Method for evaluating nerve beautifying effects of cosmetic raw materials and finished products in vitro

Abstract

The invention provides a method for evaluating nerve beautifying effects of cosmetic raw materials and finished products in vitro, and belongs to the field of cosmetic detection. The invention relates to a method for evaluating the nerve beautifying effect of a cosmetic raw material and a finished product in vitro, which utilizes human keratinocytes to evaluate the nerve beautifying activity of the cosmetic raw material and the finished product, and comprehensively evaluates and screens the nerve beautifying activity of the target raw material and the finished product in combination with the detection results of three cell models. At least one index of inhibiting cortisol generation, promoting beta-enorphin secretion and blocking TRPV1 achieves an obvious effect, and the nerve beautifying effect can be declared. The method systematically evaluates the nerve beauty activity of the target raw material and the finished product by comprehensively analyzing the test result, belongs to a systematic evaluation method, has the technical advantages of simple and comprehensive operation, rapidness, high efficiency and accuracy, and can provide a basis for research, development, compounding and screening of nerve beauty cosmetics.

Description

Technical Field

[0001] The present invention belongs to the field of cosmetics testing, and in particular relates to a method for in vitro evaluation of the neurocosmetic efficacy of cosmetic raw materials and finished products. Background Art

[0002] As the skin care mentality shifts from the outside to the inside, giving people a "feeling of pleasure and happiness" has gradually become a popular element in the cosmetics industry. In the past few years, the labels of "emotion" and "immersive" have gradually become traffic codes, and "emotional skin care" has come into being. Emotional skin care improves skin condition by regulating emotions (visual, tactile, olfactory, taste, etc.). In fact, it is more like a "consumer concept." The exquisite packaging, fragrance, soft touch, etc. of cosmetics can provide a pleasant sensory experience, and this ability to regulate emotions can easily be confused with "neuro beauty." Although there is an essential difference between "emotional skin care" and the efficacy of cosmetics, it is undeniable that people are increasingly pursuing the "emotional value" carried by cosmetics, and "neuro beauty" can do this more accurately.

[0003] The skin is an extension of the nervous system, and many skin problems are closely related to the nervous system. Therefore, in recent years, the skin's peripheral nervous system has gradually developed into a new target for anti-aging, and many neurocosmetic cosmetics have been derived from it. As a new skin care mechanism that can affect the skin-brain connection, neurocosmetics is to show the activity of the skin's nervous system by acting on the neurotransmitters of the skin, and ultimately achieve skin care effects. The main mechanism of neurocosmetics is: acting directly on nerve endings as a regulator of neurotransmitter release; acting on non-neuronal cells as an agonist or inhibitor of neuropeptide receptors and a regulator of neurotransmitter effects. Currently, neurocosmetic cosmetics on the market mainly claim to fight skin stress, inhibit inflammation, and relieve skin sensitivity to exert skin care effects. Among them, cortisol and β-endorphin, two neurotransmitters, are often used as targets of neurocosmetic cosmetics. In addition, as a skin pain and itch receptor that can mediate neural activity, TRPV1 is also one of the important targets of neurocosmetic active raw materials.

[0004] At present, there are few studies on neurocosmetology at home and abroad, and there is no systematic detection method for neurocosmetology effects and mechanisms of action. The evaluation of the efficacy of neurocosmetology cosmetics is obviously not comprehensive and objective enough. Patent document CN202110597570.3 discloses a method for evaluating neurocosmetology effects based on the human body, which evaluates the neurocosmetology effects of skin topical agents by detecting the brain waves, heart rate, etc. of the subjects, but this method has high requirements for personnel and equipment, and is not suitable for the mechanism research and efficient screening of the preliminary work of cosmetic raw material research and development. At present, in vitro alternative experiments have gradually become the mainstream method for evaluating the efficacy of cosmetics. Among them, cell models are relatively more controllable, and the experimental cycle is short and the cost is low. They can combine a variety of technical means to obtain more microscopic data, and have certain research significance in exploring the mechanism of action. Therefore, it has become an excellent experimental model in in vitro alternative experiments. Therefore, constructing an in vitro cell evaluation model helps to realize the development and high-throughput screening of neurocosmetology active raw materials and neurocosmetics more economically and efficiently.

[0005] Therefore, in order to scientifically understand the connection between nerves and skin, more accurately evaluate the skin care effects of cosmetic raw materials and finished products, standardize the neurocosmetic cosmetics market, and promote the progress of the cosmetics industry, it is necessary to establish an in vitro method that can systematically evaluate the neurocosmetic effects of cosmetics. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to propose an in vitro method for evaluating the neurocosmetic efficacy of cosmetic raw materials and finished products, so as to fill the gap in the in vitro laboratory evaluation method of the neurocosmetic efficacy of cosmetic raw materials, and to construct three cell models for evaluating the effects of combating skin stress and relieving nerve sensitivity according to the different mechanisms of neurocosmetic action. The neurocosmetic activity of the target raw materials and finished products is systematically evaluated by comprehensively analyzing the test results, which has the technical advantages of being easy to operate, comprehensive, fast, efficient and accurate, and can provide a basis for the research and development, compounding and screening of neurocosmetic cosmetics.

[0007] In order to solve the above technical problems, the technical solution adopted by the present invention is: a method for evaluating the neurocosmetic efficacy of cosmetic raw materials and finished products in vitro, comprising the following steps:

[0008] S1, evaluating the solubility of the sample to be tested and determining the dissolution scheme, wherein the sample to be tested includes cosmetic raw materials and cosmetic finished products;

[0009] S2, selected cells, said cells comprising human keratinocytes;

[0010] S3, culturing cells in the logarithmic growth phase, using CCK-8 reagent or MTT reagent to determine the toxicity of the test samples at different concentrations to the cells, and screening out the concentration of the test sample corresponding to the cell viability ≥ 90% as the dosing concentration;

[0011] S4, establishing a cortisone-induced cell cortisol secretion model, and detecting the cortisol secretion level under the administration concentration of the test sample;

[0012] S5, establishing a cell β-endorphin secretion model, and detecting the β-endorphin secretion level under the administration concentration of the test sample;

[0013] S6, establishing a capsaicin-stimulated cell TRPV1 model, and detecting the level of cellular calcium influx at the concentration of the sample to be tested;

[0014] The above steps S4, S5 and S6 are at least one optional step.

[0015] Optionally, the establishment of a cortisone-induced cellular cortisol secretion model comprises:

[0016] Cells in the logarithmic growth phase were cultured;

[0017] A blank control group, a negative control group, a positive control group and a sample group were set up, wherein the blank control group was added with a cell culture medium, the negative control group was added with a cell culture medium containing cortisone, the positive control group was added with a cell culture medium containing cortisone and the positive substance PF-915275, and the sample group was added with a cell culture medium containing cortisone and a sample of a drug concentration to be tested, and the culture was continued;

[0018] After the culture was completed, the cell supernatant was collected and the cortisol secretion level was detected.

[0019] Optionally, the establishment of a cell β-endorphin secretion model comprises:

[0020] Cells in the logarithmic growth phase were cultured;

[0021] A negative control group, a positive control group and a sample group are set, wherein the negative control group is added with a cell culture medium, the positive control group is added with a cell culture medium containing the positive substance dibutyryl cAMP sodium salt, and the sample group is added with a cell culture medium containing the sample to be tested at the administration concentration, and the culture is continued;

[0022] After the culture was completed, the cell supernatant was collected and the β-endorphin secretion level was detected.

[0023] Optionally, the establishment of a capsaicin-stimulated TRPV1 cell model comprises:

[0024] Cells in the logarithmic growth phase were cultured;

[0025] Loading cells with fluorescent probes;

[0026] A blank control group, a negative control group, a positive control group and a sample group were set up respectively, wherein the negative control group, the positive control group and the sample group used capsaicin to activate the cell TRPV1 channel to induce calcium ion influx, a balanced salt buffer was added to the blank control group, a balanced salt buffer containing capsaicin was added to the negative control group, a balanced salt buffer containing capsaicin and the positive substance capsaicin was added to the positive control group, and a balanced salt buffer containing capsaicin and the sample of the drug concentration to be tested was added to the sample group, and incubated;

[0027] After the incubation, the levels of calcium ion influx into the cells were detected.

[0028] Optionally, when the sample to be tested is a water-soluble sample, it is directly dissolved in a culture medium for subsequent experiments;

[0029] When the sample to be tested is an oil-soluble sample, dimethyl sulfoxide is used to dissolve it and then added to the culture medium for subsequent experiments. The sample to be tested cannot form a precipitate in the culture medium to interfere with the detection;

[0030] When dimethyl sulfoxide is used as a solvent, the volume percentage concentration of the solvent shall not exceed 1% of the final culture volume.

[0031] Optionally, the cells in the logarithmic growth phase are cultured in a complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37±0.5°C and (5±1)% CO. 2 concentration, saturation humidity;

[0032] The cultured cells were divided into (1.0-2.0)×10 5 The cells were inoculated at a density of 1.5 cells / mL in a 96-well plate, with 100uL of culture medium in each well, and 3 or more replicate wells were set up in each group.

[0033] Optionally, the step of loading the cells with the fluorescent probe comprises: washing the cells 2-3 times with a balanced salt buffer, incubating the cells in a cell culture medium containing the calcium ion probe, and then washing the cells 2-3 times with a balanced salt buffer after the incubation, wherein the balanced salt buffer comprises a calcium ion probe. 2+ Hanks balanced salt solution.

[0034] Optionally, the cortisol secretion level is detected by an ELISA kit and expressed as a relative cortisol content;

[0035] The cortisone-induced cell cortisol secretion model must satisfy that the relative content of cortisol in the negative control group is 10% or more higher than that in the blank control group, and the relative content of cortisol in the positive control group is 10% or more lower than that in the negative control group.

[0036] Optionally, the β-endorphin secretion level is detected by an ELISA kit and expressed as a relative content of β-endorphin;

[0037] The cell β-endorphin secretion model needs to satisfy that the relative content of β-endorphin in the positive control group is 10% or more higher than that in the negative control group.

[0038] Optionally, the cellular calcium ion influx level is detected on a fluorescence microplate reader after staining with Fluo-3AM probe and expressed as relative fluorescence intensity;

[0039] The capsaicin-stimulated cell TRPV1 model must satisfy that the relative fluorescence intensity of the negative control group is 10% or more higher than that of the blank control group, and the relative fluorescence intensity of the positive control group is 10% or more lower than that of the negative control group.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The introduction of the concept of neurobeauty has promoted people's understanding of the skin and is beneficial to more accurate and effective skin care. However, there are few related research reports at home and abroad, and they are more limited to market promotion, and lack a systematic evaluation method. Compared with the prior art, the present invention fills the gap in the in vitro laboratory evaluation method of the neurobeauty efficacy of cosmetic raw materials, innovatively combines the different action pathways of neurobeauty, and provides an in vitro method for systematically evaluating the neurobeauty efficacy of cosmetic raw materials and finished products. According to the different action mechanisms of neurobeauty, three cell models for evaluating the effects of combating skin stress and relieving nerve sensitivity were constructed respectively. The neurobeauty activity of the target raw materials and finished products is systematically evaluated by comprehensive analysis of the test results. It is a systematic evaluation method with the technical advantages of simple operation, comprehensiveness, rapidity, efficiency, and accuracy, and can provide a basis for the research and development, compounding and screening of neurobeauty cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is a schematic diagram of the relative content of cortisol in the supernatant of each group in the cortisone-induced human keratinocyte cortisol secretion model of Example 1;

[0043] Figure 2 This is a schematic diagram of the relative content of β-endorphin in the supernatant of each group in the human keratinocyte β-endorphin secretion model of Example 2;

[0044] Figure 3 This is a schematic diagram of the relative fluorescence intensity of calcium ions in each group in the capsaicin-stimulated human keratinocyte TRPV1 model in Example 3;

[0045] Figure 4The present invention is a flow chart of the method for in vitro evaluation of the neurocosmetic efficacy of cosmetic raw materials and finished products. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the technical solution of the present invention, Figure 1-4 The specific implementation methods are described in detail. It should be understood that the following examples are only for illustrative purposes and explanation of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope of protection of the present invention.

[0047] In the following examples, all results are expressed as Mean ± SD; One-way ANOVA statistical analysis was used for comparison among groups; all statistical analyses were two-tailed; p < 0.05 was considered to be significantly different, and “ * "or" # "; p < 0.01 is considered to be extremely significant, and " ** "or" ## "express.

[0048] Example 1: Evaluation of emotional skin care activity using a cortisone-induced human keratinocyte model

[0049] 1. Experimental Materials and Instruments

[0050] 1.1 Cell lines

[0051] In this example, human keratinocytes Hacat were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0052] 1.2 Main Reagents

[0053] DMEM medium (Dulbecco's Modified Eagle Medium), fetal bovine serum, penicillin-streptomycin, and PBS (Phosphate Buffered Saline) were purchased from Gibco, USA; CCK-8 reagent was purchased from Shanghai Biotech Biotechnology Co., Ltd.; human cortisol ELISA kit was purchased from Shanghai Sangon Bioengineering Technology Service Co., Ltd.; cortisone was purchased from Shanghai McLean Biochemical Technology Co., Ltd.; PF-915275 was purchased from MCE, USA.

[0054] 1.3 Instruments and Equipment

[0055] Biological Safety Cabinet, CO 2 Incubator (Thermo, USA), inverted microscope, centrifuge, constant temperature incubator, multifunctional microplate reader.

[0056] 2. Test samples

[0057] The test samples were active raw material 1 (vetiver extract) and finished product 2 (essence containing vetiver extract).

[0058] 3. Cell Culture

[0059] Hacat cells were cultured in complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C and 5% CO 2 and saturated humidity CO 2 Culture in an incubator until the cell confluence reaches 70%-80% and then pass the cells. Remove the old culture medium, wash once with PBS, add trypsin for digestion, and then terminate the reaction with complete culture medium. Gently blow and collect the cells with a pipette tip and pass them at a ratio of 1:3 or 1:6.

[0060] 4. Cytotoxicity Test

[0061] The cells in the logarithmic growth phase were collected and diluted with complete medium to a cell density of 1.5 × 10 5 cells / mL and inoculated into a 96-well cell culture plate, 100 μL per well. The culture plate was placed at 37°C and 5% CO 2 and saturated humidity CO 2 Culture in an incubator. After 24 hours, remove the old culture medium and add different concentrations of the sample to be tested. After 24±2 hours of culture, add CCK-8, detect the absorbance OD of the control group, blank group and sample group at a wavelength of 450nm, and calculate the cell viability according to the following formula:

[0062] Cell viability (%) = (OD 样品组 -OD 空白组 ) / (OD 对照组 -OD 空白组 )×100%.

[0063] The sample group contained sample-treated cells, culture medium, and CCK-8, the control group contained cells, culture medium, and CCK-8, and the blank group contained no cells but only culture medium and CCK-8.

[0064] The Hacat cytotoxicity test results of each sample are shown in Table 1.

[0065] Table 1 Hacat cytotoxicity test results of each sample

[0066]

[0067] Referring to the data results in Table 1 above, it can be considered that raw material 1 at a concentration of 1% or less and finished product 2 at a concentration of 2% or less will not inhibit the viability of Hacat cells. Therefore, raw material 1 selects a concentration of 1% as the dosing concentration for cell model detection, and finished product 2 selects a concentration of 2% as the dosing concentration for cell model detection.

[0068] 5. Detection of Cortisol Content in Supernatant of Cortisone-Induced Human Keratinocyte Model

[0069] Based on the cytotoxicity results, 1% of raw material 1 and 2% of finished product 2 were selected to test cortisone-induced cortisol secretion in the human keratinocyte model.

[0070] The cells in the logarithmic growth phase were collected and diluted with complete medium to a cell density of 1.5 × 10 5 cells / mL and inoculated into a 96-well cell culture plate, 100 μL per well. The culture plate was placed at 37°C and 5% CO 2 and saturated humidity CO 2 Culture in an incubator. After 24 hours, the old culture medium was removed and a blank control group, a negative control group, a positive control group and a sample group were set up. The blank control group only added fresh culture medium, the negative control group added fresh culture medium containing cortisone, the positive control group added fresh culture medium containing cortisone and the positive substance PF-915275, and the sample group added fresh culture medium containing cortisone and the drug concentration sample, 100 μL per well. Each experimental group was set up with 3 replicate wells and incubated at 37°C and 5% CO. 2 and saturated humidity CO 2 The cells were cultured in an incubator for 24 hours. After the culture, the cell supernatant was collected to detect the cortisol secretion level of each group. The specific test steps refer to the kit instructions.

[0071] The corresponding standard curve was drawn, and the OD value of the test sample was substituted into the standard curve to calculate the content of cortisol. The blank control group was taken as 100%, and the relative content of cortisol in each group was calculated.

[0072] Relative content of cortisol (%) = content of cortisol in the test group / content of cortisol in the blank control group × 100%.

[0073] 6. Results Analysis

[0074] The effects of the samples on cortisol secretion in the cortisone-induced human keratinocyte model are shown in Table 2 and Figure 1 shown.

[0075] Table 2 Effects of samples on cortisol secretion in cortisone-induced human keratinocyte model

[0076] Experimental Grouping Experimental treatment Cortisol relative content (%) Cortisol suppression rate (%) Blank control - 100.0±9.0 - Negative control Add cortisone <![CDATA[255.3±5.2 ## ]]> - Positive Control Add cortisone and PF-915275 <![CDATA[200.8±5.0 * ]]> 21.4±2.0 Raw material 1 Add cortisone and 1% raw material 1 <![CDATA[186.0±13.5 * ]]> 27.2±5.3 Finished product 2 Add cortisone and 2% finished product 2 <![CDATA[220.5±9.05.9 * ]]> 13.7±2.3

[0077] Note: * p<0.05, ** p < 0.01 for comparison between sample group or positive control group and negative control group; # p<0.05, ## p<0.01 compared the negative control group with the blank control group.

[0078] Compared with the blank control group, the cortisol level in the negative control group increased by more than 10%; compared with the negative control group, the cortisol level in the positive control group decreased by more than 10%, indicating that the detection model is effective and the test results are reliable.

[0079] Compared with the negative control group, the treatments of 1% raw material 1 and 2% finished product 2 can significantly inhibit the secretion of cortisol induced by cortisone, which indicates that both 1% raw material 1 and 2% finished product 2 can relieve skin stress by reducing the release of cortisol and achieve neurobeauty effects.

[0080] Example 2: Evaluation of emotional skin care activity using a human keratinocyte β-endorphin secretion model

[0081] 1.1 Cell lines

[0082] In this example, human keratinocytes Hacat were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0083] 1.2 Main Reagents

[0084] DMEM culture medium, fetal bovine serum, penicillin-streptomycin, and PBS were purchased from Gibco, USA; human β-endorphin ELISA kit was purchased from Shanghai Jianglai Biotechnology Co., Ltd.; dibutyryl-cAMP sodium salt was purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0085] 1.3 Instruments and Equipment

[0086] Biological Safety Cabinet, CO 2 Incubator (Thermo, USA), inverted microscope, centrifuge, constant temperature incubator, multifunctional microplate reader

[0087] 2. Test samples

[0088] The test samples were active raw material 1 (vetiver extract) and finished product 2 (essence containing vetiver extract).

[0089] 3. Detection of β-endorphin secretion level in human keratinocytes

[0090] According to the cytotoxicity results in Example 1, 1% of raw material 1 and 2% of finished product 2 were selected to test the secretion of β-endorphin in human keratinocytes.

[0091] The cells in the logarithmic growth phase were collected and diluted with complete medium to a cell density of 1.5 × 10 5 cells / mL and inoculated into a 96-well cell culture plate, 100 μL per well. The culture plate was placed at 37°C and 5% CO 2 and saturated humidity CO 2 Culture in an incubator. After 24 hours, remove the old culture medium and set up a negative control group, a positive control group and a sample group. Add fresh culture medium to the negative control group, add fresh culture medium containing the positive substance dibutyryl cAMP sodium salt to the positive control group, and add fresh culture medium containing the sample of the drug concentration to the sample group, and continue to culture for 24 hours. After the culture is completed, collect the cell supernatant to detect the secretion level of β-endorphin in each group. For specific test steps, refer to the instructions of the kit.

[0092] The corresponding standard curve was drawn, and the OD value of the test sample was substituted into the standard curve to calculate the content of β-endorphin, and the relative content of β-endorphin in each group was calculated with the blank control group as 100%.

[0093] Relative content of β-endorphin (%) = content of β-endorphin in the test group / content of β-endorphin in the blank control group×100%.

[0094] 4. Results Analysis

[0095] The effects of the samples on β-endorphin secretion in the human keratinocyte model are shown in Table 3 and Figure 2 shown.

[0096] Table 3 Effects of samples on β-endorphin secretion in human keratinocyte model

[0097]

[0098]

[0099] Note: * p<0.05, ** p<0.01 was used to compare the sample group or positive control group with the negative control group.

[0100] Compared with the negative control group, the β-endorphin level in the positive control group was increased by more than 10%, indicating that the detection model is effective and the test results are reliable.

[0101] Compared with the negative control group, the treatments of 1% raw material 1 and 2% finished product 2 can significantly promote the secretion of β-endorphin, which indicates that both 1% raw material 1 and 2% finished product 2 can increase the release of β-endorphin and promote nerve relaxation, thereby achieving a neurobeauty effect.

[0102] Example 3: Evaluation of the activity of capsaicin in relieving neurosensitivity by stimulating human keratinocyte TRPV1 model

[0103] 1.1 Cell lines

[0104] In this example, human keratinocytes Hacat were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0105] 1.2 Main Reagents

[0106] DMEM culture medium, fetal bovine serum, penicillin-streptomycin, and PBS were purchased from Gibco, USA; calcium ion detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.; capsaicin was purchased from Beijing Solebow Technology Co., Ltd.; and capsaicin was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0107] 1.3 Instruments and Equipment

[0108] Biological Safety Cabinet, CO 2 Incubator (Thermo, USA), inverted microscope, centrifuge, constant temperature incubator, multifunctional microplate reader

[0109] 2. Test samples

[0110] The test samples were active raw material 1 (vetiver extract) and finished product 2 (essence containing vetiver extract).

[0111] 3. Calcium ion detection in human keratinocytes

[0112] According to the cytotoxicity results in Example 1, 1% of raw material 1 and 2% of finished product 2 were selected to test the calcium ion influx in human keratinocytes.

[0113] The cells in the logarithmic growth phase were collected and diluted with complete medium to a cell density of 1.5 × 10 5 cells / mL and inoculated into a 96-well cell culture plate, 100 μL per well. The culture plate was placed at 37°C and 5% CO 2 and saturated humidity CO 2Cultured in an incubator. After 24 hours, the old culture medium was removed, washed 2-3 times with Hanks buffer, and the cells were incubated in fresh culture medium containing 0.5-5μM Fluo3AM for 30 minutes. After the incubation, they were washed 2-3 times with Hanks buffer. A blank control group, a negative control group, a positive control group and a sample group were set up respectively, among which the negative control group, the positive control group and the sample group all used capsaicin to activate the cell TRPV1 channel to induce calcium ion influx. Among them, the blank control group was added with Hanks buffer, the negative control group was added with Hanks buffer containing capsaicin, the positive control group was added with Hanks buffer containing capsaicin and the positive substance capsaicin, and the sample group was added with Hanks buffer containing capsaicin and the dosing concentration sample, and incubated for 30 minutes. After the incubation, the fluorescence intensity was detected with a multifunctional microplate reader, the excitation wavelength was 488nm, and the emission wavelength was 525nm. The relative fluorescence intensity of each group was calculated with the blank control group as 100%.

[0114] Relative fluorescence intensity (%) = fluorescence intensity of test group / fluorescence intensity of blank control group × 100%.

[0115] 4. Results Analysis

[0116] The effects of the samples on calcium influx activity in the human keratinocyte model are shown in Table 4 and Figure 3 shown.

[0117] Table 4 Effects of samples on calcium influx in human keratinocyte model

[0118] Experimental Grouping Experimental treatment Relative fluorescence intensity (%) Fluorescence inhibition rate (%) Blank control - 100.0±4.5 - Negative control Add capsaicin <![CDATA[116.0±0.8 ## ]]> - Positive Control Add capsaicin and capsaicin <![CDATA[92.4±6.4 ** ]]> 20.3±5.5 Raw material 1 Add capsaicin and 1% raw material 1 <![CDATA[79.1±2.6 ** ]]> 31.7±2.3 Finished product 2 Add capsaicin and 2% finished product 2 <![CDATA[99.4±3.1 ** ]]> 14.3±2.7

[0119] Note: * p<0.05, ** p < 0.01 for comparison between sample group or positive control group and negative control group; # p<0.05, ## p<0.01 compared the negative control group with the blank control group.

[0120] Compared with the blank control group, the fluorescence intensity of the negative control group increased by more than 10%; compared with the negative control group, the fluorescence intensity of the positive control group decreased by more than 10%, indicating that the detection model is effective and the test results are reliable.

[0121] Compared with the negative control group, the treatment with 1% raw material 1 and 2% finished product 2 can significantly reduce the calcium ion influx induced by capsaicin, which indicates that both 1% raw material 1 and 2% finished product 2 can achieve neurobeauty effect by blocking TRPV1-mediated calcium ion influx, inhibiting nerve hyperreaction and alleviating skin sensitivity.

[0122] In summary, raw material 1 and finished product 2 can reduce the release of stress hormone cortisol, promote the secretion of β-endorphin with nerve relaxation ability, inhibit calcium ion influx caused by TRPV1 activation, and avoid excessive nerve excitement. This shows that raw material 1 and finished product 2 have neurocosmetic activity to combat skin stress and relieve skin sensitivity, and can be used in the compound development of neurocosmetics.

[0123] like Figure 4 As shown, the present invention provides an in vitro method for evaluating the neurocosmetic efficacy of cosmetic raw materials and finished products, which utilizes human skin keratinocytes to evaluate the neurocosmetic activity of cosmetic raw materials and finished products, and combines the detection results of three cell models to comprehensively evaluate and screen the neurocosmetic activity of target raw materials and finished products. Neurocosmetic efficacy can only be claimed when at least one of the indicators of inhibiting cortisol production, promoting β-endorphin secretion and blocking TRPV1 achieves a significant effect.

[0124] The neurocosmetic efficacy evaluation method of cosmetic raw materials and finished products constructed based on three cell models in the present invention is a systematic evaluation method, which has the technical advantages of being easy to operate, comprehensive, fast, efficient and accurate. It fills the gap in the existing in vitro laboratory evaluation method for the neurocosmetic efficacy of cosmetic raw materials, provides a fast and reliable evaluation method for the preliminary verification and screening of neurocosmetic active raw materials in the research and development of neurocosmetic cosmetics, provides a reference basis for the compounding scheme of neurocosmetic active substances, and provides an effective basis for further research on animal replacement experiments, skin care product development and efficacy verification in the skin care industry.

[0125] Those skilled in the art should recognize that the above embodiments are only used to illustrate the present invention, and are not intended to limit the present invention. As long as they are within the spirit of the present invention, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included in the scope of the claims of the present invention.

Claims

1. A method for in vitro evaluation of the neurobeautic efficacy of cosmetic raw materials and finished products, characterized in that: The following steps are involved: S1, evaluating the solubility of the sample to be tested and determining the dissolution scheme, wherein the sample to be tested includes cosmetic raw materials and cosmetic finished products; S2, selected cells, said cells comprising human keratinocytes; S3, culturing cells in the logarithmic growth phase, using CCK-8 reagent or MTT reagent to determine the toxicity of the test samples at different concentrations to the cells, and screening out the concentration of the test sample corresponding to the cell viability ≥ 90% as the dosing concentration; S4, establishing a cortisone-induced cell cortisol secretion model, and detecting the cortisol secretion level under the administration concentration of the test sample; S5, establishing a cell β-endorphin secretion model, and detecting the β-endorphin secretion level under the administration concentration of the test sample; S6, establishing a capsaicin-stimulated cell TRPV1 model, and detecting the level of cellular calcium influx at the concentration of the sample to be tested; The above steps S4, S5 and S6 are at least one optional step.

2. The method for in vitro evaluation of the neurobeautic efficacy of cosmetic raw materials and finished products according to claim 1, characterized in that: The method of establishing a cortisone-induced cell cortisol secretion model comprises: Cells in the logarithmic growth phase were cultured; A blank control group, a negative control group, a positive control group and a sample group were set up, wherein the blank control group was added with a cell culture medium, the negative control group was added with a cell culture medium containing cortisone, the positive control group was added with a cell culture medium containing cortisone and the positive substance PF-915275, and the sample group was added with a cell culture medium containing cortisone and a sample of a drug concentration to be tested, and the culture was continued; After the culture was completed, the cell supernatant was collected and the cortisol secretion level was detected.

3. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 2, characterized in that: The method of establishing a cell β-endorphin secretion model comprises: Cells in the logarithmic growth phase were cultured; A negative control group, a positive control group and a sample group are set, wherein the negative control group is added with a cell culture medium, the positive control group is added with a cell culture medium containing the positive substance dibutyryl cAMP sodium salt, and the sample group is added with a cell culture medium containing the sample to be tested at the administration concentration, and the culture is continued; After the culture was completed, the cell supernatant was collected and the β-endorphin secretion level was detected.

4. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 3, characterized in that: The method of establishing a capsaicin-stimulated TRPV1 cell model comprises: Cells in the logarithmic growth phase were cultured; Loading cells with fluorescent probes; A blank control group, a negative control group, a positive control group and a sample group were set up respectively, wherein the negative control group, the positive control group and the sample group used capsaicin to activate the cell TRPV1 channel to induce calcium ion influx, a balanced salt buffer was added to the blank control group, a balanced salt buffer containing capsaicin was added to the negative control group, a balanced salt buffer containing capsaicin and a positive substance capsaicin was added to the positive control group, and a balanced salt buffer containing capsaicin and a sample of a drug concentration to be tested was added to the sample group, and incubated; After the incubation, the levels of calcium ion influx into the cells were detected.

5. A method for in vitro evaluation of the neurobeautic efficacy of cosmetic raw materials and finished products according to any one of claims 1 to 4, characterized in that: When the sample to be tested is a water-soluble sample, it is directly dissolved in the culture medium for subsequent experiments; When the sample to be tested is an oil-soluble sample, dimethyl sulfoxide is used to dissolve it and then added to the culture medium for subsequent experiments. The sample to be tested cannot form a precipitate in the culture medium to interfere with the detection; When dimethyl sulfoxide is used as a solvent, the volume percentage concentration of the solvent shall not exceed 1% of the final culture volume.

6. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 5, characterized in that: The cells in the logarithmic growth phase are cultured, including: using a complete culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, and the culture conditions are 37±0.5°C, (5±1)% CO2 concentration, and saturated humidity; The cultured cells were divided into (1.0-2.0)×10 5 The cells were inoculated at a density of 1.5 cells / mL in a 96-well plate, with 100uL of culture medium in each well, and 3 or more replicate wells were set up in each group.

7. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 6, characterized in that: The method of loading the cells with the fluorescent probe comprises: washing the cells 2-3 times with a balanced salt buffer, incubating the cells in a cell culture medium containing the calcium ion probe, and then washing the cells 2-3 times with a balanced salt buffer after the incubation, wherein the balanced salt buffer comprises a calcium 2+ Hanks balanced salt solution.

8. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 7, characterized in that: The cortisol secretion level was detected by ELISA kit and expressed as the relative content of cortisol; The cortisone-induced cell cortisol secretion model must satisfy that the relative content of cortisol in the negative control group is 10% or more higher than that in the blank control group, and the relative content of cortisol in the positive control group is 10% or more lower than that in the negative control group.

9. The method for in vitro evaluation of neurobeauticant efficacy of cosmetic raw materials and finished products according to claim 8, characterized in that: The β-endorphin secretion level is detected by an ELISA kit and expressed as the relative content of β-endorphin; The cell β-endorphin secretion model needs to satisfy that the relative content of β-endorphin in the positive control group is 10% or more higher than that in the negative control group.

10. The method for in vitro evaluation of neurobeauty effects of cosmetic raw materials and finished products according to claim 9, characterized in that: The cell calcium influx level is detected on a fluorescence microplate reader after staining with Fluo-3AM probe and expressed as relative fluorescence intensity; The capsaicin-stimulated cell TRPV1 model must satisfy that the relative fluorescence intensity of the negative control group is 10% or more higher than that of the blank control group, and the relative fluorescence intensity of the positive control group is 10% or more lower than that of the negative control group.

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