Cosmetic raw material in-vitro repair efficacy evaluation method based on circadian rhythm

Abstract

The invention discloses a circadian rhythm-based cosmetic raw material in-vitro repair efficacy evaluation method, and belongs to the field of cosmetic evaluation, and the method comprises the following steps: S1, HDFA human dermal fibroblast culture, and RAW264.7 mouse macrophage culture; s2, determining the detection concentration of the cosmetic raw materials through cell viability detection; s3, inducing HDFA human dermal fibroblasts with blue light, and detecting the expression quantity of the clock gene BMAL1 and / or CLOCK; s4, after cell scratches of the HDFA human dermal fibroblasts are made, the HDFA human dermal fibroblasts are induced by blue light, and the healing area of the cell scratches is detected; s5, inducing the RAW264.7 mouse macrophage model by virtue of an induction drug lipopolysaccharide, and detecting the expression quantity of a clock gene Dbp and / or Rev-erbalpha; s6, processing and analyzing detection results of the steps S3 to S5, and scoring the detection results; and summarizing all detection index scores. Different cell model detection results are combined, the repairing function of the cosmetic raw materials is subjected to multi-dimensional evaluation, the evaluation accuracy is improved, and the evaluation process is rapid and efficient.

Description

Technical Field

[0001] The invention relates to an in vitro repair efficacy evaluation method for cosmetic raw materials based on circadian rhythm, belonging to the technical field of raw material evaluation. Background Art

[0002] Circadian rhythm refers to a biological rhythm of approximately 24 hours generated by an endogenous circadian oscillator, the central pacemaker of which is located in the suprachiasmatic nucleus of the hypothalamus. The positive and negative feedback loops of the clock genes BMAL1, CLOCK, PER1, and PER2 regulate various physiological processes in the human body, and the skin also shows a complex circadian rhythm organization. Among them, CLOCK and BMAL1 contain the positive components of the clock mechanism, while PER and CRY act as negative regulators. BMAL1 and CLOCK drive the transcription of several different negative feedback genes, including Cry1, Cry2 genes and Per1, Per2, and Per3. The core components BMAL1 and CLOCK form heterodimers to activate the transcription of clock-controlled genes (CCGs), including Pers and Cries, which contain E-box elements in their promoter and / or enhancer regions. In the late cycle, PER and CRY proteins in turn inhibit the activity of CLOCK-BMAL1 by forming a complex, leading to the downregulation of Pers and Crys as well as other CCGs. Once PER and CRY proteins are degraded, a new transcription cycle mediated by CLOCK-BMAL1 can begin.

[0003] Due to the increasing pressure of life and the change of living habits, the number of people who stay up late has increased sharply in recent years, which has led to the disruption of the circadian rhythm of the skin, and then caused a series of skin problems. Studies on the relationship between DNA synthesis in human skin and the time of day have shown that compared with rodent skin, it seems to have greater variability in the circadian cycle, with more DNA synthesis in the afternoon and evening than in the morning, and keratinocytes, fibroblasts, neutrophils and macrophages all exhibit circadian rhythms and may work together to drive rhythmic functions in the skin, which is a universal behavioral and physiological change driven by the endogenous biological clock.

[0004] The pro-inflammatory factor LPS inhibits BMAL1 mRNA levels in macrophages. BMAL1 disruption has been identified as a potential pathological factor in acne-related inflammation. BMAL1 dimers also drive the expression of circadian effector genes, such as genes encoding Dbp and Rev-erbα, which are associated with a variety of physiological functions. Rev-erbα and Dbp downregulation can cause the amplitude of the cell circadian rhythm and shorten its cycle. Rev-erbα is an important regulator of inflammasomes. Rev-erbα-deficient mice show increased activation of the NLRP3 pathway, which in turn triggers skin inflammation. Dbp is a PAR domain transcription factor whose expression is controlled by the circadian rhythm. Dbp may not only be a circadian output gene, but also a component of the circadian oscillator. At the same time, when BMAL1 is overexpressed, cell migration ability is also significantly enhanced. However, the impact of miRNAs on the circadian properties of fibroblasts and macrophages has received little attention in terms of repair. At the macrotissue level, circadian control of skin physiology is necessary to enhance mechanisms that minimize daytime damage to the skin while promoting cell growth and repair mechanisms at night.

[0005] In the existing technology, a single cell model is usually used to evaluate the repair effect of cosmetic raw materials. Some studies only use human dermal fibroblasts or human immortalized keratinocytes to evaluate the repair effect of raw materials, or lack a comprehensive analysis of the results of different cell models, which may affect the accuracy and comprehensiveness of the evaluation results. The traditional evaluation methods of active raw materials based on circadian rhythm repair efficacy mainly use core gene knockout, chemical induction, physical and dietary intervention methods to establish a wide range of circadian rhythm disorder animal models. The experimental cycle is usually long and the cost is high. It is very inconvenient for high-throughput raw material screening and also involves ethical issues. In summary, the existing evaluation methods for the repair efficacy of cosmetic raw materials have certain technical defects or cost limitations. There is no low-cost, multi-dimensional, high-throughput systematic detection and screening scheme suitable for cosmetic raw materials. Therefore, it is of great significance to invent a multi-dimensional evaluation method for the in vitro repair efficacy evaluation of cosmetic raw materials based on circadian rhythm. Summary of the invention

[0006] The purpose of the present invention is to provide a method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm, so as to solve the problems of single dimension, low accuracy, long experimental cycle and high cost in the above-mentioned background technology for evaluating the repair efficacy of cosmetic raw materials.

[0007] To achieve the above object, the present invention provides the following technical solutions: A method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm, comprising the following steps: S1, HDFA human dermal fibroblast culture, RAW264.7 mouse macrophage culture; S2, evaluate the solubility of cosmetic raw materials, determine the dissolution scheme, and determine the concentration of cosmetic raw materials through cell activity detection; S3, blue light induced HDFA human dermal fibroblasts, and the expression of clock genes BMAL1 and / or CLOCK was detected; S4, after making cell scratches of HDFA human dermal fibroblasts, blue light induced HDFA human dermal fibroblasts, and the cell scratch healing area was detected; S5, induced drug lipopolysaccharide induced RAW264.7 mouse macrophage model, detected the expression of clock gene Dbp and / or Rev-erbα; S6, processing and analyzing the detection results of step S3 and scoring them; S7, processing and analyzing the detection results of step S4 and scoring them; S8, processing and analyzing the detection results of step S5 and scoring them; S9, summarizing all the detection index scores of steps S6-8, and obtaining the sum of all the detection index scores of each sample as the comprehensive score of the sample.

[0008] Furthermore, step S3 includes: taking the logarithmically growing HDFA human dermal fibroblasts in step S1 and inoculating and culturing for 18h~20h, setting a blank control group, a model group and a sample group, wherein the blank control group and the model group are added with complete culture medium, and the sample group is added with the cosmetic raw materials of the detection concentration determined in step S2 configured by the complete culture medium, culturing for 4-6h, and then washing with PBS buffer 1-2 times and then irradiating with blue light, and then adding complete culture medium and culturing for 16h~18h before collecting the cells, and then detecting the expression of the clock gene BMAL1 and / or CLOCK in each group by conventional RT-PCR method.

[0009] Furthermore, step S6 includes: summarizing the expression measurement results of the clock genes BMAL1 and / or CLOCK of each group in step S3, calculating the ratio of the expression level of the sample group to the expression level of the model group to obtain the relative expression level, using the relative expression level as the detection index, and performing analysis and scoring.

[0010] Furthermore, step S4 includes: vertically placing a cell scratch plug-in in the middle of each 12-well cell culture plate, inoculating the logarithmically growing HDFA human dermal fibroblasts in S1 into the 12-well cell culture plate and culturing for 16h~24h, and then irradiating with blue light; setting a blank control group, a positive control group and a sample group, wherein the blank control group is added with serum-free culture medium, the positive control group is added with a keratinocyte growth factor solution prepared with serum-free culture medium, and the sample group is added with a cosmetic raw material of the detection concentration determined in step S2 prepared with serum-free culture medium, observing the cell growth status at the scratches at 0 and 24h under a microscope, taking pictures and recording them respectively, and calculating the healing area of ​​each group using ImageJ software.

[0011] Further, step S7 includes: summarizing the healing areas calculated by ImageJ software in step S4, calculating the healing rate of the sample group and the healing rate of the blank control group, taking the difference between the healing rate of the sample group and the healing rate of the blank group as the healing rate compared to the blank, using the healing rate compared to the blank as the detection index, and performing analysis and scoring.

[0012] Furthermore, step S5 includes: taking the logarithmically growing RAW264.7 mouse macrophages in S1 and inoculating and culturing for 18h~20h, setting a blank control group, an induced control group and a sample group, wherein the blank control group and the induced control group are added with complete culture medium, and the sample group is added with the cosmetic raw materials of the detection concentration determined in step S2 configured with the complete culture medium, and cultured for 1h~4h, and then adding the inducing drug lipopolysaccharide solution to the induced control group and the sample group, culturing for 16h~24h, and collecting the cells, and detecting the expression of the clock gene Dbp and / or Rev-erbα in each group by conventional RT-PCR methods.

[0013] Furthermore, step S8 includes: summarizing the expression measurement results of the clock genes Dbp and / or Rev-erbα in each group in S5, calculating the ratio of the expression level of the sample group to the expression level of the induced control group to obtain the relative expression level, using the relative expression level as the detection index, and performing analysis and scoring.

[0014] Furthermore, in step S2, the method for determining the detection concentration of the cosmetic raw material is: respectively taking the logarithmically growing HDFA human dermal fibroblasts and RAW264.7 mouse macrophages in step S1, and screening the cosmetic raw material concentration corresponding to the cell survival rate of 90% as the detection concentration according to the conventional method of MTT cell activity experiment.

[0015] Furthermore, the cosmetic raw materials include chemicals, biological products and plant extracts.

[0016] Furthermore, in steps S6-S8, the analysis method is: GraphPad Prism is used for graphing, the results are expressed as Mean±SD, and One-way ANOVA statistical analysis is used for comparison among groups, and all statistical analyses are two-tailed.

[0017] The present invention has the following beneficial effects: 1. The present invention provides a method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm. Combined with the detection results of different cell models, a multi-dimensional evaluation of the repair function of the target raw materials is performed, and an in vitro evaluation method for the repair efficacy based on circadian rhythm is constructed to improve the accuracy of raw material evaluation, and the evaluation process is fast and efficient.

[0018] 2. The present invention provides a circadian rhythm-based in vitro repair efficacy evaluation method for cosmetic raw materials, which can not only improve the accuracy and efficiency of the evaluation, but also provide an effective basis for further research on animal replacement experiments in the skin care industry, skin care product development and efficacy verification, and has important innovative value.

[0019] 3. The present invention provides an in vitro repair efficacy evaluation method for cosmetic raw materials based on circadian rhythm. The skin, as the largest organ of the human body, has the ability to self-repair and regenerate, and this process is particularly active at night. The expression levels of the clock genes BMAL1 and CLOCK vary at different times of the day, usually reaching a peak at night. Therefore, the expression levels of the clock genes BMAL1 and CLOCK can more accurately reflect the repair efficacy and improve the accuracy of the evaluation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1a This is a graph showing the relationship between the survival rate of HDFA human dermal fibroblasts and the concentration of the test substances A to C of the present invention; Figure 1b The graph is a relationship between the survival rate of RAW264.7 mouse macrophages and the concentration of the test substances A to C of the present invention; Figure 2a The expression level detection results of the clock gene BMAL1 of the test substances A to C in the blue light-induced HDFA human dermal fibroblast model of the present invention; Figure 2b The expression level detection results of the clock gene CLOCK of the test substances A to C in the blue light-induced HDFA human dermal fibroblast model of the present invention; Figure 3a These are the microscope observation pictures of the test substances A to C in the HDFA human dermal fibroblast model with scratches induced by blue light of the present invention; Figure 3b The results of the healing rate test of the test substances A to C in the HDFA human dermal fibroblast model with scratches induced by blue light of the present invention; Figure 4a The results of the detection of the expression of the clock gene Dbp in the RAW264.7 mouse macrophage model test subjects A to C induced by the lipopolysaccharide-induced drug of the present invention; Figure 4b The results of the detection of the expression of the clock gene Rev-erbα in the RAW264.7 mouse macrophage model test subjects A to C induced by the lipopolysaccharide-induced drug of the present invention; Figure 5 It is a schematic diagram of the evaluation method involved in the present invention. DETAILED DESCRIPTION

[0021] A method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm, combining the test results of multiple cell models to score and screen the repair efficacy of target raw materials based on circadian rhythm, comprising the following steps: S1, HDFA human dermal fibroblast culture, RAW264.7 mouse macrophage culture; HDFA human dermal fibroblasts were cultured with ATCC complete medium, and RAW264.7 mouse macrophages were cultured with DMEM+10% FBS complete medium. All culture conditions were 37±0.5℃, (5±1)% CO2 concentration, and 95% humidity; S2, evaluating the solubility of the raw material sample, determining the dissolution scheme, and determining the detection concentration of the raw material sample by cell activity detection. The method for determining the detection concentration of the raw material sample is as follows: taking the logarithmically growing HDFA human dermal fibroblasts and RAW264.7 mouse macrophages in step S1, respectively, and screening the raw material sample concentration corresponding to the cell survival rate of 90% (CV90) according to the conventional method of MTT cell activity experiment as the detection concentration; S3, blue light induced HDFA human dermal fibroblasts, and detected the expression of clock genes BMAL1 and / or CLOCK, the specific method is as follows: The logarithmically growing HDFA human dermal fibroblasts in step S1 were inoculated and cultured for 18h~20h, and a blank control group, a model group and a sample group were set, wherein the blank control group and the model group were added with complete culture medium, and the sample group was added with the raw material sample of the detection concentration determined in step S2 configured by the complete culture medium, and cultured for 4-6h, and then washed with PBS buffer 1-2 times and irradiated with blue light, and then added with complete culture medium and cultured for 16h~18h, and then the cells were collected, and then the expression of the clock gene BMAL1 and / or CLOCK in each group was detected by conventional RT-PCR method; S4, after making cell scratches of HDFA human dermal fibroblasts, blue light induced HDFA human dermal fibroblasts, and detected the cell scratch healing area, the specific method is: A cell scratch plug was placed vertically in the middle of each 12-well cell culture plate, and the HDFA human dermal fibroblasts growing logarithmically in S1 were inoculated into the 12-well cell culture plate and cultured for 16h~24h, and then irradiated with blue light, wherein the cell suspension of the cell culture plate was prepared with complete culture medium; a blank control group, a positive control group and a sample group were set, wherein the blank control group was added with serum-free culture medium, the positive control group was added with keratinocyte growth factor (EGF) solution prepared with serum-free culture medium, and the sample group was added with the raw material sample of the detection concentration determined in step S2 prepared with serum-free culture medium, and the cell growth status at the scratch at 0 and 24h was observed under a microscope, and photographed and recorded respectively, and the healing area of ​​each group was calculated using ImageJ software; S5, induced by lipopolysaccharide (LPS) in the RAW264.7 mouse macrophage model, and detected the expression of clock genes Dbp and / or Rev-erbα. The specific method is as follows: Take the logarithmically growing RAW264.7 mouse macrophages in S1 and inoculate and culture for 18h~20h, set up a blank control group, an induced control group and a sample group, wherein the blank control group and the induced control group are added with complete culture medium, and the sample group is added with the raw material sample of the detection concentration determined in step S2 of the complete culture medium configuration, and cultured for 1h~4h, and then the inducing drug lipopolysaccharide (LPS) solution is added to the induced control group and the sample group, and the cells are collected after culturing for 16h~24h, and the expression of the clock gene Dbp and / or Rev-erbα in each group is detected by conventional RT-PCR method; S6, processing and analyzing the detection results of step S3, and scoring them, the specific method is as follows; Summarize the expression results of the clock genes BMAL1 and / or CLOCK in each group in step S3, calculate the ratio of the expression level of the sample group to the expression level of the model group to obtain the relative expression level, use the relative expression level as the detection index, and use GraphPad Prism to draw a graph. The results are expressed as Mean±SD. One-way ANOVA statistical analysis is used for comparison between groups, and all statistical analyses are two-tailed; Compared with the relative expression of the model group, which was 110-130%, it was considered that the test substance had a slight effect of promoting the expression of clock genes, indicating that the test substance had a certain repair effect, and the score was 0.3; Compared with the model group, the relative expression was 130-150%, indicating that the test substance had a certain effect in promoting the expression of clock genes, indicating that the test substance had a good repair effect, with a score of 0.7; Compared with the model group, the relative expression is ≥150%, indicating that the test substance has a significant effect of promoting clock gene expression, indicating that the test substance has a significant repair effect, and the score is 1.0; S7, processing and analyzing the detection results of step S4 and scoring them, the specific method is as follows; The healing areas calculated by ImageJ software in step S4 were summarized, and the healing rates of the sample group and the blank control group were calculated. The difference between the healing rates of the sample group and the blank group was taken as the healing rate compared with the blank. The healing rate compared with the blank was used as the detection index. GraphPad Prism was used for plotting, and the results were expressed as Mean±SD. One-way ANOVA statistical analysis was used for comparison among the groups; all statistical analyses were two-tailed; Compared with the blank healing rate of 10-25%, it is believed that the test substance has a slight effect of promoting cell migration, indicating that the test substance has a certain repair effect, with a score of 0.3; Compared with the blank, the healing rate was 25-40%, indicating that the test substance had a certain effect of promoting cell migration, indicating that the test substance had a good repair effect, with a score of 0.7; Compared with the blank, the healing rate is ≥40%, which means that the test substance has a significant effect of promoting cell migration, indicating that the test substance has a significant repair effect, and the score is 1.0; S8, processing and analyzing the detection results of step S5, and scoring them, the specific method is as follows; The expression results of clock genes Dbp and / or Rev-erbα in each group in S5 were summarized, and the ratio of the expression level of the sample group to the expression level of the induced control group was calculated to obtain the relative expression level. The relative expression level was used as the detection index, and GraphPad Prism was used for plotting. The results were expressed as Mean±SD. One-way ANOVA statistical analysis was used for comparison among the groups, and all statistical analyses were two-tailed; Compared with the relative expression of the induced control group of 110-130%, it is believed that the test substance has a slight effect of promoting the expression of clock-targeted genes, indicating that the test substance has a certain repair effect, and the score is 0.3; Compared with the relative expression of the induced control group, which was 130-150%, it was considered that the test substance had a certain effect of promoting the expression of clock-targeted genes, indicating that the test substance had a good repair effect, with a score of 0.7; Compared with the relative expression of the induced control group ≥150%, the test substance is considered to have a significant effect of promoting the expression of clock-targeted genes, indicating that the test substance has a significant repair effect, and the score is 1.0; S9, comprehensively evaluating the in vitro repair efficacy of the sample according to the scores obtained in steps S6-S8, the specific method is as follows; Summarize the scores of all test indicators in steps S6-8, and calculate the sum of all test indicator scores of each sample as the comprehensive score of the sample. If 0.9≤comprehensive score<1.5, the sample is considered to have a certain repair effect. If the comprehensive score is 1.5≤comprehensive score<3.5, the sample is considered to have a good repair effect. If the comprehensive score is ≥3.5, the sample is considered to have a significant repair effect. The cosmetic raw materials with better repair effect are comprehensively evaluated.

[0022] The sum of all test index scores of each sample is taken as the comprehensive score of the sample. By ranking the comprehensive scores of multiple samples, the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm can be evaluated and compared.

[0023] Preferably, in step S2, during the MTT cell activity test, HDFA human dermal fibroblasts were taken at (0.5-1.0) x10 5 The cells were inoculated in a 96-well plate at a density of (0.5-0.8) x10 cells / mL, with 100uL of culture medium in each well. Each raw material sample to be tested was configured with 5 to 8 concentration gradients, and 3 or more replicate wells were set for each concentration. RAW264.7 mouse macrophages were taken at a density of (0.5-0.8) x10 5 The cells were inoculated at a density of 10 cells / mL in a 96-well plate, with 100uL of culture medium in each well. Each raw material sample to be tested was configured with 5 to 8 concentration gradients, and 3 or more replicate wells were set for each concentration.

[0024] Raw material samples with a solubility in water ≥1000μg / mL were directly dissolved in culture medium for subsequent experiments; raw material samples with a solubility in water <1000μg / mL were dissolved in dimethyl sulfoxide or ethanol as solvent and then subjected to subsequent experiments; when dimethyl sulfoxide or ethanol was used as solvent, the volume percentage concentration of the solvent should not exceed 0.1% of the total volume, and in subsequent experimental steps, the volume ratio of the solvent in the control group and the sample group was the same.

[0025] Preferably, in step S3, the expression level of at least one of the clock genes BMAL1 and CLOCK is detected; in step S5, the expression level of at least one of the clock genes Dbp and Rev-erbα is detected.

[0026] Preferably, in step S3, HDFA human dermal fibroblasts are cultured at a rate of (0.5-1.5) x10 6 The density of cells / mL was inoculated in a 6-well plate, with 1000uL of culture medium in each well, the blue light irradiation intensity was 15-30J, the blue light irradiation wavelength was 470nm, and 3 or more replicate wells were set for each raw material sample.

[0027] Preferably, in step S4, 70 μL of HDFA human dermal fibroblast cell suspension with a density of (1.5-2.0) x105 cells / mL, 1000uL of culture medium in each well, and 3 or more replicate wells for each raw material sample.

[0028] Preferably, in step S5, RAW264.7 mouse macrophages are cultured at (3.0-5.0) x10 4 The cells were inoculated at a density of 10 cells / mL in a 24-well plate, with 1000uL of culture medium in each well, and 3 or more replicate wells were set for each raw material sample; the final concentration of the inducing drug lipopolysaccharide (LPS) was 1μg / mL, and the buffer was PBS buffer.

[0029] Preferably, the raw material sample is a cosmetic raw material, which includes chemicals, biological products and plant extracts.

[0030] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the reagents and materials used in the examples can be purchased from the market.

[0032] 1. Experimental Materials 1. Experimental Materials and Instruments 1.1 Cells HDFA human dermal fibroblasts and RAW264.7 mouse macrophages were obtained from ATCC, the Cell Bank of the Chinese Academy of Sciences, or the Cell Bank of China Center for Type Culture Collection, which are registered members of the World Federation of Culture Collections.

[0033] In the examples of the present application, HDFA human dermal fibroblasts and RAW264.7 mouse macrophages were purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0034] 1.2 Main Reagents (1) Cell culture-related reagents: DMEM medium, fetal bovine serum, and PBS (1x) were purchased from Gibco and the American Cell Culture Collection; ATCC special medium was purchased from ATCC. (2) Chemical reagents: MTT and lipopolysaccharide (LPS) were purchased from Sigma, USA; DMSO and ethanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (3) Primers: Shanghai Jierui Bioengineering Co., Ltd.; (4) RNA extraction: RNA extraction kit was purchased from Thermo Fisher Scientific, USA.

[0035] 1.3 Solution preparation (1) HDFA human dermal fibroblast ATCC complete medium: Take 500 mL of ATCC medium, add factors, mix thoroughly and store at 4°C. Preheat in a 37°C water bath before use. (2) RAW264.7 mouse macrophage DMEM complete medium: Take 450 mL of DMEM medium, add 50 mL of fetal bovine serum (FBS), mix thoroughly and store at 4°C. Preheat in a 37°C water bath before use. (3) MTT solution: weigh 100 mg of MTT powder, add 20 mL of PBS, vortex to dissolve, filter through a 0.22 μm filter membrane to sterilize, store at -20°C away from light, and thaw at 4°C before use; (4) Cell MTT detection solution: MTT solution was diluted to 0.5 mg / mL in DMEM.

[0036] 1.4 Instruments Microplate reader (Thermo, USA), CO2 incubator (Thermo, USA), cell counter (Cellometer, USA), inverted microscope (Olympus, Japan), biological safety cabinet (Thermo, USA), high-speed low-temperature centrifuge (Thermo, USA), low-temperature refrigerator (Meiling, China), ultra-low-temperature refrigerator (Thermo, USA), pipette (RAININ), ice machine (ZIEGRA), ultra-micro-volume spectrophotometer (Thermo, USA), real-time fluorescence quantitative PCR instrument (Roche, Switzerland).

[0037] 2. Raw material samples to be tested The names of raw material samples (test substances) are shown in Table 1: Table 1 serial number Sample name 1 Active ingredient A 2 Active ingredient B 3 Active Ingredients C 2. Blue light-induced HDFA human dermal fibroblast model to evaluate the repair efficacy of raw materials 1. Cell Culture HDFA human dermal fibroblasts were cultured with complete culture medium in a CO2 incubator at 37°C, 5% CO2 and saturated humidity until the cell confluence reached 70%-80% and then subcultured. During subculture, the original culture medium was removed, and fresh culture medium was added and gently pipetted to collect the cells, and subcultured at a ratio of 1:2 or 1:3.

[0038] 2. MTT cytotoxicity test and analysis of the test substance 2.1 MTT assay for cell viability HDFA human dermal fibroblasts with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 0.5×105 cells per 1 mL. They were inoculated in 96-well cell culture plates, 100 μL per well. No cells were inoculated in the outermost circle of the 96-well plate. 150 μL PBS was added and cultured at 37°C, 5% CO2 and saturated humidity. After 16 hours, the original culture medium was removed and the concentration gradient test substances prepared with fresh complete culture medium were added. Six concentrations were set for each test substance. The concentrations of liquid raw materials (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, and 0.0316%, respectively. The concentrations of solid raw materials were 1000 μg / mL, 316 μg / mL, 100 μg / mL, 31.6 μg / mL, 10 μg / mL, and 3.16 μg / mL, respectively. 100 μL per well was set to contain only cells. For the control group, three replicate wells were set up for each experimental well, and the cells were cultured at 37°C, 5% CO2 and saturated humidity for 18 h. The supernatant was removed and the cells were gently washed twice with PBS. 100 μL of cell MTT detection solution was added to all wells containing cells and 3 blank wells, and the cells were incubated at 37°C, 5% CO2 and saturated humidity for 4 h. The supernatant was removed and the cells were gently washed twice with PBS. 100 μL of DMSO was added to each well, and the absorbance at a wavelength of 570 nm was read on an enzyme reader after mixing. 630 nm was used as the reference wavelength, and the measurement results were recorded.

[0039] 2.2 Cell viability analysis and concentration determination Calibration OD = OD570-OD630 The cell viability calculation formula is as follows: Cell survival rate = [(As-Ab) / (Ac-Ab)] x 100% As: Experimental wells (culture medium containing cells, MTT, and test substances) Ac: control well (containing culture medium with cells, MTT, and no test substance) Ab: Blank well (medium without cells and test substance, MTT) The calculation formula for cell viability 90% (CV90) is:

[0040] Where: a: The cell viability exceeds the minimum value of 90%; c: cell viability is less than 90% of the maximum value; b and d indicate the concentrations corresponding to the cell activities in a and c.

[0041] The cell viability of the test substance was calculated according to the above formula, and the concentration corresponding to CV90 was selected as the detection concentration of the raw material sample.

[0042] 3. Use blue light to induce HDFA human dermal fibroblast model to detect the expression levels of clock genes BMAL1 and CLOCK.

[0043] HDFA human dermal fibroblasts with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 7.5×10 4 Cells were inoculated in 6-well cell culture plates, 2000 μL per well, and cultured at 37°C, 5% CO2 and saturated humidity. After 18 hours, the original culture medium was removed, and a blank control group, a model group and a sample group were set up. Complete culture medium was added to the blank control group and the model group, and the sample group was added with a test substance of the corresponding concentration of CV90 prepared by complete culture medium. The cells were cultured at 37°C, 5% CO2 and saturated humidity for 6 hours, washed with PBS buffer, and the blank control group was treated with 30 J / cm 2 HDFA human dermal fibroblasts were irradiated with a blue light dose of 1000 nm. After the irradiation, the PBS buffer in the well plate was aspirated, and complete culture medium was added to each well and cultured for 18 h under 37°C, 5% CO2 and saturated humidity. The cells were collected and the RNA was extracted using an RNA extraction detection kit. The operating steps were referred to the kit manual, and the expression of the clock genes BMAL1 and CLOCK was then detected by conventional RT-PCR methods.

[0044] Select the data software to calculate the standard curve, and calculate the expression of the clock genes BMAL1 and CLOCK of the test object, and the ratio of the expression of the sample group to the expression of the model group to obtain the relative expression. The calculation formula is as follows:

[0045] 3. Blue light-induced scratched HDFA human dermal fibroblast model to evaluate the repair efficacy of raw materials 1. Cell Culture HDFA human dermal fibroblasts were cultured with complete culture medium in a CO2 incubator at 37°C, 5% CO2 and saturated humidity until the cell confluence reached 70%-80% and then subcultured. During subculture, the original culture medium was removed, and fresh culture medium was added and gently pipetted to collect the cells, and subcultured at a ratio of 1:2 or 1:3.

[0046] 2. Determination of sample concentration The concentration corresponding to CV90 in the blue light-induced HDFA human dermal fibroblast model was selected as the detection concentration.

[0047] 3. Blue light induced HDFA human dermal fibroblast model with scratches, detected the cell scratch area, and calculated the healing rate.

[0048] HDFA human dermal fibroblasts with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 1.5×10 5 Place a cell scratch insert vertically in the middle of each 12-well cell culture plate, add 70 μL of cell suspension to each of the two chambers of each insert, moisten the insert with 200 μL of cell suspension, and culture the cells at 37°C with 5% CO2 for 24 hours. Gently remove the insert with sterilized tweezers, rinse the cells gently with 1 mL of PBS solution once or twice, and then rinse with 30 J / cm 2 The cells were irradiated with a dose of blue light radiation, and all PBS was removed. A blank control group, a positive control group, and a sample group were set up. 1 mL of the test substance with the detection concentration was accurately added to the sample group, and the test substance was prepared with serum-free culture medium; serum-free culture medium was added to the blank control group, and keratinocyte growth factor (EGF) solution prepared with serum-free culture medium was added to the positive control group. The cell growth status at the scratch site at 0 and 24 hours was observed under a microscope, and photographed and recorded respectively. The healing area was calculated using ImageJ software.

[0049] The formula for calculating the healing rate is:

[0050] Compare the calculated healing rate of the sample group with the healing rate of the blank control group to obtain the healing rate compared to the blank. The calculation formula for the healing rate compared to the blank is:

[0051] 4. Induced drug lipopolysaccharide (LPS) induced RAW264.7 mouse macrophage model to evaluate the repair effect of raw materials 1. Cell Culture RAW264.7 mouse macrophages were cultured with complete culture medium in a CO2 incubator at 37°C, 5% CO2 and saturated humidity until the cell confluence reached 70%-80% and then passaged. During passage, the original culture medium was removed, and fresh culture medium was added and gently pipetted to collect the cells, and then passaged at a ratio of 1:3 or 1:4.

[0052] 2. MTT cytotoxicity test and analysis of the test substance 2.1 MTT assay for cell viability RAW264.7 mouse macrophages with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 5×10 4Cells were inoculated in a 96-well cell culture plate, 100 μL per well, and no cells were inoculated in the outermost circle of the 96-well plate. 150 μL PBS was added and cultured at 37°C, 5% CO2 and saturated humidity. After 16 hours, the original culture medium was removed and the concentration gradient test substances prepared with fresh complete culture medium were added. Six concentrations were set for each test substance. The concentrations of liquid raw materials (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, and 0.0316%, respectively. The concentrations of solid raw materials were 1000 μg / mL, 316 μg / mL, 100 μg / mL, 31.6 μg / mL, 10 μg / mL, and 3.16 μg / mL, respectively. 100 μL per well was set to contain only cells. For the control group, three replicate wells were set up for each experimental well, and the cells were cultured at 37°C, 5% CO2 and saturated humidity for 18 h. The supernatant was removed and the cells were gently washed twice with PBS. 100 μL of cell MTT detection solution was added to all wells containing cells and 3 blank wells, and the cells were incubated at 37°C, 5% CO2 and saturated humidity for 4 h. The supernatant was removed and the cells were gently washed twice with PBS. 100 μL of DMSO was added to each well, and the absorbance at a wavelength of 570 nm was read on an enzyme reader after mixing. 630 nm was used as the reference wavelength, and the measurement results were recorded.

[0053] 2.2 Cell viability analysis and concentration determination Calibration OD = OD 570 -OD 630 The cell viability calculation formula is as follows: Cell survival rate = [(As-Ab) / (Ac-Ab)] x 100% As: Experimental wells (culture medium containing cells, MTT, and test substances) Ac: control well (containing culture medium with cells, MTT, and no test substance) Ab: Blank well (medium without cells and test substance, MTT) Calculation of 90% cell viability (CV90)

[0054] Where: a: The cell viability exceeds the minimum value of 90%; c: cell viability is less than 90% of the maximum value; b and d indicate the concentrations corresponding to the cell activities in a and c.

[0055] The cell viability of the test substance was calculated according to the above formula, and the concentration corresponding to CV90 was selected as the detection concentration.

[0056] 3. The inducing drug lipopolysaccharide (LPS) was used to induce the RAW264.7 mouse macrophage model, and the expression levels of the clock genes Dbp and Rev-erbα were detected.

[0057] RAW264.7 mouse macrophages with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 5×10 4 Cells were inoculated in 24-well cell culture plates, 1000 μL per well, and cultured at 37°C, 5% CO2 and saturated humidity. After 18 hours, the original culture medium was removed, and blank control group, induced control group and sample group were set up. Complete culture medium was added to the blank control group and induced control group, and the sample group was added with the test substance of CV90 corresponding to the concentration prepared by complete culture medium, and cultured at 37°C, 5% CO2 and saturated humidity for 4 hours. Except for the cell control group, LPS was added to each well to a final concentration of 1 μg / mL, and cultured at 37°C, 5% CO2 and saturated humidity for 20 hours. The cell culture medium was collected, centrifuged at 2000 rpm and 4°C for 10 minutes, and the cells were collected. The RNA extraction detection kit was used to extract the content RNA. The operation steps were referred to the instructions of the kit, and then the expression of clock genes Dbp and Rev-erbα was detected by conventional RT-PCR methods.

[0058] Select data software to calculate the standard curve, and calculate the relative expression of the clock genes Dbp and Rev-erbα of the samples to be tested, with the induced control group as 100%. The calculation formula is as follows:

[0059] V. Results Analysis GraphPad Prism was used for drawing, and the results were expressed as Mean ± SD. One-way ANOVA statistical analysis was used for comparison between groups. All statistical analyses were two-tailed. p < 0.05 was considered to be significantly different, indicated by "*", p < 0.01 was considered to be very significantly different, indicated by "**", and p < 0.001 was considered to be extremely significantly different, indicated by "***".

[0060] 5.1 Results of blue light-induced HDFA human dermal fibroblast model testing 5.1.1 Cytotoxicity test of test substance According to the cell viability MTT test results of sample AC, the corresponding raw material sample administration concentration when the cell viability of the HDFA human dermal fibroblast model is 90% (CV90) is calculated, and the subsequent administration is incubated according to this concentration. The test results are detailed in Table 2 and Figure 1a .

[0061] Table 2 serial number CV90 A 652.66μg / mL B 442.86 μg / mL C 260.10μg / mL 5.1.2 Expression detection results of clock genes BMAL1 and CLOCK Cells were collected and cell RNA was extracted according to the RNA extraction test kit operation steps. The RNA expression of clock genes BMAL1 and CLOCK was then detected by conventional RT-PCR methods. The relative expression was calculated with the model group as the control. ###P<0.001 is the comparison between the model group and the blank control group, *P<0.05, **P<0.01, ***P<0.001 is the comparison between the sample group and the model group. For details of the test results, see Figure 2a and Figure 2b .

[0062] 5.2 Blue light-induced detection results of HDFA human dermal fibroblast model with scratches 5.2.1 Cytotoxicity test of test substance According to the cell viability MTT test results of sample AC, the corresponding raw material sample administration concentration when the cell viability of the HDFA human dermal fibroblast model is 90% (CV90) is calculated, and the subsequent administration is incubated according to this concentration. The test results are detailed in Table 2 and Figure 1a .

[0063] 5.2.2 Observation and detection of healing rate under microscope The cell growth status at the scratch site was observed under a microscope at 0 and 24 hours, and photographed and recorded. The healing area was calculated using ImageJ software, compared with the initial area, and the healing rate was calculated and compared with the blank control. *P<0.05, **P<0.01, ***P<0.001 are the comparisons between the sample group and the blank control group. Figure 3a and Figure 3b .

[0064] 5.3 Results of the induced drug lipopolysaccharide (LPS)-induced RAW264.7 mouse macrophage model 5.3.1 Cytotoxicity test of test substance According to the cell viability MTT test results of sample AC, the corresponding raw material sample administration concentration when the cell viability of the RAW264.7 mouse macrophage model cell is 90% (CV90) is calculated, and the subsequent administration is incubated according to this concentration. The test results are detailed in Table 3 and Figure 1b .

[0065] Table 3 serial number CV90 1 474.64 μg / mL 2 880.24 μg / mL 3 95.44 μg / mL 5.3.2 Results of expression detection of clock genes Dbp and Rev-erbα Cells were collected and cell RNA was extracted according to the RNA extraction detection kit operation steps. The RNA expression of clock genes Dbp and Rev-erbα was then detected by conventional RT-PCR method. The relative expression was calculated with the induced group as the control. ###P<0.001 was compared between the induced group and the blank control group, *P<0.05, **P<0.01, ***P<0.001 was compared between the sample group and the induced group. The relative expression of Dbp was as follows Figure 4a The relative expression of Rev-erbα is shown in Figure 4b shown.

[0066] 5.4 Comprehensive evaluation results and analysis All samples were evaluated according to the method flow of the present invention. The three test substances detected in this example were based on Figure 5 The screening process was tested, and the experimental results in different cell models were summarized in Table 4. Evaluation and scoring were performed according to the standards in Table 5. The ranking of raw material repair effects was obtained according to the summary comprehensive score results in Table 6: C>B>A, among which C had a significant repair effect, B had a certain repair effect, and A had no repair effect.

[0067] Table 4 serial number Relative expression of BMAL1 (%) CLOCK relative expression (%) Compared with blank healing rate (%) Dbp relative expression (%) Relative expression of Rev-erbα (%) A 101.90 114.66 21.26 78.42 71.39 B 134.01 88.56 12.68 72.11 94.45 C 137.50 150.30 31.25 150.16 127.60 Table 5 Evaluation results No repair effect Has a certain repair effect Has good repair effect Has significant repairing effect Relative expression of genes in the blue light-induced HDFA human dermal fibroblast model (%) ＜110 110-130 130-150 ≥150 Blue light-induced HDFA human dermal fibroblast scratch repair model compared with blank healing rate (%) ＜10 10-25 25-40 ≥40 Relative expression of genes in the RAW264.7 mouse macrophage model (%) ＜110 110-130 130-150 ≥150 Points 0 0.3 0.7 1 Table 6

[0068] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm, characterized in that: The following steps are involved: S1, HDFA human dermal fibroblast culture, RAW264.7 mouse macrophage culture; S2, evaluate the solubility of cosmetic raw materials, determine the dissolution scheme, and determine the concentration of cosmetic raw materials through cell activity detection; S3, blue light induced HDFA human dermal fibroblasts, and the expression of clock genes BMAL1 and / or CLOCK was detected; S4, after making cell scratches of HDFA human dermal fibroblasts, blue light induced HDFA human dermal fibroblasts, and the cell scratch healing area was detected; S5, induced drug lipopolysaccharide induced RAW264.7 mouse macrophage model, detected the expression of clock gene Dbp and / or Rev-erbα; S6, processing and analyzing the detection results of step S3 and scoring them; S7, processing and analyzing the detection results of step S4 and scoring them; S8, processing and analyzing the detection results of step S5 and scoring them; S9, summarizing all the detection index scores of steps S6-8, and obtaining the sum of all the detection index scores of each sample as the comprehensive score of the sample.

2. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: Step S3 includes: taking the logarithmically growing HDFA human dermal fibroblasts in step S1 and inoculating and culturing for 18h~20h, setting a blank control group, a model group and a sample group, wherein the blank control group and the model group are added with complete culture medium, and the sample group is added with the cosmetic raw materials of the detection concentration determined in step S2 configured by the complete culture medium, culturing for 4-6h, and then washing with PBS buffer 1-2 times and then irradiating with blue light, and then adding complete culture medium and culturing for 16h~18h, and then collecting the cells, and then detecting the expression of clock genes BMAL1 and / or CLOCK in each group by conventional RT-PCR methods.

3. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 2, characterized in that: Step S6 includes: summarizing the expression measurement results of the clock genes BMAL1 and / or CLOCK in each group in step S3, calculating the ratio of the expression level of the sample group to the expression level of the model group to obtain the relative expression level, using the relative expression level as the detection index, and performing analysis and scoring.

4. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: Step S4 includes: vertically placing a cell scratch plug-in in the middle of each 12-well cell culture plate, inoculating the logarithmically growing HDFA human dermal fibroblasts in S1 into the 12-well cell culture plate and culturing for 16h~24h, and then irradiating with blue light; setting a blank control group, a positive control group and a sample group, wherein the blank control group is added with a serum-free culture medium, the positive control group is added with a keratinocyte growth factor solution prepared with a serum-free culture medium, and the sample group is added with a cosmetic raw material of the detection concentration determined in step S2 prepared with a serum-free culture medium, observing the cell growth status at the scratch at 0 and 24h under a microscope, taking pictures and recording them respectively, and calculating the healing area of ​​each group using ImageJ software.

5. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 4, characterized in that: Step S7 includes: summarizing the healing areas calculated by ImageJ software in step S4, calculating the healing rate of the sample group and the healing rate of the blank control group, taking the difference between the healing rate of the sample group and the healing rate of the blank group as the healing rate compared with the blank, using the healing rate compared with the blank as the detection index, and performing analysis and scoring.

6. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: Step S5 includes: taking the logarithmically growing RAW264.7 mouse macrophages in S1 and inoculating and culturing for 18h~20h, setting a blank control group, an induced control group and a sample group, wherein the blank control group and the induced control group are added with complete culture medium, and the sample group is added with the cosmetic raw materials of the detection concentration determined in step S2 configured with the complete culture medium, and culturing for 1h~4h, and then adding the inducing drug lipopolysaccharide solution to the induced control group and the sample group, culturing for 16h~24h, and collecting the cells, and detecting the expression of the clock gene Dbp and / or Rev-erbα in each group by conventional RT-PCR methods.

7. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 6, characterized in that: Step S8 includes: summarizing the expression measurement results of the clock genes Dbp and / or Rev-erbα in each group in S5, calculating the ratio of the expression level of the sample group to the expression level of the induced control group to obtain the relative expression level, using the relative expression level as the detection index, and performing analysis and scoring.

8. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: In step S2, the method for determining the detection concentration of the cosmetic raw material is: respectively taking the logarithmically growing HDFA human dermal fibroblasts and RAW264.7 mouse macrophages in step S1, and screening the cosmetic raw material concentration corresponding to the cell survival rate of 90% as the detection concentration according to the conventional method of MTT cell activity experiment.

9. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: The cosmetic raw materials include chemicals, biological products and plant extracts.

10. The method for evaluating the in vitro repair efficacy of cosmetic raw materials based on circadian rhythm according to claim 1, characterized in that: In steps S6-S8, the analysis method is: GraphPad Prism is used for graphing, the results are expressed as Mean±SD, and One-way ANOVA statistical analysis is used for comparison between groups, and all statistical analyses are two-tailed.

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