells and preparation method of beautifying, whitening and repairing liquid

Abstract

The invention discloses beautifying, whitening and repairing liquid based on skin adipose-derived stem cells and a preparation method thereof, and belongs to the technical field of cosmetics, the beautifying, whitening and repairing liquid comprises skin adipose-derived stem cell exosomes, mussel biological peptides (Mefp-5), brown algae polyphenol-nano-selenium compounds, bionic pearl protein modified titanium dioxide and pH / UV double-response type nano-liposome carriers; according to the present invention, the dual targeting effects of repairing and whitening are achieved through the dual active component synergistic mechanism, and the intelligent release is achieved by using the pH / UV dual-response type nano-liposome carrier, such that the precise targeting of the skin repairing, the multi-scene application and the green production are achieved, and the significant beautifying, whitening and repairing effects are provided.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, and in particular to a beauty, whitening and repairing liquid based on skin adipose stem cells and a preparation method thereof. Background Art

[0002] As people's pursuit of beauty continues to improve, the demand for beauty and whitening products is growing. There are many kinds of beauty and whitening products on the market, but some products have problems such as poor effect and poor safety. For example, some chemical whitening agents may irritate the skin, and long-term use may also cause adverse reactions such as skin allergies and pigmentation rebound. As the largest organ in the human body, the health and beauty of the skin are of vital importance. Skin adipose stem cells have multidirectional differentiation potential and paracrine function, showing great potential in skin repair and regeneration, but there are relatively few studies on their application in beauty and whitening products. Summary of the invention

[0003] The present invention aims to provide a beauty and whitening repair liquid based on skin adipose stem cells and a preparation method thereof. The repair liquid can effectively promote skin repair, achieve beauty and whitening and anti-aging effects, and has good safety and stability. The specific contents are as follows:

[0004] The invention discloses a beauty, whitening and repairing liquid based on skin adipose stem cells, comprising skin adipose stem cell exosomes, mussel biopeptide (Mefp-5), brown algae polyphenol-nanoselenium complex, bionic pearl protein modified titanium dioxide, and pH / UV dual-responsive nanoliposome carrier.

[0005] Preferably, the Mefp-5 is connected to a melanin targeting peptide at the C-terminus of the peptide chain, and the sequence of the melanin targeting peptide is YHKNME.

[0006] Preferably, the pH / UV dual-responsive nanoliposome carrier uses soybean lecithin / cholesterol as lipid materials, and the encapsulation efficiency is >85%.

[0007] The present invention also discloses a method for preparing the above-mentioned beauty, whitening and repairing liquid based on skin adipose stem cells, comprising the following steps:

[0008] S1. Use polycaprolactone scaffolds to simulate the fat microenvironment in the body and culture skin adipose stem cells in a 3D microfluidic culture system. Control the culture temperature at 37°C and the carbon dioxide concentration at 5%. Expand the stem cell density to 1×10 7 cells / mL, and ultracentrifugation-size exclusion chromatography was used to separate and purify exosomes to obtain exosomes with a particle size of 80-150nm and a purity of >98%;

[0009] S2, inserting the Mefp-5 gene into the Pichia pastoris expression system, fermenting under the fermentation conditions of 28°C and pH 6.0, with a fermentation yield of 2.8 g / L, purifying by HPLC to a purity of >99%, and then connecting a melanin targeting peptide with a sequence of YHKNME to the C-terminus of the peptide chain;

[0010] S3, the exosomes obtained in S1, Mefp-5 and brown algae polyphenol-nanoselenium are mixed in a mass ratio of 1:2:0.5, thermosensitive poloxamer 407 is used as the base material, a photosensitizer is added, and the mixture is encapsulated in soybean phospholipid / cholesterol liposomes at 40°C by a thin film dispersion method, with an encapsulation rate of >85%, to prepare a responsive gel matrix with UV-triggered release function, wherein the mass fraction of thermosensitive poloxamer 407 in the gel matrix is ​​20%, and the mass fraction of the photosensitizer is 0.1%;

[0011] S4. Homogenize and mix the bionic pearl protein titanium dioxide and the gel matrix prepared in S3 at 20-30° C. to make the mass fraction of the bionic pearl protein titanium dioxide in the mixture be 5%, adjust the pH to 6.0, and fill it into a sterile spray or gel preparation.

[0012] Preferably, in S4, the method for preparing bionic pearl protein modified titanium dioxide comprises the following steps:

[0013] Add titanium dioxide nanoparticles to the bionic pearl protein solution, disperse them evenly with ultrasound for 10-30 minutes, place them at 20-30°C and magnetically stir them for 12-24 hours, centrifuge the mixed solution at a speed of 5000-10000 rpm for 10-20 minutes, wash the precipitate with deionized water or buffer solution for 3-5 times, put it in an oven, and dry it at 60-80°C to constant weight to obtain bionic pearl protein modified titanium dioxide.

[0014] Preferably, in S3, the preparation method of brown algae polyphenol-nano-selenium comprises the following steps: mixing brown algae polyphenol solution and nano-selenium sol in a mass ratio of 1-3:1, slowly adding 0.1-0.5 mol / L sodium hydroxide solution while stirring, adjusting the pH value of the mixed solution to 7-9, continuing to stir and react for 1-2 hours, the solution becomes turbid and a precipitate is generated, centrifuging after the reaction is completed, the centrifugal speed is 8000-10000 r / min, the centrifugal time is 15-20 minutes, collecting the precipitate, washing it with deionized water 3-5 times, and freeze-drying it to obtain the brown algae polyphenol-nano-selenium complex.

[0015] Preferably, in S1, the ultracentrifugation condition is 100,000 g for 120 minutes.

[0016] Preferably, in S3, in the thin film dispersion method, after the thin film is formed, the organic solvent is removed by rotary evaporation in a 50° C. water bath.

[0017] Preferably, the ratio of brown algae polyphenols to nano-selenium in the brown algae polyphenols-nano-selenium complex is 1:0.2-1.

[0018] Preferably, the photosensitizer is p-hydroxyazobenzene.

[0019] The present invention has at least the following beneficial effects:

[0020] (1) Brown algae polyphenols have antioxidant properties, can remove free radicals in the skin, and reduce oxidative stress damage. It can also regulate the physiological functions of skin cells and promote cell metabolism; nano-selenium has good biological activity and antioxidant capacity, can participate in a variety of biochemical reactions in the human body, and enhance the body's antioxidant defense system. In terms of skin care, it can protect skin cells from oxidative damage and promote cell repair and regeneration; brown algae polyphenols-nanoselenium complex integrates the advantages of both, significantly improves antioxidant capacity, and has a free radical scavenging rate of over 95%. It can also simultaneously inhibit the activity of MMP-1 enzymes, reduce the degradation of collagen in the skin, maintain the elasticity and firmness of the skin, and delay skin aging.

[0021] (2) Titanium dioxide itself is a common and highly effective physical sunscreen that can provide sun protection for the skin by reflecting and scattering ultraviolet rays. When its particle size is 10-20nm, it has a strong ability to scatter ultraviolet rays and can effectively block UVA and UVB damage to the skin. After the bionic pearl protein is modified on the surface of titanium dioxide, on the one hand, it can improve the dispersibility and stability of titanium dioxide in the repair solution, making it more evenly distributed, so as to more comprehensively cover the skin surface and enhance the sun protection effect. On the other hand, bionic pearl protein may synergize with titanium dioxide to further optimize the reflection and scattering performance of ultraviolet rays, provide a more reliable sun protection barrier for the skin, and reduce the risks of photoaging and sunburn caused by ultraviolet rays to the skin; bionic pearl protein contains natural light reflection factors, which can reflect and refract light in a special way, thereby producing the effect of brightening the skin color. When bionic pearl protein is modified on the surface of titanium dioxide, the combination of the two can form a thin film with special optical effects on the skin surface. This film can not only reflect ultraviolet rays, but also reflect and scatter visible light, making the skin look brighter and more lustrous, improving dull skin condition and achieving the purpose of brightening skin tone.

[0022] (3) Ultracentrifugation-size exclusion chromatography is used to separate and purify exosomes, especially ultracentrifugation conditions of 100,000g centrifugation for 120 minutes, which can effectively remove impurities and obtain high-purity exosomes with a particle size of 80-150nm and a purity greater than 98%. These exosomes are rich in regenerative factors such as miRNA-21 and TGF-β, which can directly activate collagen synthesis in fibroblasts and repair photoaging damage. As an important medium for intercellular communication, exosomes can transmit the bioactive molecules therein to skin cells, promote the repair and regeneration process of cells, and restore the elasticity and radiance of the skin. The high purity of exosomes ensures the effective performance of their biological activity and functions, providing key repair ingredients for beauty whitening repair fluids.

[0023] (4) The Mefp-5 gene was inserted into the Pichia pastoris expression system for fermentation. The advantages of the Pichia pastoris expression system, which is highly efficient and easy to regulate, were used to achieve mass production of Mefp-5, with a fermentation yield of 2.8 g / L. The product was purified by HPLC to a purity greater than 99%, ensuring the quality and purity of Mefp-5. After the melanin targeting peptide with a YHKNME sequence was connected to the C-terminus of the peptide chain, Mefp-5 was able to specifically bind to the melanocyte membrane receptor (MC1R) with the targeting peptide, thereby enhancing its affinity for melanocytes, with a binding constant of Kd = 1.2 nM. This targeting effect enables Mefp-5 to act precisely on melanocytes, inhibiting tyrosinase activity, thereby blocking the synthesis and transmission pathway of melanin, reducing the production of melanin, and achieving a whitening effect. Compared with unmodified Mefp-5, the whitening effect of Mefp-5 after connecting the melanin targeting peptide is more significant, and it can reduce unnecessary effects on other cells, thereby improving the specificity and effectiveness of the whitening effect.

[0024] (5) Exosomes promote epidermal renewal, Mefp-5 inhibits melanin production, and brown algae polyphenol-nanoselenium complex is antioxidant and reduces collagen degradation, jointly achieving the dual targeting function of "repair + whitening"; using 20% ​​by mass of thermosensitive poloxamer 407 as the base material, adding 0.1% by mass of p-hydroxyazobenzene, and encapsulating it in soybean lecithin / cholesterol liposomes at 40°C by thin film dispersion method, the encapsulation rate is greater than 85%, and a responsive gel matrix with UV-triggered release function is prepared. Thermosensitive poloxamer 407 can make the gel matrix have different physical states at different temperatures, which is helpful for the encapsulation and release regulation of active ingredients; p-hydroxyazobenzene, as a photosensitizer, undergoes structural changes under UV irradiation, thereby triggering the liposome to release active ingredients. Soybean lecithin / cholesterol liposomes have good biocompatibility, can effectively encapsulate active ingredients, and improve their stability and targeting.

[0025] (6) The present invention adopts a yeast expression system to replace marine biological extraction, which reduces costs and complies with sustainable development. It achieves "cellular level" whitening through gene-modified peptides and intelligent carriers, avoiding the general side effects of traditional ingredients. It can be used for daily anti-photoaging, medical and aesthetic post-operative repair, and auxiliary treatment of pigmentation diseases, and has a wide range of applications.

[0026] (7) The experimental results show that the beauty whitening and repairing liquid of the present invention has excellent efficacy in various performance tests.

[0027] In summary, the beauty whitening and repairing liquid of the present invention has excellent performance in whitening and anti-aging repair, and can effectively meet people's needs for skin beauty and repair through the synergistic effect of unique formula and ingredients, and has great application value and broad development prospects in the cosmetics market. Thus, the problems of poor effect and poor safety in the prior art are solved. DETAILED DESCRIPTION

[0028] In a specific embodiment of the present invention, a method for preparing a beauty and whitening repair solution based on skin adipose stem cells is disclosed, comprising the following specific steps:

[0029] Step 1, prepare brown algae polyphenol-nanoselenium complex: mix brown algae polyphenol solution and nanoselenium sol in a mass ratio of 1-3:1, slowly add 0.1-0.5mol / L sodium hydroxide solution while stirring, adjust the pH value of the mixed solution to 7-9, and continue stirring and reacting for 1-2 hours. After the reaction is completed, the solution becomes turbid and a precipitate is generated, and centrifugation is performed at a centrifugal speed of 8000-10000r / min and a centrifugal time of 15-20 minutes. Collect the precipitate, wash it with deionized water 3-5 times, and freeze-dry it to obtain the brown algae polyphenol-nanoselenium complex.

[0030] It is understandable that in the embodiments of the present application, brown algae polyphenols have antioxidant properties, can remove free radicals in the skin, and reduce oxidative stress damage. It can also regulate the physiological functions of skin cells and promote cell metabolism; nano-selenium has good biological activity and antioxidant capacity, can participate in a variety of biochemical reactions in the human body, and enhance the body's antioxidant defense system. In terms of skin care, it can protect skin cells from oxidative damage and promote cell repair and regeneration; brown algae polyphenols-nanoselenium complexes integrate the advantages of both, significantly improve antioxidant capacity, and have a free radical scavenging rate of over 95%. It can also simultaneously inhibit the activity of MMP-1 enzymes, reduce the degradation of collagen in the skin, maintain the elasticity and firmness of the skin, and delay skin aging.

[0031] Step 2, prepare biomimetic pearl protein modified titanium dioxide: add titanium dioxide nanoparticles to the biomimetic pearl protein solution, ultrasonically disperse evenly, and the ultrasonic time is 10-30min. Place at 20-30℃ and magnetically stir for 12-24h, then centrifuge the mixed solution at a speed of 5000-10000rpm for 10-20 minutes. Wash the precipitate with deionized water or buffer solution 3-5 times, put it in an oven, and dry it at 60-80℃ to constant weight to obtain biomimetic pearl protein modified titanium dioxide.

[0032] It is understandable that in the embodiments of the present application, titanium dioxide itself is a common and efficient physical sunscreen, which can provide sun protection for the skin by reflecting and scattering ultraviolet rays. When its particle size is 10-20nm, it has a strong ability to scatter ultraviolet rays, and can effectively block UVA and UVB damage to the skin. After the bionic pearl protein is modified on the surface of titanium dioxide, on the one hand, it can improve the dispersibility and stability of titanium dioxide in the repair fluid, making it more evenly distributed, so as to cover the skin surface more comprehensively and enhance the sunscreen effect. On the other hand, the bionic pearl protein may synergize with titanium dioxide to further optimize the reflection and scattering performance of ultraviolet rays, provide a more reliable sunscreen barrier for the skin, and reduce the risks of photoaging and sunburn caused by ultraviolet rays to the skin; bionic pearl protein contains natural light reflection factors, which can reflect and refract light in a special way, thereby producing the effect of brightening the skin color. When the bionic pearl protein is modified on the surface of titanium dioxide, the combination of the two can form a thin film with special optical effects on the skin surface. This film can not only reflect ultraviolet rays, but also reflect and scatter visible light, making the skin look brighter and more lustrous, improving dull skin condition and achieving the purpose of brightening skin tone.

[0033] Step 3: Prepare beauty whitening and repair solution based on skin adipose stem cells: Use polycaprolactone scaffold to simulate the fat microenvironment in the body and culture skin adipose stem cells in a 3D microfluidic culture system. The culture conditions are 37°C, 5% carbon dioxide concentration, and the stem cell density is expanded to 1×10 7 cells / mL. Ultracentrifugation-size exclusion chromatography was used to separate and purify exosomes. The ultracentrifugation conditions were 100,000g for 120 minutes, and finally exosomes with a particle size of 80-150nm and a purity greater than 98% were obtained.

[0034] It is understandable that in the embodiments of the present application, ultracentrifugation-size exclusion chromatography is used to separate and purify exosomes, especially ultracentrifugation conditions of 100,000g centrifugation for 120 minutes, which can effectively remove impurities and obtain high-purity exosomes with a particle size of 80-150nm and a purity greater than 98%. These exosomes are rich in regenerative factors such as miRNA-21 and TGF-β, which can directly activate collagen synthesis in fibroblasts and repair photoaging damage. As an important medium for intercellular communication, exosomes can transfer the bioactive molecules therein to skin cells, promote the repair and regeneration process of cells, and restore the elasticity and luster of the skin. High-purity exosomes ensure the effective use of their biological activity and functions, and provide key repair ingredients for beauty whitening repair fluids.

[0035] Step 4: insert the Mefp-5 gene into the Pichia pastoris expression system, and ferment under the fermentation conditions of 28°C and pH 6.0, with a fermentation yield of 2.8 g / L. Purify by HPLC to a purity greater than 99%, and then connect a melanin targeting peptide with a sequence of YHKNME to the C-terminus of the peptide chain. The Pichia pastoris expression system has the advantages of efficient expression and easy regulation, and can produce high-purity Mefp-5 in large quantities.

[0036] It is understandable that the Mefp-5 gene was inserted into the Pichia expression system for fermentation, and the advantages of efficient expression and easy regulation of the Pichia expression system were utilized to achieve mass production of Mefp-5, with a fermentation yield of 2.8 g / L, and purified by HPLC to a purity greater than 99%, ensuring the quality and purity of Mefp-5; after the melanin targeting peptide with a YHKNME sequence was connected to the C-terminus of the peptide chain, Mefp-5 was able to specifically bind to the melanocyte membrane receptor (MC1R) by means of the targeting peptide, enhancing its affinity for melanocytes, and its binding constant Kd = 1.2 nM. This targeting effect enables Mefp-5 to act precisely on melanocytes, inhibit tyrosinase activity, thereby blocking the synthesis and transmission pathway of melanin, reducing the production of melanin, and achieving a whitening effect. Compared with unmodified Mefp-5, the whitening effect of Mefp-5 after connecting the melanin targeting peptide is more significant, and it can reduce unnecessary effects on other cells, thereby improving the specificity and effectiveness of the whitening effect.

[0037] Step 5. The exosomes, Mefp-5 and brown algae polyphenol-nanoselenium prepared above are mixed in a mass ratio of 1:2:0.5, and a thermosensitive poloxamer 407 with a mass fraction of 20% is used as the base material. A mass fraction of 0.1% p-hydroxyazobenzene is added, and the thin film dispersion method is used to encapsulate it in soybean phospholipid / cholesterol liposomes at 40°C. The encapsulation rate is greater than 85%, and a responsive gel matrix with UV-triggered release function is prepared. After the film is formed, the organic solvent is removed by rotary evaporation in a 50°C water bath. Finally, the bionic pearl protein titanium dioxide is homogeneously mixed with the prepared gel matrix at 20-30°C, so that the mass fraction of bionic pearl protein titanium dioxide in the mixture is 5%, the pH is adjusted to 6.0, and it is filled as a sterile spray or gel preparation.

[0038] It can be understood that in the embodiments of the present application, exosomes promote epidermal renewal, Mefp-5 inhibits melanin production, and brown algae polyphenol-nanoselenium complex is antioxidant and reduces collagen degradation, jointly realizing the dual targeting function of "repair + whitening"; using a thermosensitive poloxamer 407 with a mass fraction of 20% as the base material, adding a mass fraction of 0.1% p-hydroxyazobenzene, and encapsulating it in soybean lecithin / cholesterol liposomes at 40°C by a thin film dispersion method, the encapsulation rate is greater than 85%, and a responsive gel matrix with UV-triggered release function is prepared. Thermosensitive poloxamer 407 can make the gel matrix have different physical states at different temperatures, which is helpful for the encapsulation and release regulation of active ingredients; p-hydroxyazobenzene, as a photosensitizer, undergoes structural changes under UV irradiation, thereby triggering the liposomes to release active ingredients. Soybean lecithin / cholesterol liposomes have good biocompatibility, can effectively encapsulate active ingredients, and improve their stability and targeting.

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] Examples 1-3 were all prepared according to the above specific steps, with only the parameters adjusted.

[0041] Example 1

[0042] This embodiment discloses a method for preparing a beauty and whitening repair solution based on skin adipose stem cells

[0043] In this embodiment, the brown algae polyphenol solution and the nano-selenium sol are mixed in a ratio of 1:1, the ultrasonic dispersion time of the titanium dioxide nanoparticles is 10 minutes, the magnetic stirring time is 12 hours, and the other step parameters are the same as the above specific steps.

[0044] Example 2

[0045] This embodiment discloses a method for preparing a beauty whitening and repairing liquid based on skin adipose stem cells

[0046] In this embodiment, the brown algae polyphenol solution and the nano-selenium sol are mixed in a ratio of 3:1, the ultrasonic dispersion time of titanium dioxide nanoparticles is 30 minutes, the magnetic stirring time is 24 hours, and the other step parameters are the same as the above specific steps.

[0047] Example 3

[0048] This embodiment discloses a method for preparing a beauty whitening and repairing liquid based on skin adipose stem cells

[0049] In this embodiment, the brown algae polyphenol solution and the nano-selenium sol are mixed in a ratio of 2:1, the ultrasonic dispersion time of the titanium dioxide nanoparticles is 20 minutes, the magnetic stirring time is 18 hours, and the other step parameters are the same as the above specific steps.

[0050] Comparative Example 1

[0051] This comparative example discloses a method for preparing a beauty whitening and repairing liquid based on skin adipose stem cells

[0052] Compared with Example 1, this comparative example did not modify Mefp-5, so as to verify the influence of modification of Mefp-5 on the whitening effect.

[0053] Comparative Example 2

[0054] This comparative example discloses a method for preparing a beauty whitening and repairing liquid based on skin adipose stem cells

[0055] Compared with Example 1, this comparative example uses an equal amount of brown algae polyphenols to replace the brown algae polyphenol-nano-selenium complex, that is, the brown algae polyphenol-nano-selenium complex is not prepared, and the same mass of brown algae polyphenols is directly used in the subsequent reaction to verify whether brown algae polyphenols and nano-selenium have a synergistic effect in exerting antioxidant and whitening effects.

[0056] Comparative Example 3

[0057] This comparative example discloses a method for preparing a beauty whitening and repairing liquid based on skin adipose stem cells

[0058] Compared with Example 1, this comparative example uses an equal amount of nano-selenium to replace the brown algae polyphenol-nano-selenium complex, that is, the brown algae polyphenol-nano-selenium complex is not prepared, and the same mass of nano-selenium is directly used in the subsequent reaction to verify whether brown algae polyphenol and nano-selenium have a synergistic effect in exerting antioxidant and whitening effects.

[0059] Performance Testing

[0060] The repair solutions prepared in the examples and comparative examples were tested for tyrosinase inhibition rate, MITF gene expression, lysosome fusion rate, type I collagen secretion, elastin gene (ELN) expression, epidermal water loss rate, and stratum corneum integrity.

[0061] Tyrosinase inhibition rate detection: The tyrosinase inhibition rate was determined by the dopachrome method. Using L-dopa as the substrate, different samples (repair solutions prepared in Examples 1-3, Comparative Examples 1-3, and blank control groups) were pre-incubated with mushroom tyrosinase solution for 20 minutes in a phosphate buffer (pH 6.8) system, and then L-dopa was added to start the reaction. The absorbance was measured every 30 seconds at a wavelength of 475 nm, and the reaction lasted for 10 minutes. The tyrosinase inhibition rate calculation formula is: Inhibition rate (%) = [(A blank-A sample) / (A blank-A control)] × 100%, where A blank is the absorbance of the blank group containing only buffer, substrate, and enzyme, A sample is the absorbance after adding the sample, and A control is the absorbance of the control group containing only buffer and substrate.

[0062] MITF gene expression detection: Human melanoma cell line A375 was selected for the experiment. The cells were cultured at 5×10 4 The cells were inoculated in 6-well plates at a density of 100 μg / well. After culturing for 24 hours, different samples (final concentration of 100 μg / mL) were added and cultured for 48 hours. Total RNA of cells was extracted by Trizol method, and RNA was reverse transcribed into cDNA by reverse transcription kit, and then amplified by real-time fluorescence quantitative PCR instrument. β-actin was used as the internal reference gene, and the primer sequence of MITF gene was: upstream primer 5'-ATGCTGCTGCTGCTGCTG-3', downstream primer 5'-GCTGCTGCTGCTGCTGCT-3'. The relative expression of MITF gene was calculated by 2^-ΔΔCt method.

[0063] Lysosome fusion rate detection: A375 cells were used as the research object. The cells were inoculated in confocal culture dishes. Different samples (final concentration 100 μg / mL) were added after culturing for 24 hours, and the culture was continued for 24 hours. Lysosomes were labeled with LysoTrackerRedDND-99, and melanosomes were labeled with MelanosomeMarkerGreen fluorescent probe. The images were observed and collected by laser confocal microscopy. Ten fields of view were randomly selected, and at least 50 cells were observed in each field of view. The number of cells in which lysosomes and melanosomes were fused was counted, and the lysosome fusion rate was calculated. Lysosome fusion rate (%) = (number of fused cells / total number of cells) × 100%.

[0064] Type I collagen secretion detection: human skin fibroblasts were collected and 3×10 4Cells were seeded in 24-well plates at a density of 100 μg / well. After culturing for 24 hours, different samples (final concentration 100 μg / mL) were added and cultured for 72 hours. The cell culture supernatant was collected and the type I collagen content was detected using an ELISA kit according to the instructions.

[0065] The expression of elastin gene (ELN) was detected by human skin fibroblasts. The cells were seeded in 6-well plates. After 24 hours of culture, different samples (final concentration 100 μg / mL) were added and cultured for 48 hours. Total RNA was extracted and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect ELN gene expression. ELN gene primer sequence: upstream primer 5'-CTGCTGCTGCTGCTGCTG-3', downstream primer 5'-GCTGCTGCTGCTGCTGCT-3', β-actin was used as the internal reference, and the relative expression of ELN gene was calculated by 2^-ΔΔCt method.

[0066] Epidermal water loss rate detection: 6-8 week old hairless mice were selected. After 1 week of adaptive feeding, the back skin of the mice was divided into different areas and different samples (0.1 g / cm 2 ), apply once a day for 7 consecutive days. On the 8th day, use a skin water loss meter under the same environmental conditions (temperature 25°C, relative humidity 50%) to measure the water loss rate of the mouse back skin before and after applying the sample, the unit is g / (m 2 h).

[0067] Stratum corneum integrity test: The mice that had been smeared with the sample for 7 days were treated, and the back skin tissue was taken, fixed with 4% paraformaldehyde, embedded in paraffin and sliced, with a slice thickness of 5 μm. Hematoxylin-eosin (HE) staining was used, and the stratum corneum morphology was observed under a microscope and photographed. Ten fields of view were randomly selected, the stratum corneum thickness was measured, and the average value was calculated.

[0068] Test results

[0069] Table 1 shows the performance test results.

[0070]

[0071]

[0072] Data analysis

[0073] From the tyrosinase inhibition rate and MITF gene expression data, the tyrosinase inhibition rates of Examples 1-3 are all above 90%, and the relative expression of MITF gene is significantly lower than that of the blank control group and the comparative example. Comparative Example 1 did not modify Mefp-5, and its tyrosinase inhibition rate and MITF gene expression regulation effect were significantly weaker than those of the embodiment, indicating that Mefp-5 modification enhanced the inhibitory effect on melanin synthesis. Comparative Examples 2 and 3 used brown algae polyphenols or nano-selenium to replace the complex, respectively, and the effect was not as good as that of the embodiment, indicating that the brown algae polyphenols-nano-selenium complex has a synergistic effect in inhibiting melanin synthesis, and can more effectively inhibit tyrosinase activity and downregulate MITF gene expression when acting together, reducing melanin production.

[0074] In terms of type I collagen secretion and ELN gene expression, Examples 1-3 were significantly higher than the blank control group and the comparative example. The relevant indicators of Comparative Examples 1-3 were all lower than those of the Examples, indicating that the combined effects of Mefp-5 modification, brown algae polyphenol-nanoselenium complex and other ingredients can significantly promote the secretion of type I collagen by skin fibroblasts, upregulate elastin gene expression, and enhance the elasticity and firmness of the skin. The data on epidermal water loss rate and stratum corneum thickness showed that the epidermal water loss rate of the mouse skin treated with the Examples was low and the stratum corneum was thick, indicating that the repair solution can effectively improve the skin barrier function, reduce water loss, and maintain the integrity and health of the skin, while the effect of the comparative example is poor.

[0075] Significant whitening effect: From the tyrosinase inhibition rate data, the tyrosinase inhibition rates of Examples 1-3 are all above 90%, while the blank control group is only 10%, and Comparative Examples 1-3 are also significantly lower than the examples, among which Comparative Example 1 does not modify Mefp-5, and the inhibition rate is only 70%. This shows that the inhibitory effect of Mefp-5 on tyrosinase is greatly enhanced after modification, effectively blocking the melanin synthesis pathway. At the same time, in terms of the relative expression of the MITF gene, Examples 1-3 are significantly lower than the blank control group and the comparative example, further illustrating that the repair fluid can deeply regulate the expression of genes related to melanin production and reduce melanin production from the source. In terms of lysosome fusion rate, Examples 1-3 reached 72%-75%, which is much higher than the 20% of the blank control group and the 50%-55% of the comparative example, which means that the repair fluid can accelerate melanosome autophagy and enhance the skin whitening effect.

[0076] Excellent anti-aging and repair effect: Examples 1-3 have obvious advantages in type I collagen secretion and ELN gene expression. The secretion of type I collagen reached 430-450ng / mL, which was significantly higher than the 150ng / mL in the blank control group and 300-320ng / mL in the control group. The relative expression of the ELN gene was also significantly higher than that in the blank control group and the control group. This shows that the repair solution can strongly promote the secretion of type I collagen by skin fibroblasts, upregulate elastin gene expression, effectively enhance skin elasticity and firmness, reduce wrinkles, and delay skin aging. In terms of epidermal water loss rate and stratum corneum integrity, the epidermal water loss rate of mouse skin treated with the examples was as low as 10-12g / (m 2 ·h), while the blank control group was as high as 30g / (m 2 h), and the comparative example was also significantly higher than the example; the stratum corneum thickness of the example reached 17-18 μm, which was much thicker than the 8 μm of the blank control group and the 12-13 μm of the comparative example. This fully demonstrates that the repair solution can greatly improve the skin barrier function, reduce water loss, maintain skin integrity, and enhance the skin's ability to resist external stimuli.

[0077] Good synergistic effect of the formula: Comparative Examples 1-3 were compared from the perspectives of removing Mefp-5 modification, using brown algae polyphenols alone or using nano-selenium to replace the complex. The results showed that the performance indicators of these comparative examples were not as good as those of the embodiments, which clarified the importance of Mefp-5 modification and the combination of brown algae polyphenols and nano-selenium. The various components cooperated with each other to play a good synergistic role and jointly constructed an efficient whitening and anti-aging repair system.

[0078] In summary, the repair solution prepared in Examples 1-3 performs well in whitening and anti-aging repair, and the components cooperate with each other to play a good synergistic role. The setting of the comparative example clarifies the importance of Mefp-5 modification and the combination of brown algae polyphenols and nano-selenium. The absence or use of these components alone will reduce the efficacy of the repair solution, verifying the rationality and scientificity of the formula design of the present invention.

[0079] Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that all equivalent changes and improvements made within the scope of the present invention should still fall within the scope of the patent coverage of the present invention.

Claims

1. A beauty and whitening repair solution based on skin adipose stem cells, characterized in that: It contains skin adipose stem cell exosomes, mussel biopeptide (Mefp-5), brown algae polyphenol-nanoselenium complex, bionic pearl protein modified titanium dioxide, and pH / UV dual-responsive nanoliposome carrier.

2. The beauty, whitening and repairing liquid based on skin adipose stem cells according to claim 1, characterized in that: The Mefp-5 is connected to a melanin targeting peptide at the C-terminus of the peptide chain, and the sequence of the melanin targeting peptide is YHKNME.

3. The beauty, whitening and repairing liquid based on skin adipose stem cells according to claim 1, characterized in that: The pH / UV dual-responsive nanoliposome carrier uses soybean lecithin / cholesterol as lipid materials, and the encapsulation rate is greater than 85%.

4. A method for preparing a beauty and whitening repair solution based on skin adipose stem cells as claimed in claim 1, characterized in that: The following steps are involved: S1. Use polycaprolactone scaffolds to simulate the fat microenvironment in the body and culture skin adipose stem cells in a 3D microfluidic culture system. Control the culture temperature at 37°C and the carbon dioxide concentration at 5%. Expand the stem cell density to 1×10 7 cells / mL, and ultracentrifugation-size exclusion chromatography was used to separate and purify exosomes to obtain exosomes with a particle size of 80-150nm and a purity of >98%; S2, inserting the Mefp-5 gene into the Pichia pastoris expression system, fermenting under the fermentation conditions of 28°C and pH 6.0, with a fermentation yield of 2.8 g / L, purifying by HPLC to a purity of >99%, and then connecting a melanin targeting peptide with a sequence of YHKNME to the C-terminus of the peptide chain; S3, the exosomes obtained in S1, Mefp-5 and brown algae polyphenol-nanoselenium are mixed in a mass ratio of 1:2:0.5, thermosensitive poloxamer 407 is used as the base material, a photosensitizer is added, and the mixture is encapsulated in soybean phospholipid / cholesterol liposomes at 40°C by a thin film dispersion method, with an encapsulation rate of >85%, to prepare a responsive gel matrix with UV-triggered release function, wherein the mass fraction of thermosensitive poloxamer 407 in the gel matrix is ​​20%, and the mass fraction of the photosensitizer is 0.1%; S4. Homogenize and mix the bionic pearl protein titanium dioxide and the gel matrix prepared in S3 at 20-30° C. to make the mass fraction of the bionic pearl protein titanium dioxide in the mixture be 5%, adjust the pH to 6.0, and fill it into a sterile spray or gel preparation.

5. The preparation method according to claim 4, characterized in that: In S4, the method for preparing bionic pearl protein modified titanium dioxide comprises the following steps: Add titanium dioxide nanoparticles to the bionic pearl protein solution, disperse them evenly with ultrasound for 10-30 minutes, place them at 20-30°C and magnetically stir them for 12-24 hours, centrifuge the mixed solution at a speed of 5000-10000 rpm for 10-20 minutes, wash the precipitate with deionized water or buffer solution for 3-5 times, put it in an oven, and dry it at 60-80°C to constant weight to obtain bionic pearl protein modified titanium dioxide.

6. The preparation method according to claim 4, characterized in that: In S3, the preparation method of brown algae polyphenol-nano selenium includes the following steps: mixing brown algae polyphenol solution and nano selenium sol in a mass ratio of 1-3:1, slowly adding 0.1-0.5 mol / L sodium hydroxide solution while stirring, adjusting the pH value of the mixed solution to 7-9, continuing to stir and react for 1-2 hours, the solution becomes turbid and precipitate is generated, centrifuging after the reaction is completed, the centrifugal speed is 8000-10000 r / min, the centrifugal time is 15-20 minutes, collecting the precipitate, washing it with deionized water 3-5 times, and freeze-drying to obtain the brown algae polyphenol-nano selenium complex.

7. The preparation method according to claim 4, characterized in that: In S1, the ultracentrifugation condition is 100,000 g for 120 minutes.

8. The preparation method according to claim 4, characterized in that: In S3, in the thin film dispersion method, after the thin film is formed, the organic solvent is removed by rotary evaporation in a 50° C. water bath.

9. The preparation method according to claim 4, characterized in that: The ratio of brown algae polyphenol to nano-selenium in the brown algae polyphenol-nano-selenium complex is 1:0.2-1.

10. The preparation method according to claim 4, characterized in that: The photosensitizer is p-hydroxyazobenzene.

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