Application of tetrazole violet and skin care recommendation method based on tetrazole violet color development

Abstract

The invention discloses application of tetrazole violet and a skin care recommendation method based on tetrazole violet color development. The tetrazole violet is used for screening skin care components which keep original effects on the skin; or is used for preparing a reagent or a kit, and the reagent or the kit is used for screening skin care components which keep original effects on skin. The invention provides a new efficacy detection thought for the field of skin care products, and provides a screening method for skin care components which can quickly and ensure that the original efficacy is kept on the skin of a target crowd. And more comprehensive quality management and supervision data can be provided for research and development personnel and consumer groups by utilizing evaluation results, technical data is provided for product upgrading of production operators, and instructive data is provided for the operators and consumers to select higher-quality products.

Description

Technical Field

[0001] The present application relates to a use of tetrazolium violet and a recommended skin care method based on tetrazolium violet color development. Background Art

[0002] At present, the development of cosmetics generally adopts the method of in vitro cell test verification, that is, first verifying whether the cosmetics have a regulatory effect on the relevant targets at the cell level, and then transferring the cosmetics that have efficacy at the cell level to the human body for efficacy testing. However, due to individual differences in skin quality, there are obvious individual differences in the efficacy of cosmetics. For example, many cosmetics have cosmetic efficacy when verified in cell and mouse experiments, but are ineffective in humans. There is currently no suitable solution to this problem.

[0003] Therefore, there is an urgent need in the art to find a method for screening skin care ingredients that can be quickly targeted at a target population and ensure that the original efficacy is maintained on the skin. Summary of the invention

[0004] The technical problem to be solved by this application is to overcome the defects in the prior art, such as the obvious individual differences in the efficacy of cosmetics due to individual differences, and the lack of a method for screening skin care ingredients that maintain the original efficacy for the target population, and to provide a use of tetrazolium violet and a recommended skin care method based on tetrazolium violet color development. This application provides a new idea for efficacy testing in the field of skin care products, and provides a method for screening skin care ingredients that can be quickly targeted at the target population and ensure that the original efficacy is maintained on the skin. The evaluation results can be used to provide more comprehensive quality management and supervision data for R&D personnel and consumer groups, provide technical data for product upgrades of manufacturers and operators, and provide guiding data for operators and consumers to choose better products.

[0005] That is, the more intensely the skin care ingredients are transformed by the skin surface flora, the more electrons are released, the darker the color of the reaction system, and the lower the content of the original effective ingredients, which makes it easier to change the original skin care conditions of the skin care ingredients and affect the original effectiveness of the skin care ingredients. If no color change occurs, it means that the amount of skin care ingredients biologically converted is very small or even unconverted, and the original effective ingredients can take effect on the skin and maintain their original effectiveness.

[0006] This application adopts the following technical solutions to solve the above technical problems:

[0007] The present application provides a use of tetrazolium violet for screening skin care ingredients that maintain their original efficacy on the skin; or for preparing a reagent or a kit, wherein the reagent or the kit is used for screening skin care ingredients that maintain their original efficacy on the skin.

[0008] The present application also provides a recommended skin care method based on tetrazolium violet color development, the method comprising the following steps;

[0009] (1) collecting skin samples and preparing a bacterial solution containing skin flora;

[0010] (2) co-culturing the skin care ingredient to be screened, tetrazolium violet and the bacterial solution prepared in step (1);

[0011] (3) judging whether the skin care ingredient is a target skin care ingredient that maintains its original efficacy based on the difference in the degree of color change and / or absorbance value of the system after the co-cultivation and before the co-cultivation.

[0012] During the research and development process, it was found that individual differences in the efficacy of cosmetics may be due to the influence of skin microorganisms on skin care products. Skin microorganisms are a general term for a class of living microorganisms that exist on the surface of the skin. Skin microorganisms are essentially biological and can carry out life activities. They may also carry out biotransformation of skin secretions and foreign substances, which leads to the fact that not all skin care ingredients can produce real effects on the skin. This application uses the principle that skin flora releases electrons during biotransformation, and tetrazolium violet captures the electrons released by microorganisms and produces a color reaction, so as to screen out skin care ingredients that can maintain their original efficacy on the skin.

[0013] In step (3) of some embodiments, when the difference in absorbance between the system after co-cultivation and before co-cultivation is less than or equal to 0.082, the skin care ingredient is recommended as a target skin care ingredient that maintains the original efficacy. When the difference in absorbance between the system after co-cultivation and before co-cultivation is greater than 0.082, the skin care ingredient is not recommended as a target skin care ingredient that maintains the original efficacy.

[0014] In step (3) of some embodiments, when the color change of the system after the co-cultivation and before the co-cultivation is significant, the skin care ingredient is recommended as the target skin care ingredient to maintain the original efficacy; when the color change of the system after the co-cultivation and before the co-cultivation is not significant, the skin care ingredient is not recommended as the target skin care ingredient to maintain the original efficacy.

[0015] In some embodiments, the skin care recommendation method includes recommending skin care products or cosmetics, configuring skin care products or cosmetics based on the recommended target skin care ingredients to obtain target skin care products or target cosmetics.

[0016] In step (1) of some embodiments, the skin sample is derived from whole body skin or facial skin, preferably from at least one of the cheek, nose wing and forehead. According to the conventional art, whole body skin generally refers to the skin of the limbs and trunk.

[0017] In step (1) of some embodiments, the method for preparing the bacterial solution comprises the following steps:

[0018] A: Samples were collected from the skin using a sterile cotton swab soaked in nuclease-free water, and transferred to a culture medium for shaking culture to obtain material a;

[0019] B; collecting the bacterial cells in the material a and mixing them with a salt solution to obtain the bacterial solution.

[0020] In step A of preparing the bacterial solution, the nuclease-free water may include the following components at the following concentrations: 40-50 mM Tris (Tris), 1-1.5 mM EDTA, and 0.5 wt%-1 wt% Tween 20.

[0021] In step A of preparing the bacterial solution, the culture medium may be a substance commonly used in the art that can provide for the growth and reproduction of microorganisms, and generally may include TBS culture medium.

[0022] In step A of preparing the bacterial solution, the shaking culture time may be 1 to 3 days.

[0023] In step A of preparing the bacterial solution, the temperature of the shaking culture may be 30-37°C.

[0024] In step B of preparing the bacterial solution, the salt solution may include the following components in concentrations: 80-120 mM sodium chloride, 20-40 mM triethanolamine, 20-30 mM sodium pyruvate, 3-5 mM ammonium chloride, 1.0-5.0 mM sodium phosphate, 0.2-0.4 mM sodium sulfate, 0.05-0.10 mM magnesium chloride, 0.8-1.2 mM potassium chloride and 0.8-1.2 μM ferric chloride, preferably including 100 mM sodium chloride, 30 mM triethanolamine, 25 mM sodium pyruvate, 5.0 mM ammonium chloride, 2.0 mM sodium phosphate, 0.25 mM sodium sulfate, 0.05 mM magnesium chloride, 1.0 mM potassium chloride and 1.0 μM ferric chloride. Among them, 80-120 mM sodium chloride means that the concentration of sodium chloride contained in the salt solution is 80-120 mmol / L.

[0025] In step B of preparing the bacterial solution, the method for collecting the bacterial cells in the material a can be the centrifugation method conventionally used in the art.

[0026] Wherein, the centrifugal force of the centrifugation may be conventional in the art, generally 4300-4500g. The time of the centrifugation may be conventional in the art, generally 10-15min. After the centrifugation operation, a cleaning operation may be performed according to the conventional procedures in the art. The purpose of the cleaning is to remove the supernatant components remaining in the precipitate after the centrifugation. The number of cleanings may be 2-3 times. The cleaning solution used in the cleaning may be the saline solution as described above.

[0027] In step (2) of some embodiments, the skin care ingredients may include at least one of whitening active ingredients, anti-aging active ingredients, anti-inflammatory active ingredients and skin repairing active ingredients conventionally used in the art.

[0028] In step (2) of some embodiments, the skin care component can be added in the form of a skin care component solution according to the conventional art, and the concentration of the skin care component in the skin care component solution can be 10-20 mg / mL. The solvent used in the skin care component solution can include water.

[0029] In step (2) of some embodiments, the tetrazolium violet may be added in the form of a tetrazolium violet aqueous solution according to the conventional art, and the mass percentage of the tetrazolium violet in the tetrazolium violet aqueous solution may be 0.2-1%.

[0030] In step (2) of some embodiments, the number of viable bacteria in the bacterial solution may be 10 6 ~10 11 CFU / mL, preferably 5×10 7 ~5×10 10 CFU / mL.

[0031] In step (2) of some embodiments, the number of live bacteria in the bacterial solution inoculated in 1 mg of the skin care component is 5×10 6 ~10 10 CFU, preferably 5×10 6 ~5×10 9 CFU.

[0032] In step (2) of some embodiments, the mass ratio of the skin care ingredient to the tetrazolium violet may be 1:(0.1-1), for example, 1:0.2.

[0033] In step (2) of some embodiments, the co-cultivation time may be 60 to 90 minutes.

[0034] In step (2) of some embodiments, the co-cultivation temperature may be room temperature.

[0035] The present application also provides a kit for screening a target skin care ingredient that maintains its original efficacy on the skin, the kit comprising a culture medium, a saline solution and tetrazolium violet.

[0036] In some embodiments, the salt solution includes the following components in concentration: 80-120 mM sodium chloride, 20-40 mM triethanolamine, 20-30 mM sodium pyruvate, 3-5 mM ammonium chloride, 1.0-5.0 mM sodium phosphate, 0.2-0.4 mM sodium sulfate, 0.05-0.10 mM magnesium chloride, 0.8-1.2 mM potassium chloride and 0.8-1.2 μM ferric chloride, preferably including 100 mM sodium chloride, 30 mM triethanolamine, 25 mM sodium pyruvate, 5.0 mM ammonium chloride, 2.0 mM sodium phosphate, 0.25 mM sodium sulfate, 0.05 mM magnesium chloride, 1.0 mM potassium chloride and 1.0 μM ferric chloride.

[0037] In some embodiments, the type and content of the culture medium may be conventional in the art, and generally can be sufficient for the growth and reproduction of the skin flora.

[0038] On the basis of conforming to the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present application.

[0039] The reagents and raw materials used in this application are commercially available.

[0040] The positive and progressive effects of the present application are: the present application provides a new idea for efficacy detection in the field of skin care products, and provides a method for screening skin care ingredients that can be quickly targeted at the target population and ensure that the original efficacy is maintained on the skin. The present application uses the skin care ingredients as the only nutrient to form a co-culture system with the skin flora, that is, the skin flora releases electrons during the biotransformation process, and tetrazolium violet captures the electrons released by the microorganisms and then undergoes a color reaction, so as to screen the skin care ingredients that can maintain the original efficacy on the skin. When the skin care ingredients are transformed more violently by the skin surface flora, the more electrons are released, the darker the color of the reaction system, the less the content of the original effective ingredients, the easier it is to change the original skin care efficacy of the skin care ingredients, and affect the skin care ingredients from exerting their original efficacy; when the color change of the system is not obvious or no color change occurs, it indicates that the skin care ingredients are not easily transformed by the skin surface flora, and the original effective ingredients of the skin care ingredients can take effect on the skin and maintain their original efficacy.

[0041] This application uses the evaluation results to provide more comprehensive quality management and supervision data for R&D personnel and consumer groups, provide technical data for product upgrades of manufacturers and operators, and provide guiding data for operators and consumers to choose better quality products. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0043] in:

[0044] Figure 1 The effect of PNS on the glutathione peroxidase content in Hacat cells under UVA stimulation;

[0045] Figure 2 The effect of PNS on the expression of COL-1 protein in human skin fibroblasts under UVA stimulation;

[0046] Figure 3 To observe the effect of PNS on the expression of Col-I in skin tissue sections under an optical microscope;

[0047] Figure 4 The effect of PNS on the activity of T-SOD in the homogenate of mouse back skin tissue;

[0048] Figure 5 The effect of PNS on CAT activity in mouse back skin tissue homogenate;

[0049] Figure 6 The effect of PNS on the activity of GSH-Px in the homogenate of mouse back skin tissue;

[0050] Figure 7 The effect of PNS on the MDA content in the homogenate of mouse back skin tissue;

[0051] Figure 8 It is the average growth rate diagram of bacteria in different concentrations of bacterial solution;

[0052] Fig. 9 It is the growth curve of bacteria in different concentrations of bacterial solution;

[0053] Fig.10 The following is a comparison chart of the color reaction change patterns of each sample reaction system. DETAILED DESCRIPTION

[0054] The present application is further described below by way of examples, but the present application is not limited to the scope of the examples. The experimental methods without specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product instructions.

[0055] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0056] Example 1 Verification of the anti-aging efficacy of Panax notoginseng saponins at the cellular level

[0057] (1) Panax notoginseng saponins can prevent Hacat cells from producing antioxidant stress response due to UVA stimulation

[0058] Hacat cells in good logarithmic growth phase were counted and inoculated into 10 cm culture dishes, with 5 × 10 cells per dish. 6 cells. After culturing overnight at 37°C and 5% CO2, the culture medium was discarded, and different concentrations of PNS (total saponins of Panax notoginseng) (1 mg / mL, 0.2 mg / mL) were added to the experimental group; VC with a concentration of 0.1 g / mL was added to the positive control group; serum-free DMEM culture medium was added to the model group and the blank control group; after addition, each group acted on the cells for 6 hours, and PBS buffer (pH = 7.4) was added until the cells were just covered. The radiation dose for the experimental group, positive control group and model group was 7 J / cm 2 The cells were stimulated with UVA for 2 hours, and the blank control group was not irradiated. Three parallels were made for each sample, and each parallel sample was tested three times. The cells were taken out and placed on ice, and washed twice with PBS. 800uL of lysis buffer was added to lyse the cells, and the cells were centrifuged at 12000r / min and 4℃ for 5min, and the supernatant was taken to obtain the cell lysis supernatant.

[0059] The ELISA kit provided by Nanjing Jiancheng Technology Co., Ltd. was used to test the content of glutathione peroxidase in the supernatant prepared above.

[0060] The data of the model group showed that after UV stimulation, Hacat cells produced an antioxidant stress response and the content of glutathione peroxidase increased (see the results). Figure 1 and Table 1); the results of the experimental group treated with 1 mg / mL and 0.2 mg / mL of notoginseng total saponins and the positive control group treated with VC were the same, that is, both notoginseng total saponins and VC can reduce the damage of UV to human skin fibroblasts.

[0061] Table 1

[0062]

[0063] (2) Effect of Panax notoginseng saponins on the expression of COL-Ⅰ in human skin fibroblasts

[0064] Human skin fibroblasts in good logarithmic growth phase were counted and inoculated into 6 cm culture dishes, with the number of cells per dish controlled at 1×10 5 Cells were cultured overnight at 37°C and 5% CO2, and the culture medium was discarded. Different concentrations of notoginseng saponin samples (1 mg / mL and 0.2 mg / mL) were added to the experimental group; VC with a concentration of 0.1 g / mL was added to the positive control group; serum-free DMEM culture medium was added to the model group and the blank control group; after addition, each group acted on the cells for 6 hours, and a small amount of PBS (pH = 7.4) was added to each group to just cover the cells; UVA was used to stimulate the cells in the experimental group, the positive control group and the model group, and the UV radiation was 7 J / cm 2, irradiated for 2 hours, and the blank control group was not irradiated. Each sample was tested three times, and each parallel sample was tested three times. The cells were taken out, placed on ice, and washed twice with PBS; 500uL of lysis buffer was added to lyse the cells, centrifuged at 12000g, 4℃ for 5min, and the supernatant was taken to obtain the cell lysis supernatant.

[0065] The expression of COL-Ⅰ was measured using the ELISA kit provided by Shanghai ELISA Biotechnology Co., Ltd. The OD value of each group was measured at 450nm. According to the standard curve (y = 0.7446x + 0.0876, R 2 =0.9922) to calculate the expression level of COL-Ⅰ A, and correct it with the protein content B to calculate C=A / B. The corrected results are shown in Table 2 and Figure 2 .

[0066] Table 2

[0067] serial number COL-1 content Blank control 0.034 UV stimulation model group 0.013 Positive control group 0.057 Experimental group with 1 mg / mL PNS 0.058 Experimental group with PNS 0.2 mg / mL 0.071

[0068] COL-Ⅰ (human type I collagen) is the main non-collagenous glycoprotein in the extracellular matrix and basement membrane. According to the experimental results of the model group and the blank control group, the COL-I content in the cells was significantly reduced after ultraviolet light stimulation, indicating that the cells were damaged after ultraviolet light stimulation. Compared with the model group, the expression of COL-Ⅰ in human skin fibroblasts was significantly increased after the experimental group was treated with Panax notoginseng total saponins. The expression of COL-Ⅰ was even higher than that of the blank control group. Figure 2 and Table 2, indicating that Panax notoginseng total saponins can not only reduce the damage of ultraviolet radiation to cells, but also induce human skin fibroblasts to produce more COL-Ⅰ.

[0069] Example 2 Verification of the anti-aging efficacy of Panax notoginseng saponins at the animal level

[0070] Existing technology has verified the anti-aging effect of Panax notoginseng total saponins at the animal level, and published an article "Gao Siyu, Huo Tong, Li Xuetao, et al. Study on the anti-aging effect of Panax notoginseng extract [J]. Daily Chemical Industry, 2020, 50(11): 6.". Some experiments and results are shown below:

[0071] Preparation of model mice: subcutaneous injection of D-galactose 1g / kg per day for 45 consecutive days;

[0072] A blank control group of mice was prepared; an equal volume of normal saline was injected subcutaneously into the mice every day for 45 consecutive days.

[0073] After the model was formed, the above model mice were randomly divided into 5 groups on average, with 6 mice in each group. The 5 groups were named model group, Vc positive control group, 1% Panax notoginseng ointment group, 2% Panax notoginseng ointment group, 4% Panax notoginseng ointment group (Panax notoginseng ointment is composed of matrix and Panax notoginseng total saponins; the matrix ointment includes: 3g glycerin, 0.3g Solagum Tara thorny gum, 8g octyldodecanol olive oil ester, 4g ButerRus compound oil, 5g Zeniveg emollient, 3g Montanov L emulsifier, 0.1g glyceryl caprylate and 76.6g water; taking the 1% Panax notoginseng ointment group as an example, it represents that the mass percentage of Panax notoginseng total saponins in Panax notoginseng ointment in the matrix ointment is 1%) and blank matrix group.

[0074] Before the experiment, the back area of ​​the aged mice was shaved with electric scissors (the area was 2cm×2cm), and the drug was applied the next day. It was applied twice a day for 50 consecutive days. During the experiment, the mice in each experimental group were free to eat and drink water. Among them, the blank control group and the model group mice were not treated in any way; the Vc positive control group was applied with a concentration of 1mg / mL of Vc; the 1% Panax notoginseng ointment group, the 2% Panax notoginseng ointment group, and the 4% Panax notoginseng ointment group were respectively applied with the corresponding concentration of Panax notoginseng ointment prepared by the matrix ointment and Panax notoginseng total saponins; the blank matrix group was applied with a matrix ointment without Panax notoginseng total saponins.

[0075] (1) 50 days after application, the treated skin tissue was taken, the skin tissue sections were routinely dewaxed, and then placed in a citric acid buffer solution, heated in a microwave oven for 5 minutes for antigen repair, and repeated heating 3 times. After addition, the sections were cooled at room temperature, washed with PBS for 5 minutes, and washed 3 times; 3% H2O2 was added to the system, and incubated at room temperature for 5 minutes to block endogenous peroxidase; COL-I antibody dilution was added respectively, and incubated at 4°C overnight. The next day, HRP-labeled secondary antibody dilution was added respectively, and incubated at room temperature for 1 hour. DAB staining was performed for 10 minutes, and hematoxylin counterstaining was performed for 15 minutes. The expression of Col-I in each group of sections was observed under an optical microscope. The results are shown in Figure 3 ( Figure 3 A represents the blank control group; B represents the model group; C represents the positive control group; D represents the blank matrix group; E represents the 1% Panax notoginseng ointment group; F represents the 2% Panax notoginseng ointment group; G represents the 4% Panax notoginseng ointment group).

[0076] The experimental results showed that compared with the blank control group, the Col-I content in the skin of the model group mice was significantly reduced, and the collagen content in the dermis, epidermis and subcutaneous tissue was significantly reduced. After treatment with 4% Panax notoginseng ointment and Vc application, the Col-I content increased significantly compared with the model group and was close to that of the blank control group. The Col-I content in the blank matrix group was not much different from that in the model group.

[0077] (2) After 50 days of application, the treated skin tissue was taken and the activity of T-SOD, CAT, GSH-Px and MDA content in the homogenate of the mouse back skin tissue were determined using a kit (see the results). Figures 4 to 7 and Table 3) to evaluate the changes in antioxidant capacity of each group of mice after receiving the test drugs. The specific steps were carried out according to the instructions.

[0078] Compared with the blank control group, the T-SOD activity of the model group was significantly reduced. Compared with the blank matrix group, the T-SOD activity values ​​of the 1% Panax notoginseng ointment group, 2% Panax notoginseng ointment group, 4% Panax notoginseng ointment group and Vc positive control group increased by 12.23%, 23.51%, 36.62% and 24.42%, respectively.

[0079] Compared with the blank control group, the CAT activity value of the model group decreased. Compared with the blank matrix group, the CAT activity values ​​of the 1% Panax notoginseng ointment group, the 4% Panax notoginseng ointment group and the Vc group increased by 7.4%, 20.39% and 17.80%, respectively.

[0080] Compared with the blank control group, the GSH-Px activity value of the model group decreased by 45.36%, and the difference was statistically significant (P<0.05). Compared with the blank matrix group, the GSH-Px activity values ​​of the 1% Panax notoginseng ointment group, 2% Panax notoginseng ointment group, 4% Panax notoginseng ointment group and Vc group increased by 45.08%, 54.92%, 68.77% and 87.45%, respectively. The Panax notoginseng ointment group had a good upregulation effect on GSH-Px activity, and it was dose-dependent.

[0081] Compared with the blank control group, the MDA content in the model group increased by 501.25%, and the difference was statistically significant (P<0.05). Compared with the blank matrix group, the MDA content in the 1% Panax notoginseng ointment group, 2% Panax notoginseng ointment group, 4% Panax notoginseng ointment group and Vc group decreased by 23.16%, 15.45%, 17.11% and 67.09%, respectively. The Panax notoginseng ointment group had a certain downward effect on the MDA content.

[0082] Table 3

[0083]

[0084] Example 3

[0085] According to the results of the above-mentioned cell (Example 1) and animal level experiments (Example 2), it was found that Panax notoginseng saponins have an antioxidant effect and can regulate the expression of Col-I, and have anti-aging effects. This experiment tests the anti-aging effect of Panax notoginseng saponins on the human body.

[0086] Test conditions and subject requirements: To ensure that each group obtains more than 30 valid data, a total of 30 female subjects aged 32-55 were recruited. The subjects had healthy and normal skin, initial eye wrinkle score 1-3, initial firmness R2 value ≤0.65, normal skin color, not too white, and facial skin met TEWL>16g / h / m 2 , the moisture content of the stratum corneum is <60 c.u. The test environment temperature is (22±1℃) and the humidity is 50%±5%.

[0087] The subject cleans the face with a facial cleanser and wipes the skin dry with dry facial tissue. Use cardboard to record the area of ​​the face to be tested. Before the formal test begins, sit quietly in the above environment for 30 minutes. During the test, you cannot drink water or go to the toilet.

[0088] A double-blind trial was conducted, with a dose of 2.0 ± 0.1 mg / cm 2 Apply 2% Panax notoginseng ointment evenly on the face for 33 consecutive days, then mark an area on the cheek with an area of ​​3cm*3cm. Test the basal elasticity of cheek skin wrinkles before use, on the 22nd day, and on the 33rd day after use, and use Visia 7 and Primos for image acquisition. Each index is tested 3 times with an interval of 5 minutes each time, and the average value is taken.

[0089] After using the samples for four weeks, the improvement rate of skin elasticity R2 values ​​of 30 subjects was as low as -13.10%, and the highest improvement rate was 129.64%, of which 20% of the subjects had an improvement of less than 5%. After using the samples for four weeks, the improvement rate of skin firmness F4 values ​​of the subjects ranged from -12.61% to 58.01%, and 14% of the subjects had an improvement of less than 5%.

[0090] In terms of wrinkle area, 37% of the subjects did not see improvement in the wrinkle area at the corners of the eyes, while 30% of the subjects did not see improvement in the wrinkle area under the eyes. 40% of the subjects did not see improvement in the Sa value of wrinkles at the corners of the eyes, while 40% of the subjects did not see improvement in the Sa value of wrinkles under the eyes.

[0091] In general, although the anti-aging effects of Panax notoginseng total saponins have been confirmed at the cellular level and in animal aging models, in human experiments, it does not have an improvement effect on all subjects, and there are large individual differences. In facial studies, it was found that there are also large individual differences in the skin microbiome. We speculate that the correlation between these differences may be due to the different degrees of biotransformation of the skin care ingredients of skin care products by skin microorganisms, resulting in individual differences in efficacy.

[0092] Example 4 Experiment on Metabolism of Panax Notoginseng Saponins by Skin Microflora

[0093] A reaction system was established, and three subjects who had unsatisfactory anti-aging effects after using Panax notoginseng total saponins were selected. The three subjects were referred to as A, B and C, respectively. The following method was used to obtain bacterial solutions of different concentrations containing skin flora of the three subjects.

[0094] A sterile cotton swab was soaked in nuclease-free water containing 50mM Tris, 1mM EDTA, and 0.5wt% Tween 20 for 30s, and then the face was sampled (whole face), and the sampling was performed 3 times. Then the cotton swab was immediately cut and immersed in the pre-prepared and sterilized TSB medium, and cultured at 30°C for 2 days (divided into 3 groups). After that, it was centrifuged at 4300g for 10min, the supernatant was discarded, and then it was suspended and washed with saline solution 3 times. Finally, the precipitate was evenly dispersed with the saline solution and configured to have a viable bacterial concentration of 5×10 7 , 5×10 8 , 5×10 10 CFU / mL of bacterial liquid containing skin flora. Taking subject A as an example, the above three bacterial liquids with different concentrations are named A-1, A-2 and A-3 respectively.

[0095] The types of components and concentrations of the components in the salt solution used are as follows: 100 mM sodium chloride, 30 mM triethanolamine, 25 mM sodium pyruvate, 5.0 mM ammonium chloride, 2.0 mM sodium phosphate, 0.25 mM sodium sulfate, 0.05 mM magnesium chloride, 1.0 mM potassium chloride and 1.0 μM ferric chloride, and the solvent is water.

[0096] A tetrazolium violet aqueous solution was prepared, wherein the mass percentage of the tetrazolium violet in the tetrazolium violet aqueous solution was 0.2%.

[0097] A Panax notoginseng total saponin aqueous solution was prepared, wherein the concentration of the Panax notoginseng total saponin in the Panax notoginseng total saponin aqueous solution was 10 mg / mL.

[0098] Experimental group: The bacterial suspension containing skin flora of the three subjects prepared above was mixed with equal volumes of tetrazolium violet aqueous solution and Panax notoginseng total saponin solution.

[0099] Positive control group: the bacterial suspension containing skin flora of the three subjects prepared above was mixed with equal volumes of TSB culture medium and tetrazolium violet aqueous solution.

[0100] The negative controls were "mixing equal volumes of Panax notoginseng total saponin aqueous solution and tetrazolium violet aqueous solution ( Fig.10 Notoginseng total saponins)", "Mix A-3 bacterial solution and tetrazolium violet aqueous solution in equal volumes ( Fig.10 Mix equal volumes of saline solution and tetrazolium violet aqueous solution ( Fig.10 salt solution)”, “mix equal volumes of TSB medium and tetrazolium violet aqueous solution ( Fig.10TSB)" and "mix equal volumes of TSB medium, saline solution and tetrazolium violet aqueous solution ( Fig.10 (referred to as salt solution + TSB)".

[0101] The order of sample addition was as follows: TSB medium & Panax notoginseng total saponin aqueous solution - tetrazolium violet aqueous solution - bacterial solution. After adding the sample, 200uL was immediately aspirated into a 96-well plate, and OD590 was measured at different time periods.

[0102] The microbial activity when the reaction system was established was evaluated by measuring the OD600 of the bacterial solution of subject A at 30 minutes. The results are shown in Figure 8 and Fig. 9 . Figure 8 It represents the average growth rate of bacteria in different concentrations of bacterial solution (A-1, A-2 and A-3). The results show that at high dilution multiples, the nutritional conditions are sufficient and the microbial growth rate is faster; Fig. 9 The growth curves of bacteria in different concentrations of bacterial solution (A-1, A-2 and A-3) show that when the reaction system was established, the microorganisms had not yet reached the decay stage. During the culture process, the nutrients were sufficient and the microorganisms grew well. At the same time, the average growth rate and growth curve of different concentrations of bacterial solution of subjects B and C were tested, and the results showed that the microorganisms grew well.

[0103] After the reaction system was established, the OD590 of each group of samples was tested at 0 min after mixing, 30 min after culture, and 60 min after culture. The results are shown in Table 4. The results show that with the change of time, the OD590 in each tube gradually increases.

[0104] according to Fig.10 From the results of a (OD590 of each group of samples after 60 min of culture) and Table 4, it can be seen that for the same subject, OD590 is positively correlated with the number of viable bacteria in the bacterial solution.

[0105] according to Fig.10 b (the first row is the color comparison diagram of the system at 0 min after mixing for each group, and the second row is the color comparison diagram of the system at 60 min after mixing for each group) and the results of Table 4 show that, except for the positive control, the color of the negative control group did not change. Due to differences in microbial abundance and species between different samples, there are differences in OD590, but the difference between the absorbance at 60 min and the initial absorbance of the samples of the three subjects tested is greater than 0.082. This method is used to conclude that Panax notoginseng total saponins are not recommended as the target skin care ingredient for the above three subjects. This conclusion is consistent with the facts. In the actual test process, the anti-aging effect of the three subjects after using Panax notoginseng total saponins was not ideal.

[0106] Table 4

[0107] serial number <![CDATA[OD 0min ]]> <![CDATA[OD 30min ]]> <![CDATA[OD 30min -OF 0min ]]> <![CDATA[OD 60min ]]> <![CDATA[OD 60min -OF 0min ]]> A-1 0.082 0.356 0.274 0.382 0.108 A-2 0.081 0.448 0.367 0.510 0.143 A-3 0.082 0.636 0.554 0.723 0.169 B-1 0.080 0.289 0.209 0.305 0.096 B-2 0.079 0.370 0.291 0.407 0.116 B-3 0.080 0.497 0.417 0.550 0.133 C-1 0.080 0.532 0.452 0.694 0.242 C-2 0.082 0.643 0.561 1.047 0.486 C-3 0.079 0.864 0.785 1.308 0.523 Positive Control 0.81 0.538 0.457 0.976 0.519 Total saponins of Panax notoginseng 0.044 0.044 0 0.046 0.046 TSB 0.051 0.051 0 0.051 0.051 TSB+ Saline Solution 0.051 0.051 0 0.051 0.051 Bacterial liquid 0.076 0.076 0 0.076 0.076 Saline solution 0.046 0.046 0 0.046 0.046

[0108] When the active ingredient is used as a single carbon source, it can be used by skin microorganisms to produce redox reactions and release electrons through the respiratory chain. This also confirms that skin microorganisms degrade the active ingredients to varying degrees, thereby affecting the efficacy to varying degrees. When the OD590 before and after the reaction does not change, that is, the difference in OD590 before and after the reaction is less than or equal to 0.082, it can be regarded that the reaction system has not changed in color and no electrons are generated to cause a color reaction of tetrazolium violet. When the difference in OD590 before and after the reaction is greater than 0.082, it indicates that a metabolic reaction has occurred, and electrons are generated to cause a color change in tetrazolium violet. The darker the color, the more complete the reaction and the more substrates are reacted.

[0109] Finally, it should be noted that in this application, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements that are not explicitly listed, or also includes elements that are inherent to such process, method, article or apparatus.

[0110] Although the present application has been disclosed above through the description of the specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the attached scheme. These modifications, improvements or equivalents should also be considered to be included in the scope of protection claimed in the present application.

Claims

1. A use of tetrazolium violet, characterized in that, Used for screening skin care ingredients that maintain their original efficacy on the skin; or used for preparing a reagent or a kit, wherein the reagent or the kit is used for screening skin care ingredients that maintain their original efficacy on the skin.

2. A recommended skin care method based on tetrazolium violet color development, characterized in that: The method comprises the following steps: (1) collecting skin samples and preparing a bacterial solution containing skin flora; (2) co-culturing the skin care ingredient to be screened, tetrazolium violet and the bacterial solution prepared in step (1); (3) judging whether the skin care ingredient is a target skin care ingredient that maintains its original efficacy based on the difference in the degree of color change and / or absorbance value of the system after the co-cultivation and before the co-cultivation.

3. The recommended skin care method based on tetrazolium violet color development as claimed in claim 2, characterized in that: In step (3), when the difference in absorbance values ​​of the system after the co-cultivation and before the co-cultivation is less than or equal to 0.082, the skin care ingredient is recommended as a target skin care ingredient that maintains the original efficacy; when the difference in absorbance values ​​of the system after the co-cultivation and before the co-cultivation is greater than 0.082, the skin care ingredient is not recommended as a target skin care ingredient that maintains the original efficacy; Alternatively, in step (3), when the color change of the system after the co-culture and before the co-culture is significant, the skin care ingredient is recommended as the target skin care ingredient that maintains the original efficacy; when the color change of the system after the co-culture and before the co-culture is not significant, the skin care ingredient is not recommended as the target skin care ingredient that maintains the original efficacy.

4. The recommended skin care method based on tetrazolium violet color development according to claim 2, characterized in that: The skin care recommendation method includes recommending skin care products or cosmetics, configuring skin care products or cosmetics based on the recommended target skin care ingredients to obtain target skin care products or target cosmetics.

5. The recommended skin care method based on tetrazolium violet color development according to any one of claims 2 to 4, characterized in that: In step (1), the skin sample is derived from the whole body skin or facial skin, preferably from at least one of the cheek, nose wing and forehead.

6. The recommended skin care method based on tetrazolium violet color development according to any one of claims 2 to 4, characterized in that: In step (1), the method for preparing the bacterial solution comprises the following steps: A: Samples were collected from the skin using a sterile cotton swab soaked in nuclease-free water, and transferred to a culture medium for shaking culture to obtain material a; B; collecting the bacterial cells in the material a and mixing them with a salt solution to obtain the bacterial solution.

7. The recommended skin care method based on tetrazolium violet color development according to claim 6, characterized in that: The method for preparing the bacterial solution satisfies at least one of the following conditions: In step A of preparing the bacterial solution, the nuclease-free water comprises the following components at the following concentrations: 40-50 mM tris(hydroxymethyl)aminomethane, 1-1.5 mM EDTA and 0.5 wt%-1 wt% Tween 20; In step A of preparing the bacterial solution, the culture medium includes TBS culture medium; In step A of preparing the bacterial solution, the shaking culture time is 1 to 3 days; In step A of preparing the bacterial solution, the temperature of the shaking culture is 30-37° C.; In step B of preparing the bacterial solution, the salt solution includes the following components in the following concentrations: 80-120 mM sodium chloride, 20-40 mM triethanolamine, 20-30 mM sodium pyruvate, 3-5 mM ammonium chloride, 1.0-5.0 mM sodium phosphate, 0.2-0.4 mM sodium sulfate, 0.05-0.10 mM magnesium chloride, 0.8-1.2 mM potassium chloride and 0.8-1.2 μM ferric chloride; In step B of preparing the bacterial solution, the method for collecting the bacterial cells in the material a is centrifugation.

8. The recommended skin care method based on tetrazolium violet color development according to claim 7, characterized in that: The method for preparing the bacterial solution satisfies at least one of the following conditions: In step B of preparing the bacterial solution, the salt solution includes the following components in the following concentrations: 100 mM sodium chloride, 30 mM triethanolamine, 25 mM sodium pyruvate, 5.0 mM ammonium chloride, 2.0 mM sodium phosphate, 0.25 mM sodium sulfate, 0.05 mM magnesium chloride, 1.0 mM potassium chloride and 1.0 μM ferric chloride; In step B of preparing the bacterial solution, when the centrifugal method is used to collect the bacteria in the material a, the centrifugal force of the centrifugation is 4300-4500g; the centrifugation time is 10-15min; and the centrifugation operation also includes a washing operation.

9. The recommended skin care method based on tetrazolium violet color development according to any one of claims 2 to 4, characterized in that: The recommended skin care method meets at least one of the following conditions: In step (2), the skin care ingredients include at least one of whitening active ingredients, anti-aging active ingredients, anti-inflammatory active ingredients and skin repairing active ingredients; In step (2), the skin care ingredient is added in the form of a skin care ingredient solution, and the concentration of the skin care ingredient in the skin care ingredient solution is 10 to 20 mg / mL; In step (2), the skin care ingredient is added in the form of a skin care ingredient solution, and the solvent used in the skin care ingredient solution includes water; In step (2), the tetrazolium violet is added in the form of a tetrazolium violet aqueous solution, and the mass percentage of the tetrazolium violet in the tetrazolium violet aqueous solution is 0.2% to 1%; In step (2), the number of viable bacteria in the bacterial solution is 10 6 ~10 11 CFU / mL, preferably 5×10 7 ~5×10 10 CFU / mL; In step (2), the number of live bacteria in the bacterial solution inoculated in 1 mg of the skin care component is 5×10 6 ~10 10 CFU, preferably 5×10 6 ~5×10 9 CFU; In step (2), the mass ratio of the skin care component to the tetrazolium violet is 1:(0.1-1); In step (2), the co-cultivation time is 60 to 90 minutes.

10. A kit for screening a target skin care ingredient that maintains its original efficacy on the skin, the kit comprising a culture medium, a saline solution and tetrazolium violet.

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