A cellular bionic drying model and its application in the evaluation of moisturizing efficacy and dryness damage repair efficacy

Abstract

The present invention belongs to the field of cell biology, and specifically relates to a cell bionic drying model and its application in the evaluation of moisturizing efficacy or dryness damage repair efficacy. The present invention establishes a method for evaluating the moisturizing efficacy or dryness damage repair of cells under culture conditions that only change the humidity, and a method for detecting the cell viability after drying. The drying conditions are relatively mild, simulating the growth environment of cells in the human body, the drying modeling time is appropriate, the cell death rate is slow, the influence of operation errors is smaller, and the degree of recovery of cell viability after drying can be more intuitively reflected, which can be applied to the high-throughput screening of moisturizing active substances in cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cell biology, and in particular relates to a cell bionic drying model and an application thereof in the evaluation of moisturizing efficacy or drying damage repair efficacy. Background Art

[0002] The evaluation methods for the moisturizing effects of cosmetics and their raw materials are mainly divided into in vivo experiments and in vitro experiments according to the experimental environment and the objects involved.

[0003] In vivo experiments are experiments conducted using animal models or humans, mainly evaluating the moisturizing effect of cosmetics and their raw materials by testing the moisture content of the skin stratum corneum. Due to certain differences in physiological characteristics between humans and animals, animal experiments cannot accurately reflect the actual effects of cosmetics and their raw materials on human skin. The animal models used in the prior art include rabbits, mice, etc., which have a long breeding cycle, are time-consuming, and have high capital costs. Internationally, many countries have issued relevant regulations prohibiting animal testing of cosmetics and prohibiting the sale of cosmetics that rely on animal testing as data support. Therefore, there are problems such as large species differences, long experimental cycles, high costs, and difficulty in exporting when evaluating the efficacy of cosmetics and their raw materials through animal experiments. In addition, this type of animal experiment is difficult to use for high-throughput screening of cosmetic active substances. Similarly, human experiments also have problems such as long cycles, high costs, and difficulty in scale, as well as difficulties in recruiting volunteers and uncontrollable interference factors.

[0004] In vitro experiments mainly include moisture change rate test and in vitro cell experiment. The moisture change rate test usually adopts the weighing method, which measures the weight change of different substances over a period of time under certain humidity conditions. This method evaluates the hygroscopicity and water retention of the substance itself, and cannot evaluate whether the substance has moisturizing effect on the skin. Commonly used cell models for in vitro cell experiments include human immortalized epidermal cell line (HaCaT), etc. HaCaT is an immortal cell cultivated from keratinocytes and retains the differentiation ability of keratinocytes. It has strong proliferation, growth and differentiation ability. Keratinocytes are the main constituent cells of the epidermis, accounting for more than 80% of epidermal cells. They are related to functions such as skin barrier, aging, immune regulation, and wound healing, and are usually used as a model for skin biology research. The HaCaT cell model not only avoids the ethical issues existing in animal experiments, but also avoids the impact of interspecies differences. At the same time, it has the advantages of strong proliferation ability, short culture cycle, and low culture cost. It is widely used in in vitro experiments for the evaluation of the moisturizing effect of cosmetics.

[0005] The current method for establishing a cell drying model is to remove the cell culture medium and place it in a clean bench for 20 minutes. The humidity condition is about the clean bench environment humidity, the temperature is room temperature, and the carbon dioxide concentration is the carbon dioxide concentration in the atmosphere. A study used this method to evaluate the active lipids extracted from longan seeds. That is, a 96-well plate of HaCaT cells treated with extracts and with a cell confluence of 80% was placed in a clean bench after removing the culture medium. It was placed at room temperature for 5, 10, 15, 20, and 25 minutes to measure the cell survival rate (Ji Man. Study on the extraction and separation of active lipids from longan seeds and their moisturizing activity [D]. Wuxi: Jiangnan University, 2019). The existing technology has the following disadvantages and shortcomings: (1) The cell environment conditions vary greatly: the carbon dioxide concentration of the environment during the drying process is only 0.04%, while the normal culture environment of HaCaT cells requires a carbon dioxide concentration of 5%; the normal culture temperature of HaCaT cells is 37°C, and room temperature is used during clean bench drying, resulting in a large temperature difference. Therefore, the existing technical solutions are far from the normal cell culture conditions, and it is impossible to eliminate the influence of carbon dioxide concentration and temperature on cells. In addition, there are large differences in different laboratory environments, and the conditions are not easy to control and the results are difficult to repeat. (2) Drying humidity is uncontrollable: The existing technology dries cells in a clean bench, and the humidity of the clean bench is equivalent to the ambient humidity. Different seasons, time, precipitation and other factors will cause different ambient humidity, and experimental conditions are difficult to control. (3) The modeling time is too short, resulting in large operational errors: The existing drying technology cannot better simulate the environment in which cells grow in the human body, resulting in a too fast cell death rate. When the modeling time is 20 minutes, the cell death rate has reached about 50%. In actual operation, the impact of time errors is relatively large.

[0006] Gexianmi, scientific name is Nostoc pseudospherical Nostoc sphaeroides ), belonging to the phylum Cyanobacteria and the genus Nostoc. As a precious microalgae resource, Nostoc has a long history of medicinal and edible use and is rich in nutrients. Studies have shown that Nostoc and other Nostoc algae have a variety of biological activities, including antioxidant, anti-inflammatory, antibacterial, immunomodulatory, wound repair, etc., and have great potential for application in the fields of biomedicine, cosmetics, etc. Li et al. Nostoc commune. CarbohydratePolymers 2011;83(4):1821-1827.) showed that the polysaccharide from Nostoc algae has stronger hygroscopicity and moisturizing ability than chitosan and urea, and can promote the improvement of the water retention capacity of the stratum corneum of the skin isolated from mice, and has application potential in moisturizing. However, the existing technology does not explore whether the polysaccharide from Pueraria lobata has moisturizing effect on human skin, does not study the moisturizing effect of polysaccharide from a deeper level, and does not study its moisturizing activity on a suitable cell model, so it is impossible to deeply study the moisturizing effect mechanism of polysaccharide from Pueraria lobata. Summary of the invention

[0007] In order to overcome the deficiencies and shortcomings of the prior art, the primary purpose of the present invention is to provide a method for constructing a cell bionic dry model.

[0008] Another object of the present invention is to provide a cell bionic dry model constructed by the above construction method.

[0009] Another object of the present invention is to provide an application of the above-mentioned cell bionic dryness model in the evaluation of moisturizing efficacy or dryness damage repair efficacy.

[0010] A fourth object of the present invention is to provide a method for evaluating moisturizing efficacy using a cell bionic drying model.

[0011] The fifth object of the present invention is to provide a method for evaluating the repair efficacy of dryness damage using a cell bionic dryness model.

[0012] A sixth object of the present invention is to provide a method for determining the viability of dry cells after passage.

[0013] The purpose of the present invention is achieved through the following technical solutions:

[0014] A method for constructing a cell bionic drying model comprises the following steps:

[0015] (1) Cell plating: Incubate HaCaT cells with complete medium at 5% CO2 and 37°C for 12-24 h, then replace with fresh complete medium and continue incubation for 12-24 h.

[0016] (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the lid of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 h to obtain a cell bionic drying model;

[0017] The average humidity of the cell culture incubator during cell incubation in step (1) is 100%;

[0018] The complete culture medium in step (1) is preferably DMEM complete culture medium containing fetal bovine serum and double antibody;

[0019] The DMEM complete medium preferably contains 10% fetal bovine serum and 1% double antibody, and the double antibody includes penicillin and streptomycin;

[0020] The incubation time in step (2) is preferably 16 h;

[0021] A cell bionic drying model is obtained by the above construction method;

[0022] Application of the cell bionic drying model in the evaluation of moisturizing efficacy or dryness damage repair efficacy;

[0023] The object of the moisturizing effect and / or repairing effect evaluation is preferably cosmetics and / or its raw materials;

[0024] A method for evaluating moisturizing efficacy using a cell bionic drying model comprises the following steps:

[0025] (1) Drug administration and culture: HaCaT cells were incubated with complete culture medium at 5% CO2 and 37°C for 12-24 h, and then the cells were divided into a model group and a treatment group. The cells in the treatment group were replaced with fresh complete culture medium containing the sample to be tested, and the cells in the model group were replaced with fresh complete culture medium and incubated for another 12-24 h.

[0026] (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the lid of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 h;

[0027] (3) Compare the cell viability of the model group and the treated group to evaluate the moisturizing effect of the test samples.

[0028] The average humidity of the cell culture incubator during cell incubation in step (1) is 100%;

[0029] The complete culture medium in step (1) is preferably DMEM complete culture medium containing fetal bovine serum and double antibody;

[0030] The DMEM complete medium preferably contains 10% fetal bovine serum and 1% double antibody, and the double antibody includes penicillin and streptomycin;

[0031] The incubation time in step (2) is preferably 16 h;

[0032] The method for evaluating moisturizing efficacy using a cell bionic drying model preferably sets a control group of cells, in which no medication is given and no drying modeling is performed;

[0033] A method for evaluating the repair efficacy of dryness damage using a cell bionic dryness model comprises the following steps:

[0034] (1) Cell seeding: HaCaT cells were cultured in complete medium at 5% CO 2、 Incubate at 37°C for 12-24 h, then replace with fresh complete medium and continue incubation for 12-24 h;

[0035] (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the lid of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 h;

[0036] (3) Drug administration and culture: The cells dried in step (2) are divided into a model group and a treatment group, wherein the cells in the treatment group are added with fresh complete culture medium containing the sample to be tested, and the cells in the model group are added with fresh complete culture medium, and the cells are further incubated under the conditions of 5% CO2 and 37°C;

[0037] (4) Compare the cell viability of the model group and the treated group to evaluate the dryness damage repair efficacy of the test samples.

[0038] The complete culture medium in step (1) is preferably DMEM complete culture medium containing fetal bovine serum and double antibody;

[0039] The DMEM complete medium preferably contains 10% fetal bovine serum and 1% double antibody, and the double antibody includes penicillin and streptomycin;

[0040] The incubation time in step (2) is preferably 16 h;

[0041] The time for continued incubation in step (3) is preferably 1-24 h;

[0042] The average humidity of the cell culture incubator during cell incubation in steps (1) and (3) was 100%;

[0043] The method for evaluating the repair efficacy of dryness damage using a cell bionic dryness model preferably sets a control group of cells, in which cells are not dried and modeled, and are not given medication;

[0044] The cell viability is at least one of cell survival rate or cell adhesion rate;

[0045] A method for determining the viability of dried cells after passage, comprising the following steps:

[0046] (1) digesting the above-mentioned cell bionic drying model, the cells dried in the above-mentioned method for evaluating moisturizing efficacy using the cell bionic drying model, or the cells dried and cultured in the above-mentioned method for evaluating dryness damage repair efficacy using the cell bionic drying model with trypsin, and incubating them at 37°C and 5% CO2 for 3-5 min;

[0047] (2) After the incubation, adjust the cell density of each group to be close and inoculate them into new complete medium. Take photos 24 hours after inoculation to record the cell adhesion. Cut 1 cm from the four corners of the photo 2 cm from the border and the center. 2 Count the adherent cells in 5 small squares, calculate the average value, and calculate the cell adhesion rate according to the formula:

[0048] Cell adhesion rate after drying = average number of cells attached （干燥） Average number of cells attached to the wall （未干燥） .

[0049] Principle of the present invention:

[0050] (1) Establishment of cell bionic drying model

[0051] Establishing a cell drying model based on human skin cells and applying it to the evaluation of the moisturizing effect of cosmetics and their raw materials can overcome the problems and shortcomings of in vivo experiments. It has the advantages of strong proliferation ability, short culture cycle, and low culture cost. However, the cell environment conditions of the existing technology vary greatly. The carbon dioxide concentration in the environment of the cells during the drying process is only 0.04%, while the normal culture environment of HaCaT cells requires a carbon dioxide concentration of 5%. The effects of carbon dioxide concentration and temperature on cells cannot be ruled out; the normal culture temperature of HaCaT cells is 37°C, and room temperature is used when drying on a clean bench, and the temperature difference is large. At the same time, the relative humidity during drying with the existing technology is prone to change with the environment, the conditions are not easy to control, and the results are difficult to repeat. The existing technology cannot better simulate the environment in which cells grow in the human body, resulting in an accelerated rate of cell death. When the modeling time is 20 minutes, the cell mortality rate is about 50% ( Figure 3 ), the impact of time error is relatively large in actual operation.

[0052] The present invention also uses HaCaT cells as the modeling object and adopts the bionic drying method to simulate the growth environment of cells in the human body. Specifically, the drying environment is a cell culture box, the location is the lowest layer of the culture box, and the cells are dried under the conditions of 5% carbon dioxide concentration, 90% relative humidity, and 37°C temperature. Among them, saturated BaCl2 solution is used to accurately control the relative humidity during drying. The drying time is 16 h, and the cell mortality rate is about 50% ( Figure 1 ). Compared with the prior art, the present invention only changes the humidity of the culture environment (controls the relative humidity to 90%) while ensuring that other culture conditions are suitable, eliminating the interference of other environmental factors. At the same time, the drying conditions are mild and controllable, the cell death rate is slow, and the impact of time errors in the operation is relatively small.

[0053] (2) Application of the cell bionic drying model in evaluating the moisturizing effect or drying damage repair effect of the sample to be tested

[0054] Based on the above-mentioned cell bionic drying model, the present invention explores the protective activity of kudzu vine polysaccharide on the drying of human skin cells by administering a sample to be tested (for example, kudzu vine polysaccharide, etc.), drying the model, and evaluating its cell viability (for example, cell survival rate or cell adhesion rate, etc.). Figure 4 ).

[0055] (3) Viability determination of dried cells after subculturing

[0056] After cell drying modeling, active substance administration + cell drying modeling, or cell drying modeling + active substance administration, when the cell survival rate is determined by the CCK-8 method, some active substances are covered on the cell surface and cannot be effectively separated, and may react with the CCK-8 reagent, causing the test result of the cell survival rate to be high. The present invention establishes a method for passage detection of dry cell viability. By calculating the adhesion rate after dry cell passage, the effective vitality and vitality recovery of cells after drying can be reflected. The present invention can eliminate the interference of the color reaction between the active substance and the CCK-8 reagent on the experimental results by passage, and more directly observe the cells with better vitality recovery after drying and able to adhere to the wall for growth. The calculated cell viability is more accurate and truly reflects the degree of recovery of cell viability after drying. Among them, the cell counting area of ​​the dry cell adhesion rate is as follows: Figure 5 As shown in Figure 2, the cell adhesion after drying and passage is as follows Figure 8 shown.

[0057] Compared with the prior art, the present invention has the following advantages and effects:

[0058] The present invention establishes a bionic model that allows cells to be damaged by drying under culture conditions that only change the humidity, a method for evaluating the moisturizing efficacy and / or drying repair efficacy using the model, and a method for detecting the viability of dried cells by passage. The model construction method provided by the present invention has mild and controllable drying conditions, simulates the growth environment of cells in the human body, has a slow cell death rate, has a smaller impact on the operation time error, and can more intuitively reflect the degree of recovery of cell viability after drying, and can be applied to high-throughput screening of moisturizing active substances in cosmetics.

[0059] (1) In terms of model selection: The cell model used in the present invention is cultivated from human cells, which not only avoids the problem of ethical requirements in animal experiments, but also avoids the impact of interspecies differences. It also has the advantages of strong proliferation ability, short test cycle, and low cost. In addition, cell experiments can be carried out in 96-well plates, etc., which can realize large-scale screening of active substances.

[0060] (2) In the drying modeling method: the present invention ensures that other culture conditions are suitable when cells are dried, eliminates the interference of other environmental factors, and only changes the humidity of the culture environment. The relative humidity can be controlled at around 90%. The drying conditions are relatively mild, which better simulates the growth environment of cells in the human body, resulting in a slower cell death rate. The cell death rate only reaches about 50% after drying for 16 hours, and the impact of time errors in the operation is relatively small.

[0061] (3) Method for detecting the viability of dried cells by passage: The present invention establishes methods for calculating the cell passage, cell counting and adhesion rate after drying, which can relatively quantitatively determine the effective viability of dried cells. It eliminates the interference of the reaction between active substances and CCK-8 reagents on the experimental results, and more directly observes cells with good viability and adherence to the wall after drying. The measured cell viability is more accurate and can more truly reflect the degree of cell viability recovery after drying.

[0062] In summary, the present invention uses the degree of cell vitality recovery after drying as an indicator to evaluate the moisturizing effect of cosmetic raw materials, establishes a cell bionic drying model, a method for evaluating the moisturizing effect and drying damage repair effect, and a method for measuring the vitality of dried cells after passaging. The above model and method can be applied to high-throughput screening of moisturizing active substances. The present invention applies this method to the exploration of the moisturizing effect of Pueraria lobata polysaccharide. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 This is a result analysis diagram of the effect of the well plate cover drying time on cell viability in Example 1.

[0064] Figure 2 This is a result analysis chart of the effect of the opening and drying time of the well plate on the cell survival rate in Example 1.

[0065] Figure 3 This is an analysis chart of the effect of drying time on cell survival rate in the prior art (Comparative Example 2).

[0066] Figure 4 This is the result analysis diagram of the effect of Pueraria lobata polysaccharide on cell viability after drying for 16 hours.

[0067] Figure 5 This is a schematic diagram of the cell drying and adhesion rate counting area.

[0068] Figure 6 This is a comparison of the cell adhesion of cells cultured with Pueraria lobata extract before and after drying.

[0069] Figure 7 This is the result analysis diagram of the effect of Pueraria lobata extract on cell viability after drying for 16 hours.

[0070] Figure 8 The figures show the adhesion of cells after drying and passage in Example 4, wherein A: adhesion of cells not dried + 24 h after passage, B: adhesion of cells 16 h after drying + 24 h after passage, and C: adhesion of cells cultured with Pueraria lobata extract 16 h after drying + 24 h after passage. DETAILED DESCRIPTION

[0071] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0072] (1) Cell seeding: HaCaT cells (commercially purchased) were seeded into 96-well plates at a density of 50,000 cells / well. The cells were divided into a control group and a model group. The edge wells of the wells were sealed with PBS, and 6 replicate wells were set up for each group. The cells in the control group and the model group were incubated with 100 μL of DMEM complete medium containing 10% fetal bovine serum and 1% double antibody at 5% CO2 and 37°C for 12 h, and then replaced with fresh DMEM complete medium containing 10% fetal bovine serum and 1% double antibody and continued to incubate for 24 h.

[0073] (2) Drying modeling: The culture medium in step (1) was aspirated, and the cells were washed with 100 μL PBS. The control group was added with 100 μL DMEM culture medium containing no fetal bovine serum and 1% double antibody, and the model group was not added with culture medium. The sterile water in the water tray in the cell culture incubator was replaced with saturated BaCl2 solution, and the average humidity of the cell culture incubator was adjusted to 90%. Then, the 96-well plate was closed and placed in the cell culture incubator, where the model group was placed at the lowest level of the cell culture incubator and close to the water tray. After incubation for 12, 14, 16, 18, 20, 22, and 24 h, 100 μL DMEM culture medium containing no fetal bovine serum and 1% double antibody was added to the model group to stop drying.

[0074] (3) Determination of modeling time: 10 μL CCK-8 was added to each well and incubated for 3 h. Six wells without cells and containing 100 μL culture medium and 10 μL CCK-8 were used as the zeroing group. The absorbance A was detected at 450 nm and the cell survival rate was calculated. The calculation formula for cell survival rate is: Cell survival rate = (A 模型组 -A 调零组 ) / (A 对照组 -A 调零组 ) × 100%. The time when the cell viability was 50% was selected as the drying modeling time. The highest and lowest values ​​were removed when processing the data, and 4 valid data points were used.

[0075] (1) Cell seeding: HaCaT cells were seeded into 96-well plates at a density of 50,000 cells / well. The cells were divided into a control group and a model group. The edge wells of the wells were sealed with PBS, and 6 replicate wells were set up for each group. The cells in the control group and the model group were incubated with 100 μL of DMEM complete medium containing 10% fetal bovine serum and 1% double antibody at 5% CO2 and 37°C for 12 h, and then replaced with fresh DMEM complete medium containing 10% fetal bovine serum and 1% double antibody and continued to be incubated for 24 h.

[0076] (2) Drying modeling: The culture medium in step (1) was aspirated, and the cells were washed with 100 μL PBS. The control group was added with 100 μL DMEM culture medium containing no fetal bovine serum and 1% double antibody, and the model group was not added with culture medium. The sterile water in the water tray in the cell culture incubator was replaced with saturated BaCl2 solution, and the average humidity of the cell culture incubator was adjusted to 90%. Then, the 96-well plate was opened and placed in the cell culture incubator, where the model group was placed at the lowest level of the cell culture incubator and close to the water tray. After incubation for 15, 30, 45, and 60 min, 100 μL DMEM culture medium containing no fetal bovine serum and 1% double antibody was added to the model group to stop drying.

[0077] (3) Determination of modeling time: 10 μL CCK-8 was added to each well and incubated for 3 h. Six wells without cells and containing 100 μL culture medium and 10 μL CCK-8 were used as the zeroing group. The absorbance A was detected at 450 nm and the cell survival rate was calculated. The calculation formula for cell survival rate is: Cell survival rate = (A 模型组 -A 调零组 ) / (A 对照组 -A 调零组 ) × 100%. The time when the cell viability was 50% was selected as the drying modeling time. The highest and lowest values ​​were removed when processing the data, and 4 valid data points were used.

[0078] A cell drying model was established using HaCaT cells according to (Ji Man. Extraction and separation of active lipids from longan seeds and study on their moisturizing activity [D]. Wuxi: Jiangnan University, 2019.).

[0079] The cell survival rate results after treatment in Example 1, Comparative Example 1 and Comparative Example 2 are shown in Figure 1-3 :

[0080] from Figure 2 It can be seen that the cells in Comparative Example 1 died rapidly within 15-30 min, and the death rate was too fast. Modeling by this method may lead to large experimental errors due to differences in operation time, so it is not the best modeling method.

[0081] In the modeling process of the prior art (Comparative Example 2), the CO2 concentration was only 0.04%, the temperature was room temperature, the relative humidity during drying would vary with the environment and could not be controlled, and the cell death rate was significantly accelerated. When the drying time was 20 min, the cell death rate reached about 50% ( Figure 3 ), cannot better simulate the environment in which cells grow in the human body, and the impact of time errors in actual operations is relatively large.

[0082] Example 1 After drying for 16 h, the cell survival rate reached 54.5% ( Figure 1 ), this method only changes the humidity of the culture environment while ensuring that other culture conditions are appropriate. The drying humidity is controllable and the interference of other environmental factors is eliminated. At the same time, the drying conditions are mild and controllable, the drying rate is slow, the cell death rate is slow, and the impact of time errors in the operation is relatively small. Therefore, this method was finally selected for drying modeling, and subsequent experiments were carried out, and 16 h was determined as the drying modeling time.

[0083] 1. Preparation of Puerariae lobata polysaccharide

[0084] (1) Grinding: Take an appropriate amount of dried kudzu root and grind it;

[0085] (2) Ethanol defatting: add 20 mL of 95% ethanol solution per gram of powder, soak the powder overnight at room temperature, filter and recover the precipitate of Pueraria lobata;

[0086] (3) High-pressure homogenization: 400 mL of water was added per gram of kudzu vine precipitate, and the precipitate was soaked at room temperature for 24 h. Homogenization was performed twice under a pressure of 100 MPa to obtain a dispersion of kudzu vine precipitate.

[0087] (4) Hot water extraction: Add grade tertiary water at a solid-liquid ratio of 1:15, stir and extract at 100°C for 2 h, centrifuge (room temperature, 6000 rpm, 10 min), collect the powder and supernatant separately, repeat the powder extraction twice, discard the powder, and combine the two extracts to obtain the Pueraria extract;

[0088] (5) Enzymatic protein removal: papain was added at a ratio of 3000 U / g dry algae raw material, and enzymatic hydrolysis was performed at a temperature of 40°C and a pH value of 6.0 for 3 h. The mixture was cooled to room temperature, and then trypsin was added at a ratio of 3000 U / g dry algae raw material. The mixture was enzymatic hydrolyzed at a temperature of 45°C and a pH value of 8.0 for 3 h. The mixture was then inactivated at 100°C for 10 min and centrifuged (room temperature, 6000 rpm, 10 min) to obtain a clear extract.

[0089] (6) Microfiltration and ultrafiltration: The extract is subjected to microfiltration, and the permeate is then ultrafiltered to 1 / 10 volume using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the retentate is obtained to obtain a concentrated solution of kudzu vine polysaccharide;

[0090] (7) Concentration and drying: The concentrated solution of kudzu vine polysaccharide is concentrated under reduced pressure, and then freeze-dried to obtain kudzu vine polysaccharide.

[0091] 2. Monosaccharide composition of Pueraria polysaccharide

[0092] After testing, the monosaccharide composition of Pueraria lobata polysaccharide is shown in Table 1.

[0093] Table 1 Monosaccharide composition of Puerariae lobata polysaccharide

[0094] Monosaccharide Type glucose Galactose Mannose Xylose Glucuronic acid Content (mol%) 41.9 21.7 15.3 16.6 4.5

[0095] Example 3 Protective effect of kudzu vine polysaccharide on dried cells

[0096] (1) Administration and culture: HaCaT cells were inoculated into 96-well plates at a density of 50,000 cells / well, and the cells were divided into three groups: control group, model group, and treatment group. The edge wells of the well plate were sealed with PBS, and 6 duplicate wells were set in each group. The cells in the control group, model group, and treatment group were incubated with 100 μL of DMEM complete medium containing 10% fetal bovine serum and 1% double antibody at 5% CO2 and 37°C for 12 h. Then, the control group and the model group were replaced with 100 μL of fresh DMEM complete medium containing 10% fetal bovine serum and 1% double antibody, and the treatment group was added with 100 μL of fresh DMEM complete medium containing 1 mg / mL Pueraria polysaccharide (prepared in Example 2), 10% fetal bovine serum, and 1% double antibody. The three groups of cells were incubated for 24 h at 5% CO2 and 37°C.

[0097] (2) Drying modeling: The culture medium in step (1) was aspirated and each well of cells was washed with 100 μL PBS. After washing, the cells in the control group were replaced with DMEM culture medium without fetal bovine serum and containing 1% double antibody, and the cells in the model group and treatment group were not added with culture medium. The sterile water in the water tray in the cell culture incubator was replaced with saturated BaCl2 solution, and the 96-well plates of the three groups of cells were covered and placed in the cell culture incubator, among which the model group and treatment group were placed in the lowest layer of the cell culture incubator and close to the water tray, and incubated for 16 h. After the incubation, the cells in the model group and treatment group were added with DMEM culture medium without fetal bovine serum and containing 1% double antibody to stop drying.

[0098] (3) Cell survival rate determination: 10 μL CCK-8 was added to each well and incubated for 3 h. The well without cells and containing 100 μL culture medium and 10 μL CCK-8 was used as the zero well. The absorbance was measured at 450 nm and the cell survival rate was calculated. The cell survival rate of the model group = (A 模型组 -A 调零组 ) / (A 对照组 -A 调零组 )×100%; cell survival rate of treatment group = (A 处理组 -A 调零组 ) / (A 对照组 -A 调零组 )×100%.

[0099] The results are as follows Figure 4 As shown in the data, the cell survival rate of the model group was 55.25% after 16 h of drying, and the cell survival rate of the treatment group after administration of kudzu vine polysaccharide was 81.5%. Compared with the model group, the cell survival rate of the treatment group was significantly improved, indicating that kudzu vine polysaccharide has a certain effect in protecting cells from drying damage.

[0100] 1. Cell survival rate

[0101] (1) Drug administration and culture: HaCaT cells were inoculated into 96-well plates at a density of 50,000 cells / well and divided into three groups: control group, model group and treatment group. The edge wells of the well plate were sealed with PBS, and 6 duplicate wells were set in each group. The cells in the control group, model group and treatment group were incubated with 100 μL of DMEM complete medium containing 10% fetal bovine serum and 1% double antibody at 5% CO2 and 37°C for 12 h. Then, the control group and the model group were replaced with 100 μL of fresh DMEM complete medium containing 10% fetal bovine serum and 1% double antibody, and the treatment group was added with 100 μL of fresh DMEM complete medium containing 2 mg / mL Pueraria lobata extract (obtained by freeze-drying the Pueraria lobata extract obtained in Example 2, which has poor solubility), 10% fetal bovine serum and 1% double antibody. The three groups of cells were incubated for 24 h at 5% CO2 and 37°C.

[0102] (2) Drying modeling: The culture medium in step (1) was aspirated and each well of cells was washed with 100 μL PBS. After washing, the cells in the control group were replaced with DMEM culture medium without fetal bovine serum and containing 1% double antibody, and the cells in the model group and treatment group were not added with culture medium. The sterile water in the water tray in the cell culture incubator was replaced with saturated BaCl2 solution, and the 96-well plates of the three groups of cells were covered and placed in the cell culture incubator, among which the model group and treatment group were placed in the lowest layer of the cell culture incubator and close to the water tray, and incubated for 16 h. After the incubation, the cells in the model group and treatment group were added with DMEM culture medium without fetal bovine serum and containing 1% double antibody to stop drying.

[0103] (3) Cell survival rate determination: 10 μL CCK-8 was added to each well and incubated for 3 h. The well without cells and containing 100 μL culture medium and 10 μL CCK-8 was used as the zero well. The absorbance was detected at 450 nm and the cell survival rate was calculated. The calculation formula of cell survival rate was: Model group cell survival rate = (A 模型组 -A 调零组 ) / (A 对照组 -A 调零组 )×100%; cell survival rate of treatment group = (A 处理组 -A 调零组 ) / (A 对照组 -A 调零组 ) × 100%. At the same time, 6 wells without cells were set up and contained 100 μL of kudzu extract medium and 10 μL of CCK-8. The absorbance was detected at 450 nm and compared with the absorbance of the zero-adjusted well to determine whether the kudzu extract reacted with CCK-8.

[0104] 2. Cell Adhesion Rate

[0105] (1) Drug administration and culture: same as step 1 (1) above;

[0106] (2) Drying modeling: Same as step 1 (2) above, except that after 16 h of incubation, the cells in the model group and the treatment group do not need to be added with DMEM medium containing no fetal bovine serum and 1% double antibody;

[0107] (3) Cell passaging: The culture medium of the control group was aspirated, and 100 μL of trypsin solution was added to each well of the control group, model group, and treatment group to digest the cells. The cells were incubated at 37°C, 5% CO2 for 5 min, and then the cell density of each group was adjusted to be close and inoculated into a new 96-well plate (DMEM complete culture medium containing 10% fetal bovine serum and 1% double antibody). The cell adhesion was observed under a microscope 24 h after inoculation.

[0108] (4) Dry cell adhesion rate: Cut 1 cm from the four corners of the photo 2 cm away from the border and the center. 2The small square ( Figure 5 ), count the adherent cells in 5 small squares, calculate the average value, and calculate the cell adhesion rate according to the formula: Cell adhesion rate after drying = average number of cells adhered （干燥） Average number of cells attached to the wall （未干燥，即对照组） .

[0109] After the administration of Pueraria extract, the adhesion of cells in the treatment group before and after drying was compared. Figure 6 As shown, it is impossible to observe the difference in cell adhesion before and after drying with the naked eye.

[0110] The results of cell viability determination were as follows Figure 7 As shown in the figure, the absorbance of the cells in the group treated with culturing with kudzu extract and then drying was higher than that of the control group cells that were not dried. The average absorbance of the wells without cells inoculated and containing 100 μL kudzu extract culture medium and 10 μL CCK-8 was 1.210, and the average absorbance of the zeroed wells without cells inoculated and containing 100 μL culture medium and 10 μL CCK-8 was 0.286, indicating that the kudzu extract would react with CCK-8, resulting in a high absorbance value. Based on the above results, in the cell survival rate determination experiment, the kudzu extract covered the cell surface and could not be separated, and reacted with CCK-8, resulting in inaccurate experimental results. Therefore, it is not possible to explore whether the kudzu extract can protect cells from drying damage by evaluating the cell survival rate.

[0111] The cell adhesion status 24 h after subculturing is as follows Figure 8 As shown, the adhesion rate of the second generation cells (control group) that were not dried was 100% ( Figure 8 Figure A in the figure shows that the attachment rate of the second generation cells (modeling group) after drying for 16 h was about 42% ( Figure 8 Figure B shows that the second generation of cells (treated group) cultured with kudzu extract and then dried had an adhesion rate of about 77% ( Figure 8 Figure C in the figure shows that the extract of Pueraria lobata has a protective effect on cell drying and can improve the effective vitality of dried cells after passaging.

[0112] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for constructing a cell bionic drying model, characterized in that The following steps are included: (1) Cell plating: Incubate HaCaT cells with complete medium at 5% CO2 and 37°C for 12-24 h, then replace with fresh complete medium and continue incubation for 12-24 h. (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the lid of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 hours to obtain a cell bionic drying model; During the cell incubation in step (1), the average humidity of the cell culture incubator was 100%.

2. The method for constructing a cell bionic dry model according to claim 1, characterized in that: The complete culture medium in step (1) is DMEM complete culture medium containing fetal bovine serum and double antibody; The DMEM complete culture medium contains 10% fetal bovine serum and 1% double antibody, and the double antibody includes penicillin and streptomycin.

3. A method for evaluating moisturizing efficacy using a cell bionic drying model, characterized in that The following steps are included: (1) Drug administration and culture: HaCaT cells were cultured with complete culture medium in 5% CO 2、 Incubate at 37°C for 12-24 h, then divide the cells into a model group and a treatment group. The cells in the treatment group are replaced with fresh complete medium containing the sample to be tested, and the cells in the model group are replaced with fresh complete medium and incubated for another 12-24 h. (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the cover of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 hours; (3) Compare the cell viability of the model group and the treated group to evaluate the moisturizing effect of the test sample; During the cell incubation in step (1), the average humidity of the cell culture incubator was 100%.

4. A method for evaluating the repair efficacy of dryness damage using a cell bionic dryness model, wherein the method comprises: Follow these steps: (1) Cell seeding: HaCaT cells were cultured in complete medium at 5% CO 2、 Incubate at 37°C for 12-24 h, then replace with fresh complete medium and continue incubation for 12-24 h; (2) Drying modeling: remove the culture medium in step (1) and wash the cells with PBS; replace the water in the water tray in the cell culture incubator with a saturated BaCl2 solution and adjust the average relative humidity of the cell culture incubator to 90%; then close the cover of the culture plate containing the washed cells and place it at the lowest level of the cell culture incubator and close to the water tray, and incubate it at 5% CO2 and 37°C for 16-20 hours; (3) Drug administration and culture: The cells dried in step (2) are divided into a model group and a treatment group, wherein the cells in the treatment group are added with fresh complete culture medium containing the sample to be tested, and the cells in the model group are added with fresh complete culture medium, and the cells are further incubated under the conditions of 5% CO2 and 37°C; (4) Compare the cell viability of the model group and the treated group to evaluate the dryness damage repair efficacy of the tested samples; During the cell incubation in step (1), the average humidity of the cell culture incubator was 100%.

5. The method according to claim 3 or 4, characterized in that: The method described above sets up a control group of cells, in which cells are not dried for modeling and are not given any medication.

6. The method according to claim 3 or 4, characterized in that: The cell viability is at least one of cell survival rate or cell adhesion rate.

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