A placental / umbilical cord mesenchymal stem cell culture composition and its application in functional anti-aging.

Abstract

This invention discloses a placental / umbilical cord mesenchymal stem cell culture composition and its application in functional anti-aging. The stem cell culture composition is the culture supernatant obtained after mesenchymal stem cells are induced and cultured in a first induction medium and a second induction medium. The first induction medium contains 10-30 mg / L safflower flavonoids, 5.0-7.5 μg / L transferrin, 0.4-1.2 μg / L bovine serum albumin, and 2.0-5.5 μg / L linoleic acid. The second induction medium contains 10-30 mg / L safflower flavonoids, 60-90 mg / L tetramethylpyrazine, 5.0-7.5 μg / L transferrin, 0.4-1.2 μg / L bovine serum albumin, and 2.0-5.5 μg / L linoleic acid. The stem cell culture composition obtained by the present invention through a first induction medium containing safflower flavonoids and a second induction medium containing safflower flavonoids and tebufenozide, the safflower flavonoids and tebufenozide in the induction medium have a synergistic effect to promote the production of certain active factors of stem cells, and have significant antioxidant, tyrosine inhibition and collagen regeneration effects, and have a very outstanding anti-aging skin repair effect, and can brighten skin tone.

Description

Technical Field

[0001] This invention relates to the field of stem cell technology, and in particular to a placental / umbilical cord mesenchymal stem cell culture composition and its application in functional anti-aging. Background Technology

[0002] Mesenchymal stem cells (MSCs) are pluripotent stem cells that possess all the common characteristics of stem cells, namely, self-renewal and multi-lineage differentiation capabilities. Mesenchymal stem cell-derived exosomes (MSC-Exo) are nanovesicles responsible for the paracrine function of MSCs. They contain various contents, including mRNA, miRNA, cytokines, and proteins. They can cross the physiological barriers of body tissues, participate in intercellular material exchange and information communication, and have broad application potential in the treatment of diseases such as skin aging, wound repair, scar repair, and nerve regeneration.

[0003] Recent research on mesenchymal stem cell-derived exosomes has been very popular. Based on the potential of mesenchymal stem cells, this application develops a mesenchymal stem cell tissue culture, which can promote the secretion of a certain active cytokine by inducing culture in a first induction medium containing safflower flavonoids and a second induction medium containing safflower flavonoids and tebufenozide. This active cytokine has significant effects on whitening, anti-oxidation and promoting collagen regeneration. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a placental / umbilical cord mesenchymal stem cell culture composition and its application in functional anti-aging, so as to solve the problems mentioned in the above-mentioned technical background.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a placental / umbilical cord mesenchymal stem cell culture composition, which is a culture supernatant obtained after mesenchymal stem cells are induced and cultured in a first induction medium and a second induction medium;

[0007] The first induction medium contains red willow flavonoids, transferrin, bovine serum albumin, linoleic acid, and basal medium.

[0008] The second induction medium contains red willow flavonoids, ligustrazine, transferrin, bovine serum albumin, linoleic acid, and basal medium.

[0009] Preferably, the first induction culture medium contains 10-30 mg / L red willow flavonoids, 5.0-7.5 ug / L transferrin, 0.4-1.2 ug / L bovine serum albumin, and 2.0-5.5 ug / L linoleic acid.

[0010] More preferably, the first induction culture medium contains 20 mg / L red willow flavonoids, 6.0 ug / L transferrin, 0.8 ug / L bovine serum albumin, and 3.5 ug / L linoleic acid.

[0011] Preferably, the second induction culture medium contains 10-30 mg / L red willow flavonoids, 60-90 mg / L tebufenozide, 5.0-7.5 ug / L transferrin, 0.4-1.2 ug / L bovine serum albumin, and 2.0-5.5 ug / L linoleic acid.

[0012] More preferably, the second induction culture medium contains 20 mg / L red willow flavonoids, 75 mg / L tebufenozide, 6.0 ug / L transferrin, 0.8 ug / L bovine serum albumin, and 3.5 ug / L linoleic acid.

[0013] Preferably, the preparation method of the red willow flavonoids is as follows: dried red willow is pulverized by a pulverizer and passed through a 40-mesh sieve to obtain red willow powder; the red willow powder is weighed and placed in a 250mL round-bottom flask, and 70% volume fraction of ethanol is added at a material-to-liquid ratio of 1g:20ml, and extracted at 80℃ for 2h; the mixture is filtered while hot, and the ethanol in the filtrate is removed by rotary evaporation. Redistilled water and an equal amount of petroleum ether are added, and the mixture is extracted with a separatory funnel, and the petroleum ether layer is discarded; the aqueous layer is extracted twice with 20mL of ethyl acetate, and the ethyl acetate layers are combined and rotary evaporated to dryness to obtain red willow flavonoids.

[0014] Preferably, the mesenchymal stem cells are derived from the placenta or umbilical cord.

[0015] Preferably, the basal culture medium is DMEM / F12 or α-MEM.

[0016] Preferably, a placental / umbilical cord mesenchymal stem cell culture composition is prepared by the following method:

[0017] Mesenchymal stem cells were harvested and cultured in a basal medium containing 10% exosome-free fetal bovine serum;

[0018] The cells were transferred to the first induction medium and cultured for 24 hours. When the cell confluence reached 80%, they were transferred to the second induction medium and cultured for another 36-48 hours. The cell culture medium was then collected, centrifuged, and filtered to obtain the mesenchymal stem cell culture composition, which was stored at 4°C for later use.

[0019] In a second aspect, the present invention provides the application of the placental / umbilical cord mesenchymal stem cell culture composition described above in the preparation of epidermal whitening, skin repair and anti-aging products.

[0020] Compared with the prior art, the beneficial effects of the present invention are:

[0021] 1. The stem cell culture composition obtained by induction in the present application using a first induction medium containing safflower flavonoids and a second induction medium containing safflower flavonoids and tebufenozide has the synergistic effect of safflower flavonoids and tebufenozide in the induction medium to promote the production of certain active factors of stem cells, and has significant effects such as anti-oxidation, inhibition of tyrosine and promotion of collagen regeneration.

[0022] 2. The stem cell culture composition of this application has excellent antioxidant, tyrosine inhibition and collagen regeneration effects in in vitro experiments. It has outstanding anti-aging skin repair effects and can improve skin radiance and brighten skin tone, and has good market application prospects. Attached Figure Description

[0023] Figure 1 A bar chart showing the effect of the stem cell culture composition of the present invention on the tyrosinase inhibition rate;

[0024] Figure 2 This is a bar chart showing the effect of the stem cell culture composition of the present invention on the DPPH free radical scavenging rate. Detailed Implementation

[0025] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0026] All components and reagents used in the following examples are commercially available products. For example, DMEM / F12 medium and α-MEM medium were purchased from Gibco; red willow was collected from Karamay, Xinjiang; and chuanxiongqin was purchased from Shaanxi Guben Biotechnology Co., Ltd.

[0027] In this application's implementation scheme, mesenchymal stem cells are derived from the placenta and umbilical cord; in some implementation schemes, mesenchymal stem cells derived from mammals such as humans, cattle, pigs, pigs, and rabbits can be selected.

[0028] Example 1

[0029] A culture composition for umbilical cord mesenchymal stem cells, comprising the culture supernatant obtained after umbilical cord mesenchymal stem cells have been induced and cultured in a first induction medium and a second induction medium;

[0030] The first induction medium was DMEM / F12 medium containing 10 mg / L red willow flavonoids, 5.0 μg / L transferrin, 1.2 μg / L bovine serum albumin, and 5.5 μg / L linoleic acid. The second induction medium was DMEM / F12 medium containing 30 mg / L red willow flavonoids, 60 mg / L tetramethylpyrazine, 5.0 μg / L transferrin, 1.2 μg / L bovine serum albumin, and 5.5 μg / L linoleic acid.

[0031] Example 2

[0032] A culture composition for placental mesenchymal stem cells, comprising the culture supernatant obtained after placental mesenchymal stem cells have been induced and cultured in a first induction medium and a second induction medium;

[0033] The first induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid. The second induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 75 mg / L tetramethylpyrazine, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid.

[0034] Example 3

[0035] A culture composition for placental mesenchymal stem cells, comprising the culture supernatant obtained after placental mesenchymal stem cells have been induced and cultured in a first induction medium and a second induction medium;

[0036] The first induction medium contained 30 mg / L red willow flavonoids, 7.5 μg / L transferrin, and 0.4 μg / L bovine serum albumin.

[0037] The first induction medium was α-MEM containing 2.0 μg / L linoleic acid. The second induction medium was α-MEM containing 10 mg / L red willow flavonoids, 90 mg / L tetracycline, 7.5 μg / L transferrin, 0.4 μg / L bovine serum albumin, and 2.0 μg / L linoleic acid.

[0038] Comparative Example 1

[0039] This comparative example is similar to Example 2, except that the first induction medium does not contain red willow flavonoids; specifically, it is a placental mesenchymal stem cell culture composition, which is the culture supernatant obtained after placental mesenchymal stem cells are induced and cultured in a first induction medium and a second induction medium.

[0040] The first induction medium was DMEM / F12 medium containing 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid. The second induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 75 mg / L tetramethylpyrazine, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid.

[0041] Comparative Example 2

[0042] This comparative example is similar to Example 2, except that the second induction medium does not contain red willow flavonoids; specifically, it is a placental mesenchymal stem cell culture composition, which is the culture supernatant obtained after placental mesenchymal stem cells are induced and cultured in the first induction medium and the second induction medium.

[0043] The first induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid. The second induction medium was DMEM / F12 medium containing 75 mg / L tetramethylpyrazine, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid.

[0044] Comparative Example 3

[0045] This comparative example is similar to Example 2, except that the second induction medium does not contain tebufenozide; specifically, it is a placental mesenchymal stem cell culture composition, which is the culture supernatant obtained after placental mesenchymal stem cells are induced and cultured in a first induction medium and a second induction medium.

[0046] The first induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid. The second induction medium was also DMEM / F12 medium containing the same ingredients.

[0047] Comparative Example 4

[0048] This comparative example is similar to Example 2, except that the second induction medium does not contain red willow flavonoids and tebufenozide; specifically, it is a placental mesenchymal stem cell culture composition, which is the culture supernatant obtained after placental mesenchymal stem cells are induced and cultured in the first induction medium and the second induction medium.

[0049] The first induction medium was DMEM / F12 medium containing 20 mg / L red willow flavonoids, 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid. The second induction medium was DMEM / F12 medium containing 6.0 μg / L transferrin, 0.8 μg / L bovine serum albumin, and 3.5 μg / L linoleic acid.

[0050] Comparative Example 5

[0051] A second induction medium is a DMEM / F12 medium containing 20 mg / L red willow flavonoids, 75 mg / L ligustrazine, 6.0 ug / L transferrin, 0.8 ug / L bovine serum albumin, and 3.5 ug / L linoleic acid.

[0052] In this invention, the preparation method of the red willow flavonoids is as follows: dried red willow is pulverized by a pulverizer and passed through a 40-mesh sieve to obtain red willow powder; the red willow powder is weighed and placed in a 250mL round-bottom flask, and 70% volume fraction of ethanol is added at a material-to-liquid ratio of 1g:20ml, and extracted at 80℃ for 2h; the mixture is filtered while hot, and the ethanol in the filtrate is removed by rotary evaporation. Redistilled water and an equal amount of petroleum ether are added, and the mixture is extracted using a separatory funnel, and the petroleum ether layer is discarded; the aqueous layer is extracted twice with 20mL of ethyl acetate, and the ethyl acetate layers are combined and rotary evaporated to dryness to obtain red willow flavonoids.

[0053] In this invention, mesenchymal stem cell culture compositions were prepared according to the formulations of Examples 1-3 and Comparative Examples 1-4. The method for preparing the mesenchymal stem cell culture compositions is as follows:

[0054] Placental or umbilical cord mesenchymal stem cells were collected and cultured in basal medium containing 10% exosome-free fetal bovine serum. The cells were incubated at 37°C and 5% CO2 for 48 hours. The basal medium was DMEM / F12 or α-MEM.

[0055] The above cells were divided into 1×10 6 At a density of cells / mL, cells were transferred to the first induction medium and cultured for 24 hours. When the cell confluence reached 80%, the cells were transferred to the second induction medium and cultured for another 48 hours. The cell culture medium was then collected, centrifuged at 4000 rpm for 20 minutes, and filtered through a 0.22 μm filter to obtain the mesenchymal stem cell culture composition, which was stored at 4℃ for later use. The placental mesenchymal stem cells used were rabbit placental mesenchymal stem cells (catalog number: CP-Rb303, specification: 5×10⁻⁶). 5 Cells / T25 culture flasks were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.; rabbit umbilical cord mesenchymal stem cells (catalog number: CP-Rb041, specification: 5×10⁻⁶) were used. 5Cells / T25 culture flasks were purchased from Wuhan Pronosei Life Science Technology Co., Ltd.

[0056] To further clarify the efficacy of the mesenchymal stem cell culture composition provided in the embodiments of this application, the following experiments were also conducted in the embodiments of this application:

[0057] 1. Tyrosinase Inhibition Test

[0058] Tyrosinase is crucial in the formation of melanin in the skin, and inhibiting tyrosinase activity can have a skin-whitening effect. Therefore, a tyrosinase-catalyzed reaction system was constructed to evaluate the inhibitory effect of the stem cell culture composition of this application on tyrosinase.

[0059] Samples to be tested and grouped: 8 groups were set up, which were the stem cell culture compositions or induction culture media provided in Examples 1-3 and Comparative Examples 1-5, respectively;

[0060] Experimental Methods: The inhibition test of tyrosinase by each sample was conducted according to the tyrosinase catalytic reaction system (Table 1), with three replicates for each sample. The tyrosinase catalytic reaction system consisted of four reaction tubes, A1, A2, A3, and A4, for sample addition. After sample addition, the reaction was carried out at a constant temperature of 37℃ for 10 min, and the absorbance values ​​of A1, A2, A3, and A4 were measured at 475 nm. Tyrosinase inhibition rate (%) = [1 - (A3 - A4) / (A1 - A2)] × 100%, where A1, A2, A3, and A4 are the absorbance values ​​at 475 nm after the reaction of the corresponding reaction tubes. The tyrosinase inhibition rate of each experimental group was calculated, and the experimental results are shown in Table 2 and [Table 3]. Figure 1 As shown.

[0061] Table 1

[0062] reaction system A1 A2 A3 A4 Sample to be tested (ul) 0 0 200 200 0.2M PBS (µL) pH 6.80 1000 1200 800 1000 500 U / mL tyrosinase solution (ul) 200 0 200 0 0.5wt% tyrosine solution (ul) 300 300 300 300

[0063] Table 2

[0064]

[0065]

[0066] From Table 2 and Figure 1 It can be seen that the tyrosinase inhibition rate of the stem cell culture compositions provided in Examples 1-3 is significantly higher than that of the stem cell culture compositions and induction culture media provided in Comparative Examples 1-5. This indicates that the stem cell culture compositions provided in the examples of this application have a stronger tyrosinase inhibition rate and can play a whitening role. This also indicates that the stem cell culture compositions induced by the first induction culture medium containing red willow flavonoids and the second induction culture medium containing red willow flavonoids and tebufenozide have a more significant whitening effect.

[0067] 2. DPPH free radical scavenging test

[0068] Samples to be tested: 8 groups were set up, which were the stem cell culture compositions or induction culture media provided in Examples 1-3 and Comparative Examples 1-5, respectively.

[0069] Experimental Method: Take 200 μL of the sample solution to be tested, add 200 μL of 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, and react at room temperature in the dark for 30 min. Then, measure the absorbance of the sample at 517 nm using a microplate reader. The blank group is a mixture of 200 μL distilled water and 200 μL DPPH-anhydrous ethanol solution, and the control group is a mixture of 200 μL sample solution and 200 μL anhydrous ethanol solution. The DPPH free radical scavenging rate (Q, %) is calculated according to the formula. The results are shown in Table 3 and... Figure 2 As shown. Q / %=[1-(A1-A2) / A0]×100%, where: A1, A2, and A0 are the absorbance of the sample group, control group, and blank group, respectively.

[0070] Table 3

[0071]

[0072] From Table 3 and Figure 2 It can be seen that the DPPH free radical scavenging rate of the stem cell culture compositions provided in Examples 1-3 is significantly higher than that of the stem cell culture compositions and induction mediums provided in Comparative Examples 1-5, indicating that the stem cell culture compositions provided in this application have better antioxidant capacity, and good antioxidant performance can delay skin aging. This shows that the stem cell culture compositions induced by the first induction medium containing safflower flavonoids and the second induction medium containing safflower flavonoids and tebufenozide have more significant antioxidant and anti-aging effects.

[0073] 3. In vitro proliferation activity of human fibroblasts

[0074] Cells: Human skin fibroblasts (HSF), catalog number CB-Y1274, purchased from Shanghai Enzyme Research Biotechnology Co., Ltd.

[0075] Test samples and grouping: Nine groups were set up, including one negative control group and eight experimental groups. The experimental groups used the stem cell culture compositions or induction media provided in Examples 1-3 and Comparative Examples 1-5 of this application, respectively. DMEM medium was prepared to contain 2% v / v stem cell culture composition or induction media and 10% fetal bovine serum. The negative control group used DMEM medium containing 10% fetal bovine serum.

[0076] Experimental method: Human skin fibroblasts cultured to the 4th-6th generation logarithmic growth phase were used. The cells were cultured and prepared to a concentration of 1×10⁻⁶. 5 Cell suspension at 200 μL / well was seeded into 96-well plates, with 3 replicates per group. A blank control group was set up with only 200 μL of DMEM medium. The seeded 96-well plates were incubated overnight at 37°C and 5% CO2. After cell adhesion, the culture medium was discarded, and the cells were administered the corresponding culture medium for each experimental group. The cells were then placed in a cell culture incubator and cultured for 24 hours. The culture medium in the wells was discarded, and after washing with PBS, a mixture of 20 μL MTT reagent (5 mg / mL) and 180 μL serum-free DMEM was added to each well. After incubation for 4 hours, the culture medium was discarded, and 150 μL LDMSO was added. The mixture was shaken for 10 minutes to dissolve the crystals. The absorbance values ​​of the experimental group, negative control group, and blank control group at a wavelength of 490 nm were measured using a microplate reader. The cell proliferation rate was calculated, and the cell proliferation rate is shown in Table 4. Cell proliferation rate % = (experimental group - blank control group) / (negative control group - blank control group) × 100%, where: experimental group: HSF cells + DMEM culture medium + stem cell culture mixture; negative control group: HSF cells + DMEM culture medium; blank control group: DMEM culture medium.

[0077] Table 4

[0078] Group Cell proliferation rate (%) Example 1 205.12±6.37 Example 2 208.85±7.54 Example 3 203.27±7.82 Comparative Example 1 159.63±4.36 Comparative Example 2 152.54±5.27 Comparative Example 3 156.19±4.38 Comparative Example 4 151.67±4.52 Comparative Example 5 134.54±5.93 negative control group 100.00

[0079] As shown in Table 4, the cell proliferation rate of HSF cells cultured by the stem cell culture compositions provided in Examples 1-3 was significantly higher than that in Comparative Examples 1-5, indicating that the stem cell culture compositions provided in this application can promote cell proliferation. This demonstrates that the stem cell culture compositions induced by the first induction medium containing safflower flavonoids and the second induction medium containing safflower flavonoids and tebufenozide have a more significant effect on promoting HSF cell proliferation.

[0080] 4. Content of collagen I secreted by cells

[0081] Cells: Human skin fibroblasts (HSF), catalog number CB-Y1274, purchased from Shanghai Enzyme Research Biotechnology Co., Ltd.

[0082] Test samples and grouping: Nine groups were set up, including one negative control group and eight experimental groups. The experimental groups used the stem cell culture compositions or induction media provided in Examples 1-3 and Comparative Examples 1-5 of this application, respectively. DMEM medium was prepared to contain 2% v / v stem cell culture composition or induction media and 10% fetal bovine serum. The negative control group used DMEM medium containing 10% fetal bovine serum.

[0083] Experimental method: Human skin fibroblasts cultured to the 4th-6th generation logarithmic growth phase were used. The cells were cultured and prepared to a concentration of 1×10⁻⁶. 5 Cells were seeded at 100 μL per well in a 96-well plate with a cell suspension of 1 / ml, with three replicates per group. Cells were incubated overnight at 37°C with 5% CO2. After cell attachment, the culture medium was discarded, and cells were washed with PBS. The corresponding culture medium for each experimental group was added, and the cells were incubated at 37°C for 36 hours. Cell culture supernatant samples were then collected, and the collagen content was measured using a human type I collagen (COL-I) ELISA kit (Shanghai Jianglai Biotechnology Co., Ltd.) to assess the promoting effect on type I collagen synthesis in human fibroblasts. The results of the type I collagen content test are shown in Table 5.

[0084] Table 5

[0085]

[0086] As shown in Table 5, the stem cell culture compositions provided in Examples 1-3 significantly promoted the secretion of type I collagen by HSF cells, which was significantly higher than that in Comparative Examples 1-5. This indicates that the stem cell culture compositions provided in this application can promote collagen production, thereby making the skin smooth and elastic. They have the potential to be used in skin anti-wrinkle repair and anti-aging products. This shows that the stem cell culture compositions induced by the first induction medium containing safflower flavonoids and the second induction medium containing safflower flavonoids and tebufenozide have a good synergistic effect in promoting the secretion of type I collagen by HSF cells.

[0087] The stem cell culture composition provided in this application can be used in the preparation of skin care products, wherein the skin care products have anti-aging, skin repair, and whitening effects.

[0088] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A placental / umbilical cord mesenchymal stem cell culture composition, characterized in that, It is the culture supernatant obtained after mesenchymal stem cells are induced and cultured in the first induction medium and the second induction medium; The first induction medium consists of red willow flavonoids, transferrin, bovine serum albumin, linoleic acid, and basal medium; The second induction medium consists of red willow flavonoids, ligustrazine, transferrin, bovine serum albumin, linoleic acid, and basal medium; The first induction medium contains 10-30 mg / L red willow flavonoids, 5.0-7.5 ug / L transferrin, 0.4-1.2 ug / L bovine serum albumin, and 2.0-5.5 ug / L linoleic acid; The second induction medium contains 10-30 mg / L red willow flavonoids, 60-90 mg / L ligustrazine, 5.0-7.5 ug / L transferrin, 0.4-1.2 ug / L bovine serum albumin, and 2.0-5.5 ug / L linoleic acid; The basal culture medium is DMEM / F12 or α-MEM.

2. The placental / umbilical cord mesenchymal stem cell culture composition according to claim 1, characterized in that, The first induction medium contains 20 mg / L red willow flavonoids, 6.0 ug / L transferrin, 0.8 ug / L bovine serum albumin, and 3.5 ug / L linoleic acid.

3. The placental / umbilical cord mesenchymal stem cell culture composition according to claim 1, characterized in that, The second induction medium contains 20 mg / L red willow flavonoids, 75 mg / L ligustrazine, 6.0 ug / L transferrin, 0.8 ug / L bovine serum albumin, and 3.5 ug / L linoleic acid.

4. The placental / umbilical cord mesenchymal stem cell culture composition according to claim 1, characterized in that, The preparation method of the red willow flavonoids is as follows: take dried red willow and crush it through a 40-mesh sieve to obtain red willow powder; weigh the red willow powder and place it in a 250mL round-bottom flask, add 70% volume fraction of ethanol at a material-to-liquid ratio of 1g:20ml, and extract at 80℃ for 2h; filter while hot, remove the ethanol from the filtrate by rotary evaporation, add redistilled water and an equal amount of petroleum ether, extract with a separatory funnel, and discard the petroleum ether layer; extract the aqueous layer twice with 20mL of ethyl acetate, combine the ethyl acetate layers, and rotary evaporate to dryness to obtain red willow flavonoids.

5. The placental / umbilical cord mesenchymal stem cell culture composition according to claim 1, characterized in that, Its preparation method is as follows: Mesenchymal stem cells were harvested and cultured in a basal medium containing 10% exosome-free fetal bovine serum; The cells were transferred to the first induction medium and cultured for 24 hours. When the cell confluence reached 80%, they were transferred to the second induction medium and cultured for another 36-48 hours. The cell culture medium was then collected, centrifuged, and filtered to obtain the mesenchymal stem cell culture composition, which was stored at 4°C for later use.

6. The use of the placental / umbilical cord mesenchymal stem cell culture composition according to any one of claims 1-5 in the preparation of epidermal whitening, skin repair and anti-aging products, wherein the placental / umbilical cord mesenchymal stem cells are placental / umbilical cord mesenchymal stem cells derived from non-human mammals; and the product is a cosmetic.

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