Cytokines for removing freckles and whitening the skin, their preparation methods and applications

Abstract

The present invention discloses a preparation method of cytokines for freckle removal and skin whitening, comprising: S1, preparing a cell culture medium; S2, culturing the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts; S3, collecting the culture solution when the human skin fibroblasts grow to 80-90% confluence to obtain a human skin fibroblast culture solution; S4, digesting the human skin fibroblasts, centrifuging after terminating digestion, removing the supernatant, and retaining the precipitate; then resuspending the precipitate with the cell culture medium, centrifuging after repeated freezing and thawing, and removing cell debris to obtain a cell extract; S5, centrifuging the human skin fibroblast culture solution and removing cell debris, and then mixing it with the cell extract to obtain cytokines. The cytokines prepared by the present invention have high contents of EGF, FGF, KGF, VEGF, and TGF-β, and have good effects on freckle removal and skin whitening.

Description

Technical Field

[0001] The present invention relates to the technical field of medical beauty, and particularly relates to a cytokine for freckle removal and whitening, a preparation method thereof, and an application thereof. Background Art

[0002] Freckles are the result of pigment precipitation. The main cause of freckle formation is ultraviolet irradiation. When the skin receives too much sunlight, strong ultraviolet rays are likely to activate tyrosinase in melanocytes, accelerating melanin synthesis and resulting in pigment deposition. With the development of the medical beauty industry, methods such as laser, radiofrequency, photon, chemical peeling, and microcrystalline abrasion have become the mainstream ways to remove freckles at present. However, these technologies all achieve freckle removal by forcibly removing the freckle area, which not only causes certain damage to healthy skin, but also has a short maintenance time and cannot solve the problem from the root. At present, the conventional externally applied freckle removal products have low transdermal absorption efficiency and slow effects, which are not suitable for the current fast-paced lifestyle.

[0003] Cytokines are low-molecular-weight soluble proteins produced by cells stimulated by immunogens, mitogens, or other factors. They are bioinformation molecules and have functions such as regulating innate and adaptive immune responses, promoting hematopoiesis, and stimulating cell activation, proliferation, and differentiation. According to the main functions of cytokines, cytokines can be classified into interleukin, colony-stimulating factor, interferon, tumor necrosis factor, transforming growth factor-β family, growth factor, and chemokine family. Among them, epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), and keratinocyte growth factor (KGF) in growth factors are several commonly used growth factors in the field of skin beauty. They play a biological regulatory role in the physiological functions and metabolic activities of various cells and directly or indirectly affect the growth, division, differentiation, proliferation, and migration of various types of cells. And transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily that regulates cell growth and differentiation and can promote wound repair. In the existing preparation methods of cytokines, the contents of EGF, FGF, KGF, VEGF, and TGF-β in the prepared cytokines are low, and good beauty effects cannot be achieved. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a preparation method of a cytokine for freckle removal and whitening, in which the contents of EGF, FGF, KGF, VEGF, and TGF-β in the prepared cytokine are high, which is beneficial to biological beauty.

[0005] The technical problem to be solved by the present invention is also to provide a cytokine for freckle removal and whitening, which has good biological beauty effects and can remove freckles and whiten the skin.

[0006] The technical problem to be solved by the present invention further lies in providing the application of the cytokine for removing freckles and whitening the skin in microneedle beauty treatment.

[0007] To solve the above problems, the present invention provides a preparation method of a cytokine for removing freckles and whitening the skin, comprising the following steps:

[0008] S1. Prepare a cell culture medium, which is the culture solution when umbilical cord mesenchymal stem cells grow to 80-90% confluence;

[0009] S2. Culture the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts;

[0010] S3. When the human skin fibroblasts grow to 80-90% confluence, collect the culture solution to obtain the culture solution of human skin fibroblasts;

[0011] S4. Digest the human skin fibroblasts, centrifuge after terminating digestion, remove the supernatant, and retain the precipitate;

[0012] Resuspend the precipitate with the cell culture medium, freeze-thaw repeatedly and then centrifuge to remove cell debris to obtain a cell extract;

[0013] S5. After centrifuging the culture solution of human skin fibroblasts and removing cell debris, mix it with the cell extract to obtain a cytokine.

[0014] In one embodiment, in step S1, the cell culture medium is prepared by the following method:

[0015] Collect the culture solution when umbilical cord mesenchymal stem cells grow to 80-90% confluence, freeze and store the collected culture solution until the culture solutions of P1-(P5-P6) generations are collected, thaw and then centrifuge to remove cell debris, and filter to obtain the cell culture medium.

[0016] In one embodiment, collect the culture solution when umbilical cord mesenchymal stem cells grow to 80-90% confluence, place the collected culture solution at -80°C for storage until the culture solutions of P1-P5 generations are collected, then thaw at 4°C and centrifuge at 3000 rpm for 5 min to remove cell debris, and filter through a 0.22 μm filter membrane to obtain the cell culture medium.

[0017] In one embodiment, in step S3, when the human skin fibroblasts grow to 80-90% confluence, collect the culture solution, freeze and store the collected culture solution until the culture solutions of P1-(P4-P5) generations are collected to obtain the culture solution of human skin fibroblasts.

[0018] In one embodiment, in step S3, when the human skin fibroblasts grow to 80 - 90% confluence, the culture medium is collected, and the collected culture medium is cryopreserved until the culture media of passages P1 - P4 are collected to obtain the human skin fibroblast culture medium;

[0019] In step S4, the passage P4 human skin fibroblasts are digested, centrifuged after terminating the digestion, the supernatant is removed, and the precipitate is retained;

[0020] Then, the precipitate is resuspended with the cell culture medium, centrifuged after repeated freezing and thawing, and the cell debris is removed to obtain a cell extract.

[0021] In one embodiment, the passage P4 human skin fibroblasts are digested with trypsin, centrifuged after terminating the digestion with the cell culture medium, the supernatant is removed, and the precipitate is retained;

[0022] Then, the precipitate is resuspended with the cell culture medium, centrifuged after repeated freezing and thawing, and the cell debris is removed to obtain a cell extract.

[0023] In one embodiment, the passage P4 human skin fibroblasts are digested with 0.2 - 0.3% trypsin for 3 - 4 min, centrifuged at 1300 rpm for 6 min after terminating the digestion with the cell culture medium, the supernatant is removed, and the precipitate is retained;

[0024] Then, the precipitate is resuspended with the cell culture medium, frozen at -20°C and thawed at 4°C, and after repeating the freezing and thawing 3 times, it is ultracentrifuged to remove the cell debris to obtain a cell extract.

[0025] In one embodiment, in step S5, the human skin fibroblast culture medium is centrifuged at 3000 rpm for 5 min to remove the cell debris, and then mixed with the cell extract to obtain cytokines.

[0026] Correspondingly, the present invention also provides cytokines for freckle removal and skin whitening prepared by the above - mentioned method for preparing cytokines for freckle removal and skin whitening. In addition, the application of the cytokines for freckle removal and skin whitening in freckle removal and skin whitening products.

[0027] Implementing the present invention has the following beneficial effects:

[0028] A method for preparing a cytokine for freckle removal and skin whitening provided by the present invention uses the culture medium of umbilical cord mesenchymal stem cells when they grow to 80-90% confluence as a cell culture medium to culture human skin fibroblasts; and mixes the culture medium of the human skin fibroblasts when they grow to 80-90% confluence with the cell extract obtained by digesting, freezing and thawing the human skin fibroblasts to obtain the cytokine. The cytokine prepared by the method provided by the present invention has high contents of EGF, FGF, KGF, VEGF, and TGF-β. When combined with the microneedle beauty treatment method, it can achieve good freckle removal and skin whitening effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the test result of the melanin content of Example 1-3 and Comparative Example 1-2 groups. DETAILED DESCRIPTION OF THE INVENTION

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments.

[0031] To solve the above problems, the present invention provides a method for preparing a cytokine for freckle removal and skin whitening, including the following steps:

[0032] S1. Prepare a cell culture medium, and the cell culture medium is the culture medium of umbilical cord mesenchymal stem cells when they grow to 80-90% confluence;

[0033] In one embodiment, in step S1, the following method is used to prepare the cell culture medium:

[0034] Collect the culture medium of umbilical cord mesenchymal stem cells when they grow to 80-90% confluence, and the collected culture medium is frozen and stored until the culture media of P1-(P5-P6) generations are collected. After thawing, centrifuge to remove cell debris, and filter to obtain the cell culture medium.

[0035] Preferably, collect the culture medium of umbilical cord mesenchymal stem cells when they grow to 80-90% confluence, and store the collected culture medium at -80°C until the culture media of P1-P5 generations are collected. Then thaw at 4°C and centrifuge at 3000 rpm for 5 min to remove cell debris, and filter through a 0.22 μm filter membrane to obtain the cell culture medium.

[0036] More preferably, according to the method for preparing human umbilical cord Wharton's jelly mesenchymal stem cells in CN110438071A, prepare and culture the umbilical cord mesenchymal stem cells.

[0037] S2. Culture the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts;

[0038] Preferably, it specifically includes the following steps:

[0039] Obtain the dermal tissue of human skin, then wash it with a tissue cleaning solution and cut it into tissue fragments after cleaning; digest the tissue fragments, after terminating the digestion, sieve out the tissue blocks, centrifuge, remove the supernatant, and retain the precipitate; resuspend the precipitate with the cell culture medium prepared in step S1, aliquot and culture to obtain human skin fibroblasts. The present invention uses the cell culture medium to replace the conventional culture medium, which can promote the proliferation of human skin fibroblasts and the secretion of cytokines.

[0040] S3. When the human skin fibroblasts grow to 80-90% confluence, collect the culture medium to obtain the human skin fibroblast culture medium;

[0041] In one embodiment, in step S3, when the human skin fibroblasts grow to 80-90% confluence, collect the culture medium, and freeze and store the collected culture medium until the culture media of P1-(P4-P5) generations are collected to obtain the human skin fibroblast culture medium.

[0042] Preferably, in step S3, when the human skin fibroblasts grow to 80-90% confluence, collect the culture medium, and freeze and store the collected culture medium until the culture media of P1-P4 generations are collected to obtain the human skin fibroblast culture medium.

[0043] S4. Digest the human skin fibroblasts, after terminating the digestion, centrifuge, remove the supernatant, and retain the precipitate;

[0044] Then resuspend the precipitate with the cell culture medium, centrifuge after repeated freezing and thawing to remove cell debris, and obtain a cell extract.

[0045] Correspondingly, in step S4, digest the P4 generation of human skin fibroblasts, after terminating the digestion, centrifuge, remove the supernatant, and retain the precipitate;

[0046] Then resuspend the precipitate with the cell culture medium, centrifuge after repeated freezing and thawing to remove cell debris, and obtain a cell extract.

[0047] In one embodiment, digest the P4 generation of human skin fibroblasts with trypsin, terminate the digestion with the cell culture medium, centrifuge, remove the supernatant, and retain the precipitate;

[0048] Then resuspend the precipitate with the cell culture medium, centrifuge after repeated freezing and thawing to remove cell debris, and obtain a cell extract.

[0049] Preferably, digest the passage 4 human skin fibroblasts with 0.2 - 0.3% trypsin for 3 - 4 minutes, terminate digestion with the cell culture medium, then centrifuge at 1300 rpm for 6 minutes, remove the supernatant, and retain the precipitate;

[0050] Resuspend the precipitate with the cell culture medium, place it at -20°C for freezing, and thaw at 4°C. Repeat the freeze-thaw cycle 3 times, then perform ultracentrifugation to remove cell debris and obtain a cell extract.

[0051] S5. After centrifuging the human skin fibroblast culture medium to remove cell debris, mix it with the cell extract to obtain cytokines.

[0052] In one embodiment, in step S5, centrifuge the human skin fibroblast culture medium at 3000 rpm for 5 minutes to remove cell debris, then mix it with the cell extract to obtain cytokines.

[0053] It should be noted that in the present invention, a cell extract is obtained by digesting, freezing, and thawing the human skin fibroblasts, and then mixed with the human skin fibroblast culture medium obtained in step S3 to obtain cytokines. Compared with simply centrifuging the human skin fibroblast culture medium to remove impurities to obtain cytokines, the contents of EGF, FGF, KGF, VEGF, and TGF-β are significantly increased, and at the same time, the freckle-removing and whitening effects are also significantly improved.

[0054] Correspondingly, the present invention also provides cytokines for freckle-removing and whitening prepared by the above method for preparing cytokines for freckle-removing and whitening. And the application of the cytokines for freckle-removing and whitening in freckle-removing and whitening products.

[0055] In summary, in the present invention, the culture medium of umbilical cord mesenchymal stem cells when growing to 80 - 90% confluence is used as the cell culture medium to culture human skin fibroblasts; and the culture medium of the human skin fibroblasts when growing to 80 - 90% confluence is mixed with the cell extract obtained by digesting, freezing, and thawing the human skin fibroblasts to obtain cytokines. The cytokines prepared by the method provided by the present invention have high contents of EGF, FGF, KGF, VEGF, and TGF-β. When combined with the microneedle beauty treatment method, good freckle-removing and whitening effects can be obtained.

[0056] The following further illustrates the present invention with specific examples:

[0057] Example 1

[0058] A method for preparing cytokines for freckle-removing and whitening includes the following steps:

[0059] S1. Preparation of cell culture medium: Collect the culture medium when umbilical cord mesenchymal stem cells grow to 80 - 90% confluence. The collected culture medium is stored at -80°C until the culture media of P1 - P5 generations are collected. Then, it is thawed at 4°C and centrifuged at 3000 rpm for 5 min to remove cell debris. After filtration through a 0.22 - μm filter membrane, the cell culture medium is obtained. The umbilical cord mesenchymal stem cells are prepared by the method for preparing human umbilical cord Wharton's jelly mesenchymal stem cells disclosed in CN110438071A.

[0060] S2. Culture the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts.

[0061] S3. When the human skin fibroblasts grow to 80 - 90% confluence, collect the culture medium. The collected culture medium is cryopreserved until the culture media of P1 - P4 generations are collected to obtain the culture medium of human skin fibroblasts.

[0062] S4. Digest the P4 - generation human skin fibroblasts with 0.2 - 0.3% trypsin for 3 - 4 min. After terminating the digestion with the cell culture medium, centrifuge at 1300 rpm for 6 min to remove the supernatant and retain the precipitate.

[0063] Resuspend the precipitate with the cell culture medium, freeze it at -20°C, and thaw it at 4°C. Repeat the freeze - thaw cycle 3 times and then perform ultra - centrifugation to remove cell debris to obtain a cell extract.

[0064] S5. Centrifuge the culture medium of human skin fibroblasts at 3000 rpm for 5 min to remove cell debris, and then mix it with the cell extract to obtain cytokines.

[0065] Example 2

[0066] A method for preparing cytokines for freckle removal and skin whitening

[0067] The difference from Example 1 is as follows: In step S1, for the preparation of the cell culture medium: Collect the culture medium when umbilical cord mesenchymal stem cells grow to 80 - 90% confluence. The collected culture medium is stored at -80°C until the culture media of P1 - P6 generations are collected. Then, it is thawed at 4°C and centrifuged at 3000 rpm for 5 min to remove cell debris. After filtration through a 0.22 - μm filter membrane, the cell culture medium is obtained. The rest is the same as in Example 1.

[0068] Example 3

[0069] A method for preparing cytokines for freckle removal and skin whitening

[0070] The difference from Example 1 is as follows: In step S3, when the human skin fibroblasts grow to a confluence of 80-90%, the culture medium is collected, and the collected culture medium is cryopreserved until the culture media of P1-P5 generations are collected to obtain the human skin fibroblast culture medium;

[0071] In step S4, the P5-generation human skin fibroblasts are digested with 0.2-0.3% trypsin for 3-4 minutes. After terminating the digestion with the cell culture medium, the mixture is centrifuged at 1300 rpm for 6 minutes to remove the supernatant and retain the precipitate; then the precipitate is resuspended with the cell culture medium, placed at -20°C for freezing, and thawed at 4°C. After repeating the freeze-thaw cycle 3 times, it is ultracentrifuged to remove cell debris to obtain the cell extract. The rest is the same as in Example 1.

[0072] Comparative Example 1

[0073] A method for preparing a cytokine, comprising the following steps:

[0074] S1. Prepare a cell culture medium: Collect the culture medium when the umbilical cord mesenchymal stem cells grow to a confluence of 80-90%. The collected culture medium is stored at -80°C until the culture media of P1-P5 generations are collected, then thawed at 4°C and centrifuged at 3000 rpm for 5 minutes to remove cell debris, and filtered through a 0.22-μm filter membrane to obtain the cell culture medium; the umbilical cord mesenchymal stem cells are prepared by the method for preparing human umbilical cord Wharton's jelly mesenchymal stem cells disclosed in CN110438071A.

[0075] S2. Culture the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts;

[0076] S3. When the human skin fibroblasts grow to a confluence of 80-90%, collect the culture medium, and cryopreserve the collected culture medium until the culture media of P1-P4 generations are collected to obtain the human skin fibroblast culture medium;

[0077] S4. Centrifuge the human skin fibroblast culture medium at 3000 rpm for 5 minutes to remove cell debris to obtain the cytokine.

[0078] Comparative Example 2

[0079] A method for preparing a cytokine, comprising the following steps:

[0080] S1. Preparation of cell culture medium: Collect the culture medium when umbilical cord mesenchymal stem cells grow to 80 - 90% confluence. The collected culture medium is stored at -80°C until the culture media of passages P1 - P5 are collected. Then, it is thawed at 4°C and centrifuged at 3000 rpm for 5 min to remove cell debris. It is filtered through a 0.22 μm filter membrane, and the retentate collected is the cytokine. The umbilical cord mesenchymal stem cells are prepared by the method for preparing human umbilical cord Wharton's jelly mesenchymal stem cells disclosed in CN110438071A.

[0081] Determination of cytokine content (ELISA method)

[0082] Use the enzyme - linked immunosorbent assay (ELISA method) to determine the contents of EGF, FGF, KGF, VEGF, and TGF - β in the cytokines obtained in Examples 1 - 3 and Comparative Examples 1 - 2. The specific steps of the test method are as follows:

[0083] a) Coating antibody: Dilute the coating antibody with 1×PBS buffer. In the ELISA plate, add 100 μL to each well using a pipette to make a solid - phase antibody - coated plate, and place it in a 4°C refrigerator overnight.

[0084] b) Washing: Set the program of the plate washer. After the program starts, the instrument automatically sucks out the liquid in the plate and washes the ELISA plate three times with the washing buffer PBS buffer T.

[0085] c) Blocking: Dilute the 5× concentrated diluent to 1× with ultrapure water in advance, add 200 μL of the diluent to each well, and place it at room temperature for 1 h. After 1 h, wash it with the plate washer, and the operation is the same as step b).

[0086] d) Sample addition: Dilute the sample 100 - fold with 1× diluent, add 100 μL of the sample diluent to each well. Note that when adding the sample, do not touch the bottom of the well plate and avoid wall - hanging. Place the well plate at room temperature for 2 h, and wash it with the plate washer. The operation is the same as step b).

[0087] e) Detection antibody: Dilute the detection antibody with the prepared 1× diluent, add 100 μL to each well, place the well plate at room temperature for 1 h, and wash it with the plate washer. The operation is the same as step b).

[0088] f) Adding enzyme - labeled antibody: According to the dilution rule, dilute the enzyme - labeled reagent Avidin - HRP with the prepared 1× diluent, add 100 μL to each well, place it at room temperature for 30 min, and then wash it with the plate washer. The operation is the same as step b).

[0089] g) Adding chromogenic agent: Add 100 μL of 1× TMB chromogenic agent to each well and place it at room temperature for 15 min.

[0090] h) Termination reaction: Add 100 μL of termination solution (2N H2SO4) to each well;

[0091] i) Detection: Immediately after step h), use an ELISA reader to detect the absorbance value of each well at a wavelength of 450 nm;

[0092] j) Data processing: Using the different concentrations of the standard product required in the kit as the abscissa and the corresponding OD450 values as the ordinate, plot a standard curve and calculate the regression equation (an R value of 0.99 or above can be used). According to the standard curve, substitute the experimental data into the standard curve, calculate the corresponding concentration, and finally multiply by the corresponding dilution factor to obtain the actual concentration of the sample.

[0093] Kit: Human Epidermal Growth Factor (EGF) ELISA Kit, product number: ZK-H238;

[0094] Human Fibroblast Growth Factor (FGF) ELISA Kit, product number: ZK-H3049;

[0095] Human Keratinocyte Growth Factor (KGF) ELISA Kit, product number: ZK-H3050;

[0096] Human Vascular Endothelial Growth Factor (VEGF) ELISA Kit, product number: ZK-H008;

[0097] Human Transforming Growth Factor β (TGF-β) ELISA Kit, product number: ZK-H042.

[0098] The measured results of the contents of EGF, FGF, KGF, VEGF, and TGF-β in the cytokines are shown in Table 1:

[0099] Table 1 shows the test results of the cytokine contents obtained in Examples 1-3 and Comparative Examples 1-2

[0100]

[0101] According to the measurement results in Table 1, the contents of EGF, FGF, KGF, VEGF, and TGF-β in the cytokines prepared in Examples 1-3 are all higher than those of the cytokines prepared in the comparative examples.

[0102] Evaluation of the whitening effect of the obtained cytokines by cell biology culture method

[0103] Determine the absorbance values of the cytokines prepared in Examples 1-3 and Comparative Examples 1-2 by the tetrazolium salt colorimetric method (MTT), calculate the cell proliferation index according to the absorbance values, and observe the inhibitory effect of the cytokines on the growth of melanoma cells.

[0104] Normal human melanocytes are the first choice of cells for screening whitening products. Or an epidermal model can be constructed with growing and differentiating human keratinocytes. After being exposed to air for a certain period of time, a dense stratum corneum can be formed, which is very close to the human epidermis. Based on this epidermal model, melanoma cells and Langerhans cells are inoculated. The production of melanin and its transfer to adjacent keratinocytes can be clearly observed in this model. The formation of melanin can be detected by the following means.

[0105] The test method is as follows: The whitening effect of cytokines is determined by inhibiting the degree of melanin production in melanoma. The cultured melanoma cells (SK23 human melanoma cells, product number EY-X64022, Shanghai Yiyan Biotechnology Co., Ltd.) are inoculated in a 96-well plate at a density of 1×10 5 cells / well, and the cytokines prepared in Examples 1 to 3 and Comparative Examples 1 to 2 are used to replace the culture medium for culturing for 3 days. The one without cytokines is used as a blank control, and only the culture medium is used for treatment. After 3 days, the culture medium is recovered and centrifuged at 4500 rpm for 10 minutes, and the absorbance is measured at 405 nm to calculate the amount of melanin released from the cells. Trypsin is used to remove the cells adhered to the plate, and the cell number is measured, and then centrifuged to recover the cells. The cells are washed once with PBS and centrifuged to obtain a cell pellet. 1 ml of sodium hydroxide solution containing 10% dimethyl sulfoxide is added to the cell pellet, and the melanin is dissolved at 80 °C for 2 hours, and then the obtained product is added to a 96-well plate, and the absorbance is measured at 405 nm. The measured absorbance is used to quantify the melanin and normalized to the protein concentration of the sample to determine the concentration of synthesized melanin.

[0106] The test results are as Figure 1 shown. It can be seen from Figure 1 that the cytokines prepared in Examples 1 to 3 and Comparative Examples 1 to 2 can all inhibit the formation of melanin, but the cytokines prepared in Examples 1 to 3 have a better effect of inhibiting melanin. Among them, in the group applying Example 1, the extracellular melanin content is less than 10 μg / mg, which is significantly lower than that of the blank control group.

[0107] Test effect of cytokine facial skin treatment

[0108] The test steps are as follows:

[0109] A. Select 30 volunteers aged 20 - 45, all of whom are indoor workers. The exclusion criteria for the subjects comply with GB171492 - 1997 "Diagnostic Criteria and Management Principles for Cosmetic Contact Dermatitis", and during the clinical observation period, they are prohibited from consuming spicy and stimulating foods, smoking, and drinking alcohol. Starting from 2 weeks before the clinical trial, the volunteers do not use hormonal drugs and anti - allergy drugs, nor do they use auxiliary cosmetics. The volunteers volunteer to participate in the experiment and sign an informed consent form. According to the Chardon grouping method, the skin color is between II and IV.

[0110] B. Test indicators: Skin melanin content (MI value). The MI value represents the melanin index, with a numerical range of 0 - 999. The higher the measured value, the higher the melanin content in the skin. The determination of the Mexameter MX18 skin melanin and heme test probe is based on the principle of spectral absorption. By measuring the reflection amount of light with a specific wavelength on the human skin, the melanin content in the skin is determined. Skin whiteness (L* value), the L* value is the white balance. The larger the value, the more the color tends to white, and vice versa, it tends to black. Skin brightness (ITA° value), the ITA° value is the skin individual type angle, which is a numerical value related to the L* value and b* value representing skin brightness. The larger the ITA° value, the brighter the skin, and vice versa, the darker the skin.

[0111] The specific test methods are as follows:

[0112] ① Control the temperature at (20 ± 2) °C and the relative humidity at 55% ± 3%. After slightly cleaning the tester's face, let the tester sit quietly in this environment for 30 minutes and then conduct the test. Use the skin melanin and heme test probe to detect the MI value of the zygomatic region of the volunteer's face before using the sample; use the skin color difference test probe to test the L* value and ITA° value of the volunteer's face before using the sample; use the VISIA facial image analysis system to take a photo of the entire face of the subject before using the sample, and try to avoid the laugh lines, reflective areas, moles, and hair in the analysis area.

[0113] ② According to the MI value, L* value, and ITA° value measured before the volunteers use the sample, divide all the volunteers into five groups to ensure that there are no significant differences in the initial values of each group of volunteers.

[0114] ③ After the volunteers wash their faces, use 3 mL of cytokines on the entire face in the form of microneedles.

[0115] ④ At 1, 2, and 4 weeks after microneedling, under the conditions described in step ①, respectively detect the MI value, L* value, and ITA° value of each group of volunteers. Measure each time 3 times, take the average value, and use the VISIA facial image analysis system to take photos of the entire face of the experimental group volunteers.

[0116] The test results are shown in Tables 2 - 4, specifically as follows:

[0117] 1) Skin MI value

[0118] The test results of the skin MI value are shown in Table 2. As can be seen from Table 2, within 4 weeks of the test, after the volunteers were treated with the cytokine microneedles prepared in Examples 1 to 3, the MI values all showed a decreasing trend. And in the 1st, 2nd, and 4th weeks, the group of Example 1 was significantly lower than the group of Comparative Example 2 (p < 0.05), indicating that the cytokines prepared in Examples 1 to 3 can all reduce the melanin content in the skin, and the cytokine of the group of Example 1 has the most significant effect. Compared with the 0th week, the MI value of the volunteers decreased by 17.73% after using the product for 4 weeks, with a highly significant difference (P < 0.001).

[0119] Table 2 Changes in skin MI values of each group (mean ± SD, n = 6)

[0120]

[0121] 2) Skin L* value

[0122] The test results of the skin L* value are shown in Table 3. As can be seen from Table 3, within 4 weeks of the test, after the subjects were treated with the cytokine microneedles prepared in Examples 1 to 3, the L* values all showed an increasing trend. And in the 1st, 2nd, and 4th weeks, the group of Example 1 was significantly higher than the group of Comparative Example 2 (p < 0.05), indicating that the cytokines prepared in Examples 1 to 3 can all improve the skin color and enhance the skin whiteness, and the cytokine of the group of Example 1 has the most significant effect.

[0123] Table 3 Changes in skin L* values of each group (mean ± SD, n = 6)

[0124]

[0125] 3) Skin ITA° value

[0126] Table 4 shows the test results of the skin ITA° value. As can be seen from Table 4, within 4 weeks of the test, after the volunteers were treated with the cytokine microneedles prepared in Examples 1 to 3, the ITA° values all showed an increasing trend. And in the 1st, 2nd, and 4th weeks, the group of Example 1 was significantly higher than the group of Comparative Example 2 (p < 0.05). The statistical results show that in the 2nd and 4th weeks, there was a significant difference in the ITA° value between the groups of Examples 1 to 3 and before applying the sample (p < 0.05), indicating that the cytokines prepared in Examples 1 to 3 can all significantly improve the dullness of the skin and enhance the skin brightness, having a whitening effect.

[0127] Table 4 Changes in skin ITA° values of each group (mean ± SD, n = 6)

[0128]

[0129] 4) VISIA Effect Analysis before and after Treatment with Cytokine Microneedles

[0130] Before treatment with cytokine microneedles and 4 weeks after treatment, full-face photos of the subjects were taken using the VISIA facial image analysis system. The results showed that before treatment with the cytokine microneedles prepared in Examples 1 to 3, the subjects had dull yellow skin with scattered small spots; while after 4 weeks of use, the skin texture became finer than before, the color became whiter, and the spots became lighter. Among them, the number of spots in the Example 1 group decreased by 21%, and the number of brown spots decreased by 18%. Compared with the effects of other groups, the effect was relatively significant, indicating that this cytokine has obvious advantages.

[0131] The above are the preferred embodiments of the invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements are also regarded as the protection scope of the present invention.

Claims

1. A preparation method of a cytokine for removing freckles and whitening the skin, characterized in that, It includes the following steps: S1. Prepare a cell culture medium, which is the culture solution when umbilical cord mesenchymal stem cells grow to 80%-90% confluence; S2. Culture the dermal tissue of human skin with the cell culture medium to obtain human skin fibroblasts; S3. When the human skin fibroblasts grow to 80%-90% confluence, collect the culture solution to obtain the culture solution of human skin fibroblasts; S4. Digest the human skin fibroblasts, centrifuge after terminating digestion, remove the supernatant, and retain the precipitate; Resuspend the precipitate with the cell culture medium, centrifuge after repeated freezing and thawing, and remove cell debris to obtain a cell extract; S5. After centrifuging the culture solution of human skin fibroblasts and removing cell debris, mix it with the cell extract to obtain cytokines; Among them, in step S1, the cell culture medium is prepared by the following method: Collect the culture solution when umbilical cord mesenchymal stem cells grow to 80%-90% confluence, freeze and store the collected culture solution until the culture solutions of P1-P5 / P6 generations are collected, thaw and centrifuge, remove cell debris, and filter to obtain the cell culture medium; In step S3, when the human skin fibroblasts grow to 80%-90% confluence, collect the culture solution, freeze and store the collected culture solution until the culture solutions of P1-P4 generations are collected to obtain the culture solution of human skin fibroblasts; Digest the P4 generation of human skin fibroblasts with trypsin, centrifuge after terminating digestion with the cell culture medium, remove the supernatant, and retain the precipitate; Resuspend the precipitate with the cell culture medium, centrifuge after repeated freezing and thawing, and remove cell debris to obtain a cell extract.

2. The preparation method of the cytokine for freckle removal and skin whitening according to claim 1, characterized in that, Collect the culture solution when umbilical cord mesenchymal stem cells grow to 80%-90% confluence, place the collected culture solution at -80°C for storage until the culture solutions of P1-P5 generations are collected, then thaw at 4°C and centrifuge at 3000 rpm for 5 min, remove cell debris, and filter through a 0.22 μm filter membrane to obtain the cell culture medium.

3. The preparation method of the cytokine for freckle removal and skin whitening according to claim 1, characterized in that, Digest the P4 generation of human skin fibroblasts with 0.2%-0.3% trypsin for 3-4 min, centrifuge at 1300 rpm for 6 min after terminating digestion with the cell culture medium, remove the supernatant, and retain the precipitate; Resuspend the precipitate with the cell culture medium, freeze at -20°C and thaw at 4°C, repeat the freezing and thawing 3 times and then ultracentrifuge to remove cell debris to obtain a cell extract.

4. The preparation method of the cytokine for freckle removal and skin whitening according to claim 1, characterized in that, In step S5, centrifuge the culture solution of human skin fibroblasts at 3000 rpm for 5 min, remove cell debris, and then mix it with the cell extract to obtain cytokines.

5. A cytokine for removing freckles and whitening the skin, characterized in that, The cytokines for freckle removal and skin whitening are prepared by the preparation method of the cytokines for freckle removal and skin whitening according to any one of claims 1 to 4.

6. The application of the cytokines for freckle removal and skin whitening according to claim 5 in freckle removal and skin whitening products.

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