Anti-wrinkle skin care solution, freeze-dried product thereof and preparation method thereof

Abstract

The present invention discloses an anti-wrinkle skin care solution and its freeze-dried product and preparation method, comprising the following steps: S1, pretreatment of ginseng roots; S2, preparation of ginseng tissue; S3, induction culture: in an induction culture medium, the ginseng tissue obtained in step S2 is induced and cultured to prepare an initial ginseng cambium meristem cell line; S4, suspension culture: the initial ginseng cambium meristem cell line prepared in step S3 is suspended and cultured on a suspension culture medium, filtered through a 40 μm cell sieve, and the filtrate is collected to obtain the anti-wrinkle skin care solution. The anti-wrinkle skin care solution is freeze-dried to obtain an anti-wrinkle skin care freeze-dried product; the present invention uses cell culture fluid cultured with ginseng cells for freeze-drying, which can be applied to skin anti-wrinkle and anti-aging, and has a promoting effect on human cell activity and collagen expression. The method has mild and simple process conditions, no special requirements for equipment, and improves resource utilization, which is conducive to large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of cosmetics, in particular to an anti-wrinkle skin care solution and a freeze-dried product thereof and a preparation method thereof. Background Art

[0002] Skin aging refers to the age-related damage to skin function, which reduces its ability to protect and regulate the body. This leads to the skin's inability to adapt to changes in the internal and external environment, resulting in changes in its overall appearance, including color, hue, shape, and texture. Skin aging can be categorized as either intrinsic or extrinsic. Intrinsic aging refers to the natural aging of the skin with age, manifested by whitening, fine wrinkles, decreased elasticity, and sagging. Extrinsic aging is primarily caused by photoaging due to sun exposure, manifested by wrinkles, sagging skin, roughness, yellowish or grayish-yellow discoloration, dilated capillaries, and the formation of pigmented spots. Environmental influences on the skin generate free radicals, which damage collagen and active substances within normal cell membranes, oxidizing cells and causing fine lines and wrinkles. Wrinkles gradually appear.

[0003] The cambium is the plant's meristem. It is composed of a small number of cells with very thin cell walls and exists in extremely small quantities in the plant body. Due to this structural characteristic, it is easily damaged during the separation process if physical external force is applied. Cambial meristematic cells (CMCs) have a strong self-renewal ability and can differentiate into other types of plant cells. CMCs produce a large number of cell products, and these ingredients can be used in the cosmetics, pharmaceutical, food and other industries. Taking ginseng cambium meristematic cells (ginseng CMCs) as an example, its cell products are used to treat various syndromes, prevent or treat liver disease, treat cancer and delay aging.

[0004] Currently, the isolation and production of ginseng CMCs through liquid culture is still at a small scale, failing to meet the requirements for scale-up production. Furthermore, the inability to ensure genetic stability during the culture process results in extremely low or even no cell product content. These factors significantly impact the application of ginseng CMCs and increase the economic cost of their use. While developing ginseng CMCs products, the inventors discovered that cell culture fluids derived from ginseng CMCs significantly promote collagen production. However, existing techniques typically treat these culture fluids as waste.

[0005] Therefore, the present invention is devoted to developing an anti-wrinkle skin care solution and a freeze-dried product of cultured ginseng cambium meristem for use in skin anti-wrinkle. Summary of the Invention

[0006] To overcome the aforementioned problems in the prior art, the present invention provides an anti-wrinkle skincare solution, a freeze-dried product thereof, and a preparation method thereof. This method utilizes mild and simple process conditions and requires no special equipment, making it suitable for large-scale industrial production. The culture fluid obtained from culturing ginseng cambium meristem cells promotes human cell activity and collagen expression.

[0007] The technical solutions of the present invention are as follows:

[0008] A method for preparing an anti-wrinkle skin care solution comprises the following steps:

[0009] S1, ginseng root pretreatment;

[0010] S2. preparing ginseng tissue;

[0011] S3, induction culture: induction culture of the ginseng tissue obtained in step S2 in an induction medium to obtain an initial ginseng cambium meristem cell line;

[0012] S4. Suspension culture: The initial ginseng cambium meristem cell line prepared in step S3 is suspended and cultured on a suspension culture medium, filtered, and the filtrate is collected to obtain the anti-wrinkle skin care solution.

[0013] In step S4, filtration can be performed using a 40-100 μm cell sieve.

[0014] Step S1 is specifically as follows: select ginseng roots, remove fibrous roots, rinse with tap water for 3 hours, then soak in ethanol for 5 minutes, rinse with sterile water 3 times, soak in sodium hypochlorite for 5 minutes, and rinse with sterile water 3 times to obtain crude ginseng roots.

[0015] Preferably, in step S1, the ginseng roots used are artificially grown one-year-old "Changbai Mountain Ginseng" ginseng seedlings, which are transplanted and hardened in the laboratory and collected after the ginseng seedling spores begin to grow (about 1 week).

[0016] Preferably, the volume percentage of ethanol used in step S1 is 70%, and the volume percentage of sodium hypochlorite is 1%.

[0017] Step S2 is specifically as follows: the crude ginseng root obtained in step S1 is cut into 0.5 cm long and 0.1 cm thick threads, soaked in ethanol for 5 minutes, rinsed with sterile water 3 times, soaked in sodium hypochlorite for 15 minutes, rinsed with sterile water for 5 minutes, soaked in sucrose solution A for 24 hours to remove other cells outside the cambium cells; soaked in sucrose solution B for 15 minutes to reduce the cell fluid concentration for easy addition to culture medium for cultivation, crushed, rinsed with citric acid solution, and obtained ginseng tissue for later use.

[0018] Preferably, the ethanol concentration used in step S2 is 70%, the sodium hypochlorite concentration is 1%, the concentration of sucrose solution A is 0.6 mol / L, the concentration of sucrose solution B is 0.3 mol / L, and the concentration of citric acid solution is 150 mg / L.

[0019] The induction medium is a liquid basal medium supplemented with 0.5-10 mg / L IAA (indoleacetic acid), 0.5-10 mg / L IBA (indolebutyric acid), 50-100 mg / L L-ascorbic acid, 100-150 mg / L citric acid, and 0.5-10 g / L sodium benzoate; the liquid basal medium is B5 medium. In some embodiments, the induction suspension medium is B5 liquid basal medium supplemented with 2.5 mg / L IAA, 2.5 mg / L IBA, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, and 2 g / L sodium benzoate.

[0020] As an embodiment of the present invention, the induction culture conditions are: 22-30°C, 50-100 rpm shaking, and dark culture for 10-20 days. In some embodiments, the induction culture conditions are: 24.6°C, 100 rpm shaking, and dark culture for 14 days.

[0021] In one embodiment of the present invention, the suspension culture comprises a liquid basal medium supplemented with 0.5-10 mg / L IAA, 0.5-10 mg / L IBA, 0.5-10 mg / L NAA, 0.5-10 mg / L sodium nitrophenolate, 50-100 mg / L L-ascorbic acid, 100-150 mg / L citric acid, and 0.5-10 g / L sodium benzoate; the liquid basal medium is B5 medium. In some embodiments, the suspension culture comprises B5 liquid basal medium supplemented with 2.9 mg / L IAA, 2.6 mg / L IBA, 5.9 mg / L NAA, 5 mg / L sodium nitrophenolate, 100 mg / L L-ascorbic acid, 150 mg / L citric acid, and 2 g / L sodium benzoate.

[0022] As an embodiment of the present invention, the suspension culture conditions are: 22-30°C, 50-200 rpm shaking, and dark culture for 10-20 days. In some embodiments, the suspension culture conditions are: 24.6°C, 100 rpm shaking, and dark culture for 11 days.

[0023] The preparation method of the present invention is based on the advantages that the culture steps are simple and quick, and the obtained culture fluid can be reused after being processed, thus saving costs, reducing resource waste, improving resource utilization, and being green, environmentally friendly and pollution-free.

[0024] The anti-wrinkle skin care solution prepared by the above-mentioned preparation method also falls within the protection scope of the present invention.

[0025] The present invention also provides a method for preparing an anti-wrinkle skin care freeze-dried product, comprising the following steps:

[0026] Step 1, freeze-drying the anti-wrinkle skin care lotion to obtain a crude freeze-dried product, which is light white;

[0027] Step 2: Take the crude freeze-dried product and dry it to remove volatile substances, and after drying, obtain a light yellow paste M;

[0028] Step 3: redissolve the light yellow paste M to obtain substance N. After freeze-drying N, a white product is obtained, which is the anti-wrinkle skin care freeze-dried product.

[0029] Preferably, the freeze-drying conditions of step 1 and step 3 are: freezing at -120°C to -60°C overnight, followed by freeze-drying at -100°C to -40°C for 3-7 days;

[0030] Preferably, the specific operation of drying in step 2 is vacuum drying at 20-60° C. for 12-48 hours, which can remove volatile substances in the crude freeze-dried product.

[0031] Preferably, the reconstitution condition in step 3 is the original volume of pure water or sterile water.

[0032] The anti-wrinkle skin care freeze-dried product prepared by the preparation method of the anti-wrinkle skin care freeze-dried product also falls within the protection scope of the present invention.

[0033] The use of the anti-wrinkle skin care freeze-dried product in skin anti-wrinkle and anti-aging products also falls within the protection scope of the present invention.

[0034] When the concentration of anti-wrinkle skin care freeze-dried products is 0.001mg / ml-0.03mg / ml, it can promote cell activity, and more significantly, it can promote the expression of collagen.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The key point of the present invention is that the suspension culture time is 10-20 days under dark conditions; if the culture time is too short, the prepared freeze-dried product has no obvious promoting effect on cell viability and type I collagen expression; when the culture time is too long, the prepared freeze-dried product has an inhibitory effect on cell activity and does not promote collagen synthesis.

[0037] 2. The cultivation steps of the present invention are simple, the culture medium required is simple and of few types, and the cultivation time is fast; the anti-wrinkle skin care liquid obtained by the present invention conforms to the green and recyclable economic concept, reduces the generation of waste, can be scaled up for production, and greatly reduces production costs.

[0038] 3. The anti-wrinkle skin care freeze-dried product prepared by the present invention has an inhibitory effect on the aging of human skin cells.

[0039] 4. Another key point of the present invention is that the cell culture fluid cultured with ginseng CMCs is freeze-dried to obtain a freeze-dried product that can be used for anti-wrinkle and anti-aging of the skin, and has a promoting effect on human cell activity and collagen expression. In the preparation of the anti-wrinkle skin care freeze-dried product, a freeze-drying-vacuum drying-freeze-drying method is adopted. The step of secondary freeze-drying after vacuum drying is crucial, and the cell culture fluid that has not been treated has no obvious promoting effect on cell viability and type I collagen expression. The process conditions of this method are mild and simple, there are no special requirements for equipment, and at the same time, resource utilization is improved, which is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0041] Figure 1 This is a sample diagram of the crude freeze-dried product in Example 1;

[0042] Figure 2 This is a sample image of the light yellow paste M in Example 1;

[0043] Figure 3 This is a sample diagram of N in Example 1;

[0044] Figure 4 This is a sample of the N anti-wrinkle skin care freeze-dried product in Example 1;

[0045] Figure 5 The performance test diagram of the anti-wrinkle skin care freeze-dried product in Example 1 is shown, wherein the left diagram shows the test results of cell viability; the right diagram shows the test results of type I collagen expression;

[0046] Figure 6 The left figure is a graph showing the performance test of the freeze-dried product in Comparative Example 1, wherein the left figure is a graph showing the test results of cell viability; the right figure is a graph showing the test results of type I collagen expression;

[0047] Figure 7 The left figure is a graph showing the performance test of the freeze-dried product in Comparative Example 2, wherein the left figure shows the test results of cell viability; the right figure shows the test results of type I collagen expression;

[0048] Figure 8 These are performance test diagrams of the crude freeze-dried product for anti-wrinkle skin care in Example 1, wherein the left diagram shows the test results of cell viability; the right diagram shows the test results of type I collagen expression;

[0049] Figure 9These are blank culture medium performance test diagrams, where the left diagram is the test result diagram of cell viability; the right diagram is the test result diagram of type I collagen expression. DETAILED DESCRIPTION

[0050] The present invention will be described in detail below with reference to the examples. The following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that those skilled in the art may make several adjustments and improvements without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0051] The B5 culture medium used in the following examples and comparative examples was prepared according to the following formula:

[0052] Potassium dihydrogen phosphate 150mg / L

[0053] Anhydrous calcium chloride 113.24 mg / L

[0054] Potassium nitrate 2500mg / L

[0055] Ammonium sulfate 134mg / L

[0056] Anhydrous magnesium sulfate 122.09 mg / L

[0057] Disodium EDTA 37.3 mg / L

[0058] Ferrous sulfate 27.8 mg / L

[0059] Manganese sulfate 10.0 mg / L

[0060] Zinc sulfate 2.0 mg / L

[0061] Boric acid 3.0 mg / L

[0062] Sodium molybdate 0.25mg / L

[0063] Copper sulfate 0.025 mg / L

[0064] Cobalt chloride 0.025 mg / L

[0065] Potassium iodide 0.75 mg / L

[0066] Vitamin B1 10.0mg / L

[0067] Vitamin B6 1.0mg / L

[0068] Niacin 1.0mg / L

[0069] Inositol 100.0 mg / L.

[0070] Example 1

[0071] (1) Preparation of anti-wrinkle skin care lotion

[0072] 1.1 Disinfection of ginseng roots

[0073] Ginseng roots were selected, the fibrous roots were removed, and they were rinsed with tap water for 3 h, then soaked in 70% ethanol for 5 min, rinsed with sterile water 3 times, soaked in 7.5% sodium hypochlorite for 5 min, and rinsed with sterile water 3 times.

[0074] 1.2 Ginseng tissue acquisition

[0075] The ginseng roots were cut into 0.5 cm long and 0.1 cm thick threads, soaked in 70% ethanol for 5 min, rinsed 3 times with sterile water, soaked in 1% sodium hypochlorite for 15 min, rinsed with sterile water for 5 min, soaked in 0.6 mol / L sucrose solution A (penetrant) for 24 h, soaked in 0.3 mol / L sucrose solution B for 15 min, crushed, and rinsed with 150 mg / L citric acid solution to obtain ginseng tissue.

[0076] 1.3 Preparation of induction medium

[0077] The basic culture medium was B5 medium supplemented with 2.5 mg / L IAA (indoleacetic acid), 2.5 mg / L IBA (indolebutyric acid), 100 mg / L L-ascorbic acid, 150 mg / L citric acid, and 2 g / L sodium benzoate.

[0078] 1.4 Induction culture

[0079] Take 1 g of ginseng tissue prepared in step 1.2 and add it to the induction medium prepared in step 1.3 (the culture medium volume is 10 ml). Culture it in the dark at 24.6°C, 100 rpm, and shake for 14 days. Then filter it through sterile gauze to remove large pieces of tissue, and then filter it through a 40 μm cell sieve to remove the culture medium to obtain the successfully induced initial ginseng cambium meristem cell line (120 mg).

[0080] 1.5 Suspension culture

[0081] The ginseng cambium meristem cell line obtained by induced culture (at a ratio of wet cells to culture medium = 1:50 (mg / ml)) was placed in a suspension culture medium (based on culture medium B5, with the addition of IAA, IBA, NAA (naphthaleneacetic acid), sodium nitrophenolate, L-ascorbic acid, citric acid, and sodium benzoate to obtain a suspension culture medium) for suspension culture under the conditions of 24.6°C, 100 rpm shaking, and dark culture for 11 days. The cell was filtered through a 40 μm cell sieve, and the filtrate was collected to obtain an anti-wrinkle skin care solution.

[0082] 1.6 Preparation of freeze-dried anti-wrinkle skin care products

[0083] 1.6.1 Take 1000ml of anti-wrinkle skin care solution, freeze it at -80℃ overnight, and then freeze-dry it at -60℃ for 4 days. The crude freeze-dried product is light white and visible. Figure 1 ;

[0084] 1.6.2 Take the above crude freeze-dried product and vacuum dry it at 40℃ for 24h to remove volatile substances to obtain a light yellow paste M. Figure 2 ;

[0085] 1.6.3 Dissolve the light yellow paste M (28.9 g) obtained in 1.6.2 in 1000 ml of pure water to obtain N. Figure 3 ;

[0086] 1.6.4 Freeze the N obtained in 1.6.3 at -80°C overnight, and then freeze-dry at -60°C for 4 days to obtain a white powder, which is the anti-wrinkle skin care freeze-dried product. Figure 4 .

[0087] (2) Performance test

[0088] 1. Test of cell viability of the anti-wrinkle skin care freeze-dried product prepared in step 1.6.4

[0089] 1.1 Sample preparation: Dissolve the anti-wrinkle skin care freeze-dried product in DMEM culture medium (unit: mg / ml);

[0090] 1.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well;

[0091] 1.3 Positive control: cell proliferation control TGF-β (25 ng / mL);

[0092] 1.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0093] 1.5 Experimental process: Cells were plated and allowed to adhere for 24 hours before adding samples and incubating for another 48 hours. Cells were stained with neutral red for cell viability testing.

[0094] according to Figure 5 The results confirm that anti-wrinkle skin care freeze-dried products have a promoting effect on cell vitality.

[0095] 2. Test of the anti-wrinkle skin care freeze-dried product prepared in step 1.6.4 on the expression of type I collagen

[0096] 2.1 Sample preparation: Dissolve the anti-wrinkle skin care freeze-dried product in DMEM culture medium (unit: mg / ml);

[0097] 2.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well;

[0098] 2.3 Positive control: Collagen I expression test control vitamin C (100 μg / mL);

[0099] 2.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0100] 2.5 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples were then added and incubated for another 48 hours. The supernatant was frozen and used for Collagen I ELISA test.

[0101] according to Figure 5 The results confirmed that the anti-wrinkle skin care freeze-dried product had a significant difference in the expression of type I collagen compared with the negative control, and had a significant promoting effect.

[0102] 3. Test of cell viability of the anti-wrinkle skin care crude freeze-dried product prepared in 1.6.1 in step (1)

[0103] 3.1 Dissolve the crude freeze-dried product of anti-wrinkle skin care prepared in 1.6.1 in DMEM medium (unit: mg / ml);

[0104] 3.2 Fibroblast P6, cell plating density: cells / 100 μL / well;

[0105] 3.3 Positive control: cell proliferation control TGF-β (25 ng / mL);

[0106] 3.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0107] 3.5 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples were added and incubated for 48 hours. The supernatant was frozen for ELISA test of Collagen I. Cells were stained with neutral red for cell viability test.

[0108] according to Figure 8 It can be confirmed that the anti-wrinkle skin care crude freeze-dried product has a significant killing effect on cell vitality.

[0109] 4. Test of the Anti-wrinkle Skin Care Crude Freeze-dried Product Prepared in 1.6.1 in Step (I) on the Promotion of Type I Collagen Expression

[0110] 4.1 Dissolve the crude freeze-dried product of anti-wrinkle skin care prepared in 1.6.1 in DMEM medium (unit: mg / ml);

[0111] 4.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well;

[0112] 4.3 Positive control: Collagen I expression test control vitamin C (100 μg / mL);

[0113] 4.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0114] 4.5 Experimental procedure: Cells were plated and allowed to adhere for 24 hours. Samples were then added and incubated for another 48 hours. The supernatant was frozen for Collagen I ELISA testing. Cells were stained with neutral red for cell viability testing.

[0115] according to Figure 8 It can be confirmed that the anti-wrinkle skin care crude freeze-dried product has no promoting effect on the expression of type I collagen.

[0116] Comparative Example 1

[0117] The difference between this comparative example and Example 1 is that the suspension culture time in step 1.5 is different; the details are as follows:

[0118] The ginseng cambium meristem cell line obtained by induced culture was placed in a suspension culture medium for suspension culture at 24.6° C., 100 rpm shaking, and dark culture for 7 days. The culture medium was filtered through a 40 μm cell sieve to obtain the ginseng cambium meristem cell culture fluid.

[0119] The remaining steps are the same as in Example 1.

[0120] Performance Testing

[0121] 1. Cell viability test

[0122] 1.1 The lyophilized product obtained in Comparative Example 1 was dissolved in DMEM medium (unit: mg / ml)

[0123] 1.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well

[0124] 1.3 Positive control: cell proliferation control TGF-β (25 ng / mL);

[0125] 1.4 Culture conditions: DMEM + 1% PS serum-free medium as negative control

[0126] 1.5 Experimental process: Cells were plated and waited for 24 hours for cells to adhere to the wall. Samples were added and incubated for 48 hours. Cells were stained with neutral red for cell viability test.

[0127] 2. Test for promotion of type I collagen expression

[0128] 2.1 The lyophilized cell culture fluid was dissolved in DMEM medium (unit: mg / ml);

[0129] 2.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well

[0130] 2.3 Positive control: Collagen I expression test control vitamin C (100 μg / mL);

[0131] 2.4 Culture conditions: DMEM + 1% PS serum-free medium as negative control

[0132] 2.5 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples were added and incubated for 48 hours. The supernatant was frozen for ELISA test of Collagen I. Cells were stained with neutral red for cell viability test.

[0133] according to Figure 6 It can be confirmed that the freeze-dried product prepared after 7 days of culture in suspension culture medium has no obvious promoting effect on cell viability and type I collagen expression, and the effect is significantly different from that of Example 1

[0134] Comparative Example 2

[0135] The difference between this comparative example and Example 1 is that the suspension culture time in step 1.5 is different; the details are as follows:

[0136] The ginseng cambium meristem cells obtained by induced culture were placed in a suspension culture medium for suspension culture at 24.6° C., 100 rpm shaking, and dark culture for 22 days. The culture fluid was filtered through a 40 μm cell sieve to obtain the ginseng cambium meristem cell culture fluid.

[0137] The remaining steps are the same as in Example 1.

[0138] 1. Test of cell viability using freeze-dried cell culture medium

[0139] 1.1 The lyophilized product obtained in Comparative Example 1 was dissolved in DMEM medium (unit: mg / ml);

[0140] 1.2 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well;

[0141] 1.3 Positive control: cell proliferation control TGF-β (25 ng / mL);

[0142] 1.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0143] 1.5 Experimental process: Cells were plated and allowed to adhere for 24 hours before adding samples and incubating for another 48 hours. Cells were stained with neutral red for cell viability testing.

[0144] 2. Test for promotion of type I collagen expression

[0145] 2.1 The lyophilized product prepared in Comparative Example 2 was dissolved in DMEM medium (unit: mg / ml);

[0146] 2.2 Fibroblast P6, cell plating density: cells / 100 μL / well;

[0147] 2.3 Positive control: Collagen I expression test control vitamin C (100 μg / mL);

[0148] 2.4 Culture conditions: DMEM + 1% PS serum-free medium was used as a negative control;

[0149] 2.5 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples were then added and incubated for another 48 hours. The supernatant was frozen for Collagen I ELISA test. Cells were stained with neutral red for cell viability test.

[0150] according to Figure 7 It was confirmed that the freeze-dried product obtained when the cells were cultured in the suspension medium for 22 days had an inhibitory effect on cell activity and did not promote collagen synthesis.

[0151] Comparative Example 3

[0152] Preparation of blank culture medium: The basic culture medium is B5 medium, supplemented with IAA 2.9 mg / L, IBA 2.6 mg / L, NAA (naphthaleneacetic acid) 5.9 mg / L, sodium nitrophenolate 5 mg / L, L-ascorbic acid 100 mg / L, citric acid 150 mg / L, and sodium benzoate 2 g / L.

[0153] 1. Test of human cell viability using blank culture medium

[0154] 1.1 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well

[0155] 1.3 Positive control: cell proliferation control TGF-β (25 ng / mL);

[0156] 1.4 Culture conditions: DMEM + 1% PS serum-free medium as negative control

[0157] 1.5 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples (blank culture medium) were added and incubated for 48 hours. The supernatant was frozen for ELISA test of Collagen I. Cells were stained with neutral red for cell viability test.

[0158] according to Figure 9 It was proved that blank culture medium had some effect on cell activity but no significant difference was found.

[0159] 2. Test of blank culture medium on type I collagen expression

[0160] 2.1 Fibroblast P6, cell plating density: 8000 cells / 100 μL / well

[0161] 2.2 Positive control: Collagen I expression test control vitamin C (100 μg / mL);

[0162] 2.3 Culture conditions: DMEM + 1% PS serum-free medium as negative control

[0163] 2.4 Experimental process: Cells were plated and allowed to adhere for 24 hours. Samples (blank culture medium) were added and incubated for 48 hours. The supernatant was frozen for ELISA test of Collagen I. Cells were stained with neutral red for cell viability test.

[0164] according to Figure 9 It was proved that blank culture medium had a certain effect on the expression of type I collagen but there was no significant difference.

[0165] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing an anti-wrinkle skin care solution, comprising the following steps: S1, ginseng root pretreatment; S2. preparing ginseng tissue; Step S2 specifically comprises cutting the ginseng root obtained in step S1 into 0.5 cm long and 0.1 cm thick threads, soaking them in ethanol for 5 minutes, rinsing them with sterile water three times, soaking them in sodium hypochlorite for 15 minutes, rinsing them with sterile water for 5 minutes, and soaking them in sucrose solution A for 24 hours to remove other cells outside the cambium cells; Soak in sucrose solution B for 15 min, grind, and rinse with citric acid solution to obtain ginseng tissue for later use; The concentration of sucrose solution A is 0.6 mol / L, and the concentration of sucrose solution B is 0.3 mol / L; S3, induction culture: induction culture of the ginseng tissue obtained in step S2 in an induction medium to obtain an initial ginseng cambium meristem cell line; S4, suspension culture: culturing the initial ginseng cambium meristem cell line prepared in step S3 on a suspension culture medium, filtering, and collecting the filtrate to obtain the anti-wrinkle skin care solution; In step S4, the suspension culture is performed by adding IAA 0.5-10 mg / L, IBA 0.5-10 mg / L, NAA 0.5-10 mg / L, sodium nitrophenolate 0.5-10 mg / L, L-ascorbic acid 50-100 mg / L, citric acid 100-150 mg / L, and sodium benzoate 0.5-10 g / L to a liquid basal culture medium; the liquid basal culture medium is B5 medium; In step S4, the suspension culture conditions are: 22-30° C., 50-200 rpm shaking, and dark culture for 10-20 days.

2. The method for preparing the anti-wrinkle skin care solution according to claim 1, wherein: In step S3, the induction medium is a liquid basal medium supplemented with IAA 0.5-10 mg / L, IBA 0.5-10 mg / L, L-ascorbic acid 50-100 mg / L, citric acid 100-150 mg / L, and sodium benzoate 0.5-10 g / L; the liquid basal medium is B5.

3. The method for preparing the anti-wrinkle skin care solution according to claim 1, wherein: In step S3, the induction culture conditions are: 22-30° C., 50-100 r / min shaking, and dark culture for 10-20 days.

4. An anti-wrinkle skin care solution obtained according to the preparation method according to any one of claims 1 to 3.

5. A method for preparing an anti-wrinkle skin care freeze-dried product, characterized in that: The following steps are involved: Step 1, freeze-drying the anti-wrinkle skin care solution according to claim 4 to obtain a crude freeze-dried product, which is light white; Step 2: taking the crude freeze-dried product, drying it to remove volatile substances, and obtaining a light yellow paste M after drying; Step 3: redissolve the light yellow paste M to obtain substance N. After freeze-drying the substance N, a white product is obtained, which is the anti-wrinkle skin care freeze-dried product.

6. The preparation method according to claim 5, characterized in that The freeze-drying conditions of step 1 and step 3 are: freezing at -120°C to -60°C overnight, followed by low-temperature freeze-drying at -100°C to -40°C for 3-7 days; the specific drying operation in step 2 is vacuum drying at 20-60°C for 12-48 hours.

7. An anti-wrinkle skin care freeze-dried product prepared by the method for preparing an anti-wrinkle skin care freeze-dried product according to claim 5 or 6.

8. Use of the anti-wrinkle skin care freeze-dried product according to claim 7 in the preparation of skin anti-wrinkle and anti-aging products.

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