Preparation method of dedifferentiated saussurea involucrata cells and anti-wrinkle tightening application of dedifferentiated saussurea involucrate cells

Abstract

The invention discloses a preparation method of dedifferentiated saussurea involucrata cells and anti-wrinkle tightening application of the dedifferentiated saussurea involucrata cells. The preparation method comprises the following specific steps: S1, transferring root tips of aseptic seedlings to an induction culture medium, and proliferating to form cell clusters; culturing the cell cluster in a culture dish containing a proliferation culture medium to obtain primary saussurea involucrata stem cells; s2, transferring the obtained primary saussurea involucrata stem cells into a liquid MS culture medium for suspension culture, and meanwhile, changing the illumination condition, and culturing to obtain the saussurea involucrata stem cells of which the total flavone content and the chlorogenic acid content are obviously increased. According to the method, large-scale production of saussurea involucrata stem cells is realized through a plant cell culture technology, the problem of scarcity of natural resources is solved, and rare and sustainable raw materials are realized; according to the method, unique culture conditions and inducer combination are adopted, so that the activity and the active ingredient content of the saussurea involucrata stem cells are improved, and process innovation is realized; meanwhile, the saussurea involucrata stem cells prepared by the method provide a safe raw material with anti-wrinkle and firming effects for the skin care product industry.

Description

Technical Field

[0001] The present invention relates to the field of medical and health technology, and in particular to a preparation method of dedifferentiated Saussurea involucrata cells and anti-wrinkle and firming applications thereof. Background Art

[0002] Tianshan Snow Lotus is known as the "King of Herbs". Its roots, stems and leaves are rich in alkaloids, flavonoids, volatile oils, lactones, steroids, polysaccharides and reducing substances. It has the effects of promoting blood circulation, dredge meridians, dispel cold and remove dampness, and can treat a variety of diseases, such as rheumatoid arthritis.

[0003] Due to over-collection and habitat destruction, the number of wild Tianshan Saussurea has dropped sharply and has been listed as a national second-class protected plant. In order to protect this rare resource, dedifferentiated Tianshan Saussurea cells are cultivated under optimal conditions of artificial control through plant cell culture technology, which not only retains the chemical composition and pharmacological effects of wild Tianshan Saussurea, but also surpasses wild varieties in some active ingredients. In addition, cell culture technology can avoid pesticide residues and heavy metal pollution, ensuring the safety of the product. Dedifferentiated plant cells originated from Haberland's research in 1902. In the past 40 years, plant cell cultures have been used for the production of metabolites of interest and the proliferation of exactly the same plants (somatic embryogenesis). This plant biotechnology is based on the concept of cell totipotency: "Any plant cell can dedifferentiate and regenerate into another new individual with the same source as the cell." Dedifferentiated plant cells are plant cells derived from organs (leaves, stems, roots, petals, etc.), which are cultured and regenerated into their dedifferentiated form, that is, they lose their leaf, stem, root, petal specificity and can regenerate a complete plant. Undifferentiated plant cells are equivalent to true plant stem cells derived from plant meristem cells and do not have an organ-specific biological history.

[0004] The skin care effects of Tianshan Snow Lotus include moisturizing, anti-oxidation, anti-inflammation, lightening of spots, anti-aging and anti-wrinkle. These properties make snow lotus extract a valuable ingredient in the cosmetics industry. Tianshan Snow Lotus stem cells produced by plant cell culture technology can be used as a natural ingredient to develop anti-wrinkle and firming skin care products, providing a new option for the cosmetics market.

[0005] It can be seen that the protection and sustainable utilization of Tianshan Saussurea involucrata is not only of great significance to the protection of biodiversity, but also has great economic potential for the development of new natural medicines and cosmetics. Through the application of modern biotechnology, the effective protection and rational utilization of this rare plant resource can be achieved. For example, the prior art CN104498425B discloses a method for the separation and culture of Saussurea involucrata stem cells and its application in the preparation of anti-aging products. Specifically, the young leaves of Tianshan Saussurea involucrata are taken, and after disinfection, the Tianshan Saussurea involucrata stem cells are first separated using a solid culture medium, and then the liquid culture medium is used for subculture and culture; the solid culture medium contains inorganic salts, vitamins, amino acids, auxins, sugars and activated carbon; the liquid culture medium contains inorganic salts, vitamins, amino acids, auxins, sugars and additives. This patent focuses on protective eye cream. CN110923190A is a method for increasing the flavonoid phenylpropanoid compounds in Saussurea involucrata cell culture. In the culture medium used in the method, the phosphorus concentration is 0.5-3mmol / L, NO3 - Ion concentration 20-35mmol / L, NH4 + Ion concentration 0-30mmol / L, Ca 2+ Ion concentration 0.5-3mmol / L, Mg 2+ The ion concentration is 0.2-1.5mmol / L, and the boron ion concentration is 0.02-0.1mmol / L; an inducer or precursor or bypass metabolic inhibitor can be added to the culture medium; and finally the total flavonoid content and the content of rutin, chlorogenic acid, syringin, and 1,5-dicaffeoylquinic acid in the snow lotus cell culture are significantly increased. CN116103219B relates to a method for preparing snow lotus stem cell powder and an antibacterial agent prepared therefrom, belonging to the field of medical and health care. The method for preparing the snow lotus stem cell powder has the following preparation steps: selecting the undifferentiated root end of Tianshan snow lotus as an explant, first performing cleaning and disinfection pretreatment, then performing primary culture and subculture under certain conditions, then performing expansion culture under certain conditions, and finally performing post-processing steps such as filtering and drying, so as to obtain snow lotus root stem cell freeze-dried powder with better healing effect. CN106867958A A large-scale subculture process of Tianshan Saussurea cells, including the following steps: 1) Tianshan Saussurea cell line screening: using natural Saussurea test tube seedlings for explant induction to obtain callus tissue; 2) Shake flask scale subculture: select solid cells with good growth state, bright color and firm texture and put them into a shake flask containing culture solution, and subculture once every 7-10 days; 3) Shake flask to reactor subculture: transfer the cells in the shake flask to the reactor for subculture, and the culture cycle of each subculture is 7-10 days; by optimizing the culture conditions and controlling the dissolved oxygen content, the uniform dispersion of cells and the increase of the content of effective ingredients are achieved. This process is more conducive to the balanced production of effective ingredients in Tianshan Saussurea cells, and provides a more efficient and stable Tianshan Saussurea cell culture method for researchers, etc.

[0006] Although the preparation methods and applications of Tianshan Snow Lotus stem cells have been improved based on previous research, more methods are still needed from researchers to meet the needs of industrial production. Summary of the invention

[0007] In view of this, the purpose of the present invention is to provide a preparation method of dedifferentiated Saussurea involucrata cells and their anti-wrinkle and firming applications. The present invention utilizes plant cell culture technology to produce Saussurea involucrata stem cells, which have high active ingredient content, short culture cycle, and high cell yield. These active ingredient-rich Saussurea involucrata stem cell freeze-dried powders have anti-wrinkle and firming effects, bringing a new, naturally derived anti-wrinkle and firming ingredient to the skin care industry.

[0008] In one aspect, the present invention provides a method for preparing dedifferentiated Saussurea involucrata cells, the specific steps of which are as follows:

[0009] S1: Take the root tip of the sterile seedling and transfer it to the induction medium to proliferate and form cell clusters; take the cell clusters and culture them in a culture dish containing the proliferation medium to obtain primary Tianshan Saussurea involucrata stem cells;

[0010] S2: The obtained primary Saussurea involucrata stem cells are transferred to liquid MS culture medium for suspension culture, and at the same time, the lighting conditions are changed to obtain Saussurea involucrata stem cells with significantly increased total flavonoids content and chlorogenic acid content.

[0011] Furthermore, the root tips of the sterile seedlings are taken from the sterile seedlings of Saussurea involucrata from the growth stage to the 10-day seedling stage. Furthermore, the cultivation process of the sterile seedlings of Saussurea involucrata is as follows: taking the seeds of Saussurea involucrata, washing them, pre-treating them, soaking them, rinsing them, and then inoculating and cultivating them. Furthermore, the washing is performed by rinsing with tap water for 20 to 30 minutes. Furthermore, the pretreatment refers to pre-treatment with 100 mg / L GA3 (gibberellic acid) solution for 12 hours. Furthermore, the soaking refers to soaking with 70% ethanol for 30 seconds. Furthermore, the rinsing refers to rinsing with 1% sodium hypochlorite solution for 8 to 10 minutes by shaking. Furthermore, the culture medium for inoculation and cultivation is an MS culture medium containing 30 g / L sucrose and 6 g / L agar.

[0012] Further, the MS medium of the present invention is a medium containing the components shown in Table 1, and its basic material composition is: NH4NO3 412.5mg / L, Na2MoO4·2H2O 0.25mg / L, CaCl2 332.2mg / L, KI 0.83mg / L, MgSO4180.7 mg / L, ZnSO4·7H2O 8.6mg / L, KNO3 475mg / L, CuSO4·5H2O 0.025mg / L, KH2PO4170mg / L, FeNaEDTA 36.7mg / L, H3BO3 6.2mg / L, MnSO4·H2O 16.9mg / L, CoCl2·6H2O 0.025mg / L, Glycine 2mg / L, Inositol 100mg / L, Nicotinic acid

[0013] 0.5mg / L, Pyridoxine HCl 0.5mg / L, Thiamine HCl 0.1mg / L.

[0014] Furthermore, the inoculation and cultivation conditions are a temperature of 25° C. and a photoperiod of 12 h light / 12 h dark.

[0015] Further, the induction medium is an MS medium containing 1.0 mg / L IBA (indolebutyric acid), 0.5 mg / L KT (kinetin), 30 g / L sucrose and 6 g / L Agar (agar), and the pH value is adjusted to 5.8-6.2. Further, the culture condition for proliferation and formation of cell clusters in S1 is to culture in a dark environment at 25° C. for 14 days.

[0016] Furthermore, the proliferation medium is an MS medium containing sucrose and hormones. Furthermore, the hormones are selected from at least one of 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, indolebutyric acid, IAA, and NAA. Further, the hormone is selected from at least one of the combination of 0.5mg / L 6-BA+1.0mg / L 2,4-D, 0.5mg / L 6-BA+2.0mg / L 2,4-D, 1.0mg / L 6-BA+1.0mg / L 2,4-D, 1.0mg / L 6-BA+2.0mg / L 2,4-D, 0.5mg / L 6-BA+2.0mg / L IAA, 0.5mg / L 6-BA+2.0mg / L NAA, 2.0mg / L 2,4-D, and 0.5mg / L 6-BA. Further, the proliferation medium is an MS medium containing 30g / L sucrose, 0.5mg / L 6-benzylaminopurine, and 2.0mg / L 2,4-dichlorophenoxyacetic acid. Furthermore, the proliferation culture medium needs to be sterilized in an autoclave at a temperature of 121° C. and a pressure of 0.1 MPa for 20 min. Furthermore, in S1, the proliferation culture condition is to culture the Tianshan Saussurea involucrata stem cells in a dark environment at 25° C. for 14 days.

[0017] Further, the liquid MS culture medium contains an inducer, sucrose, 6-BA (6-benzylaminopurine) and 2,4-D (2,4-dichlorophenoxyacetic acid). Further, the inducer is selected from at least one of L-phenylalanine and MT (melatonin). Further, the liquid MS culture medium contains 50mg / L L-phenylalanine, 20mg / L MT (melatonin), 30g / L sucrose, 0.5mg / L 6-BA (6-benzylaminopurine) and 2.0mg / L 2,4-D (2,4-dichlorophenoxyacetic acid). Further, the pH value of the liquid MS culture medium is 5.8-6.2. Further, the liquid MS culture medium is sterilized by high pressure before inoculating primary Tianshan Saussurea involucrata stem cells. Further, the high pressure sterilization conditions are sterilization for 20 minutes at a temperature of 121°C and a pressure of 0.1MPa in an autoclave. High pressure sterilization ensures that the environment for induced culture is safe, reliable and sterile.

[0018] Furthermore, the illumination condition is to select different lights, and the total illumination intensity is 32 μmol / (m 2 ·s), and the illumination time is 12h / d. Further, the different light is selected from at least one of red light, white light, blue light or darkness. Further, the different light refers to the use of red light and blue light in a 1:1 manner, wherein the central wavelength of the blue light is 457nm and the central wavelength of the red light is 660nm.

[0019] Furthermore, the culture conditions in S2 are a temperature of 25° C., a rotation speed of 100 rpm, and culture to the 4th to 6th day.

[0020] In another aspect, the present invention provides a use of the involucral stem cells prepared by the method of the present invention in products with anti-wrinkle and firming effects, which may be cosmetics, freeze-dried powders, and the like.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention adopts the root tips of aseptic seedlings of Tianshan Saussurea involucrata as explants. Under the preparation method of the present application, products (such as cosmetics, freeze-dried powder, etc.) with good anti-wrinkle and firming effects can be obtained. The products have good effects of inhibiting the secretion of matrix metalloproteinase 1 (MMP-1), and can increase the content of type I collagen (Collagen I) protein, type III collagen (Collagen III) protein and type IV collagen (Collgen IV) protein, thereby achieving anti-wrinkle and firming effects.

[0023] The method of the invention realizes the large-scale production of Tianshan Saussurea stem cells through plant cell culture technology, solves the problem of scarce natural resources, and realizes that the raw materials are rare but sustainable.

[0024] The present invention adopts unique culture conditions and inducer combination to improve the activity and active ingredient content of snow lotus stem cells and achieve process innovation.

[0025] The Tianshan Saussurea stem cells prepared by the invention provide a safe raw material with anti-wrinkle and firming effects for the skin care product industry.

[0026] The technical solution of the present invention is further described in detail below in conjunction with embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Summary of average optical density values ​​of Collagen I, Collagen III and Collagen IV. Note: When using t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **. DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific examples. These examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0029] Example 1

[0030] Step 1: Seed disinfection and sterile seedling acquisition:

[0031] Select healthy, full-grained mature seeds of Saussurea tianshanensis and place them in a small beaker, cover the cup mouth with absorbent gauze, and rinse under the tap for 20 to 30 minutes to reduce the risk of microbial contamination. Transfer the seeds of Saussurea tianshanensis to a 100 mg / L GA3 (gibberellic acid) solution for pretreatment for 12 hours to break the seed dormancy and promote germination. The pretreated seeds were transferred to a clean bench, soaked in 70% ethanol for 30 seconds for surface sterilization, washed with sterile water for 3 to 4 times to remove residual ethanol, and rinsed with 1% sodium hypochlorite solution for 8 to 10 minutes for full contact with the seeds. The seeds were washed with sterile water for 3 to 4 times to remove residual sodium hypochlorite solution, and then transferred to sterile filter paper to absorb water. The disinfected Tianshan Saussurea seeds were inoculated into tissue culture bottles. The culture medium in the tissue culture bottles was MS medium containing 30 g / L sucrose and 6 g / L agar, with a pH of 5.8 to 6.2. The culture conditions were set at a temperature of 25°C and a photoperiod of 12 h light / 12 h dark to ensure their normal growth and development. The sterile seedlings obtained can be used as explants for induced stem cells.

[0032] Table 1: MS medium composition

[0033] Components Content (mg / L) Components Content (mg / L) <![CDATA[NH4NO3]]> 412.5 <![CDATA[Na2MoO4·2H2O]]> 0.25 <![CDATA[CaCl2]]> 332.2 KI 0.83 <![CDATA[MgSO4]]> 180.7 <![CDATA[ZnSO4·7H2O]]> 8.6 <![CDATA[KNO3]]> 475 <![CDATA[CuSO4·5H2O]]> 0.025 <![CDATA[KH2PO4]]> 170 FeNaEDTA 36.7 <![CDATA[H3BO3]]> 6.2 <![CDATA[MnSO4·H2O]]> 16.9 <![CDATA[CoCl2·6H2O]]> 0.025 Glycine 2 Inositol 100 Nicotinic acid 0.5 Pyridoxine HCl 0.5 Thiamine HCl 0.1

[0034] Step 2: Induction and proliferation culture of Tianshan Snow Lotus stem cells:

[0035] When the aseptic seedlings of Saussurea tianshanica grew to the 10-day seedling age, the root tips of the aseptic seedlings of Saussurea tianshanica were cut in a clean bench and the root crowns were removed as explants and transferred to a disposable plastic cell culture dish containing an induction medium. The induction medium used for the stem cells of Saussurea tianshanica was an MS medium containing 1.0 mg / L IBA (indolebutyric acid), 0.5 mg / L KT (kinetin), 30 g / L sucrose and 6 g / L Agar (agar), wherein the specific formula and dosage of the MS medium are shown in Table 1, the pH value was adjusted to 5.8-6.2, and the culture was placed in a dark environment at 25°C for 14 days, and the stem cell layer of the root tip explant of Saussurea tianshanica proliferated and grew to form a cell cluster.

[0036] In an ultra-clean workbench, the Saussurea involucrata stem cells induced in the above steps were transferred to a disposable plastic cell culture dish containing a proliferation medium for culture. The Saussurea involucrata stem cells selected for proliferation culture were small particles, uniform shape, good dispersibility, vigorous growth, and bright color. The high proliferation multiples of the Saussurea involucrata stem cell culture medium used was 30 g / L sucrose + different hormones (the hormone ratio is shown in Table 2) of MS medium (pH 5.8-6.2), the pH value was adjusted to 5.8-6.2, the culture medium was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.1 MPa for 20 min, and the Saussurea involucrata stem cells were cultured in a dark environment at 25°C for 14 days.

[0037] Table 2 Investigation of different hormone ratios in proliferation culture medium

[0038] Different hormone ratios Multiplication times 1 0.5mg / L 6-BA+1.0mg / L 2,4-D 6.8 2 0.5mg / L 6-BA+2.0mg / L 2,4-D 9.1 3 1.0mg / L 6-BA+1.0mg / L 2,4-D 4.2 4 1.0mg / L 6-BA+2.0mg / L 2,4-D 6.7 5 0.5mg / L 6-BA+2.0mg / L IBA 6.5 6 0.5mg / L 6-BA+2.0mg / L IAA 8.5 7 0.5mg / L 6-BA+2.0mg / L NAA 8.2 8 2.0mg / L 2,4-D 7.3 9 0.5mg / L 6-BA 1.4

[0039] Through the selection and cultivation of different hormones and different hormone ratios, the proliferation multiples of stem cell culture vary greatly. The hormone combination of 0.5mg / L 6-BA (6-benzylaminopurine) + 2.0mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) has the best proliferation multiple effect.

[0040] Step 3: Tianshan Snow Lotus Stem Cell Suspension Culture:

[0041] The stem cells of Saussurea involucrata were transferred from the solid culture medium to the liquid culture medium to form a suspension culture system. The vigorously growing stem cells of Saussurea involucrata were transferred to the bioreactor for culture. The culture medium was an MS culture medium (pH 5.8-6.2) containing an inducer (as shown in Table 3) + 30 g / L sucrose + 0.5 mg / L 6-BA (6-benzylaminopurine) + 2.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid). The culture medium was sterilized in an autoclave at a temperature of 121°C and a pressure of 0.1 MPa for 20 min. The culture conditions of the stem cells of Saussurea involucrata were as follows: different light conditions were used (as shown in Table 4), and the total light intensity was 32 μmol / (m 2 ·s), the illumination time was 12h / d, the temperature was 25°C, the rotation speed was 100rpm, and the culture was carried out to the 4th to 6th day, and the Tianshan Saussurea stem cells were harvested. The total flavonoids content in the Tianshan Saussurea stem cells was determined by spectrophotometer, and the chlorogenic acid content in the Tianshan Saussurea stem cells was determined by high performance liquid chromatography HPLC.

[0042] Table 3 Effects of different inducers on the content of flavonoids and chlorogenic acid in stem cells of Saussurea involucrata

[0043]

[0044]

[0045] Table 4 Effects of different light conditions on the contents of flavonoids and chlorogenic acid in stem cells of Saussurea involucrata

[0046]

[0047] Through the screening of the results in Tables 3 and 4, it can be seen that the inducer uses 50 mg / L L-phenylalanine + 20 mg / L MT (melatonin), the illumination conditions use red and blue light (intensity ratio of 1:1), the central wavelength of blue light is 457nm, and the central wavelength of red light is 660nm; the obtained Tianshan Saussurea flavonoids content is the highest. The specific results show that the content of flavonoids in Tianshan Saussurea flavonoids accounts for 27.19% of the dry weight of stem cells, and the content of chlorogenic acid accounts for 3.51% of the dry weight of stem cells, which far exceeds the "Chinese Pharmacopoeia 2020 Edition" that the content of chlorogenic acid in Tianshan Saussurea shall not be less than 0.15%.

[0048] Example 2 Preparation of Tianshan Snow Lotus Stem Cell Lyophilized Powder

[0049] The harvested Saussurea involucrata stem cells intercepted by a 1 μm filter are placed in a freeze dryer for freeze drying. The resulting material is the Saussurea involucrata stem cell freeze-dried powder. After freeze drying, up to 20 g of Saussurea involucrata stem cell freeze-dried powder can be obtained for each liter (L) of culture medium containing the Saussurea involucrata stem cells.

[0050] Table 5 Freeze drying procedure

[0051]

[0052] Example 3 Efficacy Testing

[0053] (1) Based on fibroblasts, the secretion of matrix metalloproteinase 1 (MMP-1) was detected after UVA irradiation of fibroblasts to evaluate the anti-wrinkle effect of Tianshan Saussurea stem cell freeze-dried powder.

[0054] Table 6 Test plan

[0055]

[0056] Table 7 Summary of MMP-1 content detection results

[0057] Group Mean(pg / mL) SD P-value Inhibition rate BC 209800.05 489.53 / / NC 246907.60 4326.27 0.000## / PC 208018.34 2084.48 0.000** 15.75％ SI 203125.52 2682.94 0.000** 17.73％

[0058] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P-value < 0.05 was indicated by #, and P-value < 0.01 was indicated by ##: Compared with the NC group, the significance was indicated by *, P-value < 0.05 was indicated by *, and P-value << 0.01 was indicated by **.

[0059] Based on fibroblasts, compared with the control group, the content of matrix metalloproteinase 1 (MMP-1) in the sample Tianshan Snow Lotus A stem cell freeze-dried powder (SI) at a concentration of 0.01% was significantly decreased, and the inhibition rate was 17.73%. It is believed that the sample at this concentration achieves anti-wrinkle effect by inhibiting the secretion of matrix metalloproteinase 1 (MMP-1).

[0060] (2) This test uses in vitro cultured Exvivo skin tissue as the research object. By detecting the changes in the content of type I collagen (Collagen I), type III collagen (Collagen III), and type IV collagen (Collagen IV) in the ex vivo skin tissue, the anti-wrinkle and firming effects of the test samples are evaluated.

[0061] Table 8 Test plan

[0062]

[0063] The results are as follows Figure 1 As shown, based on Ex vivo skin tissue, compared with the control group, the content of type I collagen (Collagen I) protein, type III collagen (Collagen III) protein and type IV collagen (Collgen IV) protein in the sample Saussurea Involucrata stem cells at a concentration of 0.01% were significantly increased, with the increase rates being 20.69%, 28.17% and 24.19%, respectively. It is believed that at this concentration, the sample achieves anti-wrinkle and firming effects by increasing the content of type I collagen (Collagen I), type III collagen (Collagen III) protein and type IV collagen (Collgen IV) protein.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing dedifferentiated Saussurea involucrata cells, characterized in that: The specific steps are as follows: S1: Take the root tip of the sterile seedling and transfer it to the induction medium to proliferate and form cell clusters; take the cell clusters and culture them in a culture dish containing the proliferation medium to obtain primary Tianshan Saussurea involucrata stem cells; S2: The obtained primary Saussurea involucrata stem cells are transferred to liquid MS culture medium for suspension culture, and at the same time, the lighting conditions are changed to obtain Saussurea involucrata stem cells with significantly increased total flavonoids content and chlorogenic acid content.

2. The preparation method according to claim 1, characterized in that: The root tips of the sterile seedlings are taken from the sterile seedlings of Saussurea schrenkiana from the growth stage to the 10-day seedling stage; the cultivation process of the sterile seedlings of Saussurea schrenkiana is as follows: the seeds of Saussurea schrenkiana are taken, cleaned, pre-treated, soaked, rinsed and then inoculated and cultivated.

3. The preparation method according to claim 2, characterized in that: The cleaning refers to rinsing with tap water for 20 to 30 minutes; the pretreatment refers to pretreatment with 100 mg / L GA3 solution for 12 hours; the soaking refers to soaking with 70% ethanol for 30 seconds; the rinsing refers to shaking and rinsing with 1% sodium hypochlorite solution for 8 to 10 minutes; the culture medium for inoculation and cultivation is MS culture medium containing 30 g / L sucrose and 6 g / L agar; the conditions for inoculation and cultivation are a temperature of 25°C and a photoperiod of 12 hours light / 12 hours dark.

4. The preparation method according to claim 1, characterized in that: The induction medium is an MS medium containing 1.0 mg / L IBA, 0.5 mg / L KT, 30 g / L sucrose and 6 g / L Agar, and the pH value is adjusted to 5.8-6.2; further preferably, the culture condition for proliferation and formation of cell clusters in S1 is to culture in a dark environment at 25° C. for 14 days.

5. The preparation method according to claim 1, characterized in that: The proliferation medium is an MS medium containing sucrose and hormones; further preferably, the hormone is selected from at least one of 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, indolebutyric acid, IAA, and NAA; further preferably, the hormone is selected from at least one of a combination of 0.5 mg / L 6-BA+1.0 mg / L 2,4-D, a combination of 0.5 mg / L 6-BA+2.0 mg / L 2,4-D, a combination of 1.0 mg / L 6-BA+1.0 mg / L 2,4-D, a combination of 1.0 mg / L 6-BA+2.0 mg / L 2,4-D, a combination of 0.5 mg / L 6-BA+2.0 mg / L IBA, a combination of 0.5 mg / L 6-BA+2.0 mg / L IAA, a combination of 0.5 mg / L 6-BA+2.0 mg / L NAA, 2.0 mg / L 2,4-D, and 0.5 mg / L 6-BA.

6. The preparation method according to claim 5, characterized in that: The proliferation culture medium is an MS culture medium containing 30 g / L sucrose, 0.5 mg / L 6-benzylaminopurine, and 2.0 mg / L 2,4-dichlorophenoxyacetic acid; further preferably, the proliferation culture medium needs to be sterilized in an autoclave at a temperature of 121° C. and a pressure of 0.1 MPa for 20 min; further preferably, in S1, the conditions for proliferation culture are to culture the Tianshan Snow Lotus stem cells in a dark environment at 25° C. for 14 days.

7. The preparation method according to claim 1, characterized in that: In S2, the liquid MS culture medium contains an inducer, sucrose, 6-BA and 2,4-D; further preferably, the inducer is selected from at least one of L-phenylalanine and MT.

8. The preparation method according to claim 7, characterized in that: The liquid MS culture medium contains 50 mg / L L-phenylalanine, 20 mg / L MT, 30 g / L sucrose, 0.5 mg / L 6-BA and 2.0 mg / L 2,4-D; further, the pH value of the liquid MS culture medium is 5.8-6.2; further, the liquid MS culture medium is subjected to high-pressure sterilization before inoculating primary Tianshan Saussurea involucrata stem cells; further, the high-pressure sterilization conditions are sterilization in a high-pressure sterilization pot at a temperature of 121° C. and a pressure of 0.1 MPa for 20 minutes.

9. The preparation method according to claim 1, characterized in that: The illumination conditions are to select different lights, and the total illumination intensity is 32 μmol / (m 2 ·s), the illumination time is 12h / d; further preferably, the different light is selected from at least one of red light, white light, blue light or darkness; further preferably, the different light refers to the use of red light and blue light in a 1:1 intensity ratio, wherein the central wavelength of the blue light is 457nm, and the central wavelength of the red light is 660nm; The culture conditions in S2 are a temperature of 25°C and a rotation speed of 100 rpm, and the culture is carried out to the 4th to 6th day.

10. Use of the dedifferentiated Saussurea involucrata cells prepared by the method according to any one of claims 1 to 9 in products with anti-wrinkle and firming effects; further, the products are cosmetics or freeze-dried powders.

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