Combined method for evaluating in-vitro anti-acne efficacy of cosmetic raw materials

Abstract

The invention discloses a cosmetic raw material in-vitro anti-acne efficacy evaluation combination method which comprises the following steps: S1, establishing an in-vitro antibacterial efficacy model of a to-be-evaluated cosmetic raw material and scoring, and recording the score as T1; s2, establishing a to-be-evaluated cosmetic raw material in-vitro keratinocyte anti-acne model and performing scoring, wherein the score is recorded as T2; s3, establishing an in-vitro sebaceous gland cell oil control model of the to-be-evaluated cosmetic raw material, and performing scoring, wherein the score is marked as T3; s4, establishing a macrophage anti-inflammatory soothing effect model of the cosmetic raw material RAW264.7 to be evaluated, and scoring, wherein the score of the score is marked as T4; and S5, calculating the score of the total score and giving an evaluation result of the in-vitro anti-acne efficacy of the cosmetic raw materials. The in-vitro preliminary fast screening requirement is met, and an effective basis is provided for deep research on development and efficacy verification of anti-acne related skin care products on the basis of following the 3R principle of the cosmetic industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and more specifically, relates to a combined method for evaluating the in vitro anti-acne efficacy of cosmetic raw materials. Background Art

[0002] Acne is a chronic skin disease that is common on the face, chest and back, and is mainly manifested as acne, papules, nodules or cysts. Due to the current situation of people generally experiencing increased stress, disordered work and rest, high-sugar and high-fat diet, improper skin cleaning or genetics, the incidence of acne is relatively high. According to research, more than 82% of people will have acne to varying degrees. The pain and discomfort associated with acne, as well as the erythema, residual skin lesions and scars that may be caused by subsequent inflammatory lesions, will bring a great physical and mental burden to patients.

[0003] At present, the exact pathogenesis of acne has not been fully elucidated, but relevant experts generally believe that the main factors causing acne are: 1. Excessive secretion of oil by sebaceous glands; 2. Abnormal keratinization of the sebaceous gland ducts of the hair follicles; 3. Colonization of hair follicle microorganisms such as Propionibacterium acnes; 4. Inflammation and immune response. In addition to conventional drug treatment, the reasonable use of anti-acne functional skin care products on a daily basis can also help to achieve the effect of auxiliary prevention and treatment of acne.

[0004] Traditional in vivo efficacy evaluation of anti-acne raw materials is mainly achieved through animal models, including rabbit ear models, rat models, Mexican hairless dog models, etc., because the above evaluation methods are too expensive and have not been promoted. However, the cosmetics industry is currently actively promoting the "3R" principle of animal testing, and the use of cosmetics for in vitro anti-acne operations is simple and easy, and is highly praised by consumers. A large number of literature shows that Propionibacterium acnes and Staphylococcus aureus are widely present in lipids on the surface of human skin and are important pathogens of acne. They can release hormones related to sebum secretion, and can also damage the integrity of epidermal cells to cause excessive keratinization and form acne. At the same time, acne is also closely related to inflammation and tissue damage. After keratinocytes are stimulated by Propionibacterium acnes, the expression of Toll-like receptors, especially TLR2 and TLR4, increases, which can activate downstream signaling pathways and induce the production of chemokines and cellular inflammatory factors. A large number of inflammatory mediators are found in all stages of acne. As the main source of acne, in the early stage of acne, the lesion first produces pro-inflammatory factors, which prompt various inflammatory cells to gather locally in the lesion, and then produce a large number of inflammatory factors under the action of microorganisms, triggering a series of inflammatory reactions. The production of inflammation can also directly lead to pigmentation after acne formation, forming acne marks. Therefore, anti-acne functional cosmetics need to achieve the desired effect by improving the inhibition of related bacteria during the occurrence of acne, related downstream reactions after bacterial stimulation, and abnormal sebum secretion and inflammatory reactions throughout the occurrence of acne.

[0005] There are some existing evaluation methods for the anti-acne efficacy of cosmetics and their raw materials, such as: the patent application with application number CN202210890208.X discloses an in vitro reconstructed epidermal model and construction method of an acne-like phenotype, the defects of which are that the construction time of the in vitro reconstructed epidermal model is long, the cultivation difficulty and cost are high; the patent application with application number CN202310542376.4 discloses an in vitro acne inflammation model and construction method, the in vitro acne inflammation model used is a single cell model, the evaluation means are relatively single, and missed screening may occur. It is obvious that the above-mentioned in vitro anti-acne efficacy evaluation model of cosmetic raw materials has the problem that the efficacy evaluation is not comprehensive enough, the implementation cost is high, and it is easy to have wrong screening and missed screening.

[0006] Therefore, it is urgent to propose a combined method for evaluating the in vitro anti-acne efficacy of cosmetic raw materials to solve the technical problems of the existing in vitro anti-acne efficacy evaluation model for cosmetic raw materials, such as high implementation cost, incomplete efficacy evaluation, and easy misscreening and missed screening. This will help to achieve a more comprehensive development and screening of anti-acne cosmetic raw materials under the premise of high throughput. Summary of the invention

[0007] In view of the shortcomings of the prior art, the main purpose of the present invention is to propose a combined method for evaluating the in vitro anti-acne efficacy of cosmetic raw materials, aiming to solve at least one technical problem of the existing in vitro anti-acne efficacy evaluation model of cosmetic raw materials, namely, high implementation cost, incomplete efficacy evaluation, and easy occurrence of misscreening and missed screening.

[0008] To achieve this object, the present invention adopts the following technical solutions: The present invention provides a combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro, the combined method comprising the following steps: Step S1: Establishing an in vitro antibacterial efficacy model for the cosmetic raw material to be evaluated and scoring it, where the score is recorded as T1; Step S2: establishing an in vitro keratinocyte anti-acne model for the cosmetic raw material to be evaluated and scoring the model, wherein the score is recorded as T2; Step S3: establishing an in vitro sebaceous gland cell oil control model for the cosmetic raw material to be evaluated and scoring it, where the score is recorded as T3; Step S4: establishing a model for the anti-inflammatory and soothing effect of the RAW264.7 macrophages of the cosmetic raw material to be evaluated and scoring the model, wherein the score is recorded as T4; Step S5: Calculate the total score and give the in vitro anti-acne efficacy evaluation result of the cosmetic raw material. The total score calculation formula is as follows: ;in, a1 is the scoring weight coefficient of the in vitro antibacterial efficacy model, a2 is the scoring weight coefficient of the in vitro keratinocyte anti-acne model, a3 is the scoring weight coefficient of the in vitro sebaceous gland cell oil control model, and a4 is the scoring weight coefficient of the RAW264.7 macrophage anti-inflammatory and soothing effect model. Here, a1, a2, a3, and a4 are all 0.25; if the total score T is not less than the preset score, the in vitro anti-acne efficacy evaluation result of the cosmetic raw material is valid, and the preset score is 0.5.

[0009] Preferably, the step S1 comprises: Step S11: Stably culturing Propionibacterium acnes and Staphylococcus aureus; Step S12: setting test tubes for cosmetic raw material test substance groups and test tubes for positive control groups, wherein each group of test tubes for the cosmetic raw material test substance groups consists of test tubes No. 1 to No. 9, and the sterilized MH culture medium is divided into test tubes No. 1 to No. 9 of the cosmetic raw material test substance group, and 3840 μg / mL of the cosmetic raw material test substance is added to test tube No. 1 of the cosmetic raw material test substance group, and test tubes No. 2 to No. 7 are sequentially added with the cosmetic raw material test substance diluted by half, and test tubes No. 8 to No. 9 are not added with the cosmetic raw material test substance, then tube No. 8 is the negative control, and tube No. 9 is the blank control; in the positive control group, the cosmetic raw material test substances in test tubes No. 1 to No. 7 of the raw material test substance group are replaced with antibiotics; Step S13: The activated strains of Propionibacterium acnes and Staphylococcus aureus were prepared into bacterial suspensions with physiological saline, and added to test tubes 1 to 8 of different cosmetic raw material test groups, respectively. After culturing for 18 to 24 hours, 100 μL of liquid was taken from test tubes 1 to 9 of the cosmetic raw material test group to a 96-well cell culture plate to measure the OD600nm absorbance, and the relative antibacterial rate of the cosmetic raw material test at each concentration was calculated. The calculation formula of the relative antibacterial rate is as follows: Relative inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100%, where: As is the average absorbance of the liquid in the well plate, which involves the liquid taken out from test tubes 1 to 7 of the cosmetic raw material test group; Ac is the average absorbance of the liquid in the negative control well, which involves the liquid taken out from test tube 8; Ab is the average absorbance of the blank control well, which involves the liquid taken out from test tube 9; Step S14: Calculate the As value of the liquid taken out of each tube, and calculate the relative inhibition rate value of the corresponding test substance against Propionibacterium acnes and Staphylococcus aureus. For Propionibacterium acnes or Staphylococcus aureus, if the relative inhibition rate corresponding to any test tube from 2 to 4 is less than 40%, 0 points are assigned; if 40%≤the relative inhibition rate corresponding to any test tube from 2 to 4 is less than 80%, 0.3 points are assigned; if the relative inhibition rate corresponding to any test tube from 2 to 4 is ≥80%, 1 point is assigned; when the sum of the points assigned to the cosmetic raw material test substance in any test tube from 2 to 4 against Propionibacterium acnes and Staphylococcus aureus is greater than 0.5 points, T1 is 1 point.

[0010] It should be noted that the reason for selecting test tubes 2 to 4 of the cosmetic raw material test substance group (i.e., the concentration of the cosmetic raw material test substances is 480μg / mL to 1920μg / mL) as the basis for considering the relative inhibition rate and then scoring the in vitro antibacterial efficacy model of cosmetic raw materials is that the concentrations of the cosmetic raw material test substances corresponding to test tubes 2 to 4 are in the middle position in the model, which can statistically exclude the extreme situations that may occur at the endpoints, thereby ensuring the reference value of the scoring of the in vitro antibacterial efficacy model of cosmetic raw materials; in addition, the cost of the above operation method is controllable.

[0011] It should be noted that the relative antibacterial rate of less than 40% indicates that the antibacterial effect is not obvious, the relative antibacterial rate of not less than 40% and not more than 80% indicates that there is a certain antibacterial effect, and the relative antibacterial rate of not less than 80% indicates that the antibacterial effect is excellent. Therefore, if the sum of the scores of the cosmetic raw material test substance against Propionibacterium acnes and Staphylococcus aureus is greater than 0.5 points, it means that at least the cosmetic raw material test substance exhibits an excellent antibacterial effect against at least one of Propionibacterium acnes and Staphylococcus aureus, or exhibits a certain antibacterial effect against both Propionibacterium acnes and Staphylococcus aureus.

[0012] Preferably, the step S2 comprises: Step S21: Pick the Propionibacterium acnes colonies after stable culture in the culture medium and add 2-5wt% of sebum mixture by mass percentage, culture at 37°C and 220r / min for 3-7 days, collect the supernatant by centrifuge, filter with 0.22μm filter membrane to obtain Propionibacterium acnes culture fluid, and store in a -80°C refrigerator; Step S22: culturing Hacat cells and collecting Hacat cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating them in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using the CCK8 method to screen and obtain the corresponding dosage concentration of the cosmetic raw material test substance when the Hacat cell survival rate is 90%, taking the Hacat cells in the logarithmic growth phase and inoculating them in a 12-well cell culture plate for culture, setting up a blank control group, an induced model group, a positive control group and a sample group, using 5% by mass of the Propionibacterium acnes culture solution prepared in step S21 to model, and adding a preset concentration of the cosmetic raw material test substance to each group for culture for 16h~24h, and then collecting cell RNA to detect the expression levels of related genes TLR-2, TLR-4, IL-8, and TSLP; Step S23: Calculate the expression results of relevant genes of each group in step S22, use GraphPad Prism to draw a graph, and use One-way ANOVA statistical analysis to compare each group with the induced model group: for each gene, when p < 0.05 and the inhibition rate ≥ 15%, it indicates that it has a significant inhibitory effect compared with the induced model group; when a cosmetic raw material test substance has an inhibitory effect on at least 1 gene in the model, T2 is 1 point.

[0013] Preferably, step S3 comprises: Step S31: culturing SZ95 cells and collecting SZ95 cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating them in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using the CCK8 method to screen and obtain the corresponding dosage concentration of the cosmetic raw material test substance when the SZ95 cell survival rate is 90%; taking the logarithmic growth phase SZ95 cells and inoculating them in a 24-well cell culture plate, setting a blank control group, an induced model group, a positive control group and a sample group, using dihydrotestosterone DHT as an inducer, adding the preset concentration of the cosmetic raw material test substance to the remaining groups and culturing for 16h~24h, and then using the Nile red staining method to detect the lipid droplet secretion level of each group; Step S32: Image J was used to statistically analyze the lipid droplet secretion levels of each group in step S31, and GraphPad Prism was used to draw graphs. One-way ANOVA statistical analysis was used to compare each group with the induced model group: when p < 0.05 and the inhibition rate ≥ 10%, it was considered that the cosmetic raw material test substance had a significant inhibitory effect on lipid droplet secretion, and T3 was 1 point.

[0014] Preferably, step S4 comprises: Step S41: culturing raw264.7 cells and collecting raw264.7 cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using CCK8 method to screen and obtain the corresponding test substance administration concentration when the raw264.7 cell survival rate is 90%; taking raw264.7 cells in the logarithmic growth phase and inoculating in a 24-well cell culture plate for culture, setting up a blank control group, an induced model group, a positive control group and a sample group, using lipopolysaccharide LPS to model and adding a preset concentration of the cosmetic raw material test substance to each group for culturing for 18h~24h, collecting the cell supernatant, and detecting the protein expression levels of NO gene, IL-6 gene and TNF-α gene by ELISA; Step S42: Calculate the expression results of NO gene, IL-6 gene and TNF-α gene of each group in step S41, and use GraphPad Prism to draw graphs. One-way ANOVA statistical analysis is used to compare each group with the induced model group: when p<0.05 and the inhibition rate is ≥10%, it is considered that the cosmetic raw material test substance has an inhibitory effect on the expression of NO gene, IL-6 gene and TNF-α gene, and is assigned 0.3 points. When the total score of the cosmetic raw material test substance in this model is greater than 0.5 points, the cosmetic raw material test substance gets 1.0 points in the in vitro macrophage anti-inflammatory soothing model, and T4 is 1 point.

[0015] Preferably, the cosmetic raw materials include one or more of chemical products, biological products, and plant extracts.

[0016] It should be noted that the cosmetic raw materials can be synthesized through chemical reactions, obtained through biological reactions, or extracted from plants.

[0017] Preferably, the cosmetic raw material test substance can be uniformly dissolved in the culture medium, and when dimethyl sulfoxide or ethanol is used as the solvent, the volume percentage concentration of the solvent shall not exceed 0.5% of the total volume of the complete culture medium, and the volume ratio of the solvent in the positive control group and the sample group is the same.

[0018] Preferably, the amount of bacterial suspension added in step S13 is (1.0-2.0)×10 6 CFU / mL; Hacat cells in step S22 were (1.0-4.0)×10 4 Cells were seeded in 96-well cell culture plates at a density of (2.0-5.0) × 10 5 cells / mL in a 12-well cell culture plate; in step S31, SZ95 cells were seeded at a density of 1.0×10 4Cells were inoculated into 96-well cell culture plates at a density of (5.0-8.0)×10 4 cells / mL in a 24-well cell culture plate; in step S41, RAW264.7 cells were seeded at a density of 1.0×10 4 Cells were seeded in 96-well cell culture plates at a density of (2.0-5.0) × 10 5 The cells were seeded at a density of cells / mL in 24-well cell culture plates.

[0019] Preferably, the test tubes of the positive control group in step S12 are composed of test tubes 1 to 9, the positive drug in the positive control group is an antibiotic, the antibiotics are gentamicin and crilimycin, and no antibiotics are added to test tubes 8-9; the positive control group in step S22 is DEX with a concentration of 10~50μM; the final concentration of DHT in S31 is 50~200μM, and the positive control group is DEX with a concentration of 10~50μM; the final concentration of LPS in S41 is 10~50μg / mL, and the positive control group is DEX with a concentration of 10~50μM, and 3 or more replicate wells are set for each cosmetic raw material test substance.

[0020] Preferably, the lipid droplet secretion amount in step S31 is detected using a Nile red staining kit; the inflammatory factor NO content in step S41 is detected using a NO detection kit, and the inflammatory factors TNF-α and IL-6 content are detected using an ELISA kit.

[0021] Preferably, the evaluation index analyzed in step S1 is the antibacterial effect on Propionibacterium acnes and Staphylococcus aureus; the evaluation index analyzed in step S2 is the relative expression of TLR-2, TLR-4, IL-8, and TSLP after Propionibacterium acnes is modeled in keratinocytes; the evaluation index analyzed in step S3 is the relative oil control rate of sebaceous gland cells; the evaluation index analyzed in step S4 is the relative expression of NO, IL-6, and TNF-α; through comprehensive evaluation of the above 10 indicators, the total score T of the tested substance is finally obtained, and when T≥0.5 points, the sample is said to have anti-acne efficacy; at the same time, the in vitro anti-acne efficacy of multiple cosmetic raw materials can be compared by ranking the total scores of the tested substances.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: The combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro of the present invention includes four in vitro models, covers multiple key nodes in the process of acne occurrence, and constructs an in vitro evaluation method for the anti-acne efficacy of cosmetic raw materials, which has the advantages of being comprehensive, efficient, and inexpensive. It provides data support for the initial screening of anti-acne raw materials, and also provides an effective basis for further research on animal replacement experiments in the skin care industry, skin care product development, and efficacy verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 One of the present application is the inhibition rate of Propionibacterium acnes in the AD test of the cosmetic raw material test substance in the embodiment; Figure 2 One of the present application is the inhibition rate of Staphylococcus aureus in the AD test of the cosmetic raw material test substance in the embodiment; Figure 3 One of the present application is the Hacat cell viability of the cosmetic raw material test substance AD ​​in the embodiment; Figure 4 One of the present application is the gene expression levels of TLR-2, TLR-4, IL-8, and TSLP of the cosmetic raw material test substance AD ​​in the Hacat in vitro anti-acne model; Figure 5 One of the present application is the SZ95 cell viability of the cosmetic raw material test substance AD ​​in the embodiment; Figure 6 One of the present application is the relative lipid droplet inhibition rate in the SZ95 cell oil control model of the cosmetic raw material test substance AD ​​in the embodiment; Figure 7 One of the present application is the RAW264.7 cell viability of the cosmetic raw material test substance AD ​​in the embodiment; Figure 8 One of the present application is NO, IL-6, and TNF-α in the supernatant of the cosmetic raw material test substance AD ​​in the in vitro RAW264.7 macrophage anti-inflammatory and soothing effect model in the embodiment; Fig. 9 One of the present application is a screening flow chart of the combination scheme for evaluating the anti-acne efficacy of cosmetic raw material subjects in vitro in the embodiments. DETAILED DESCRIPTION

[0024] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0025] Unless otherwise specified, the reagents and materials used in the examples can be purchased from the market.

[0026] Example 1

[0027] 1. Experimental Materials and Instruments 1.1 Strains and cells Propionibacterium acnes, Staphylococcus aureus, Hacat human keratinocytes, and RAW264.7 mouse macrophages were from ATCC, and SZ95 sebaceous gland cells were from Guangdong Boxi Biotechnology Co., Ltd.

[0028] 1.2 Main Reagents (1) Cell culture related reagents: BHI medium was purchased from Thermo Company, USA; DMEM medium, MEM medium, fetal bovine serum, and PBS were all purchased from Gibco Company, USA; (2) Gentomycin and criminicin were purchased from Aladdin; dexamethasone sodium phosphate (DEX) and lipopolysaccharide (LPS) were purchased from Sigma; dihydrotestosterone (DHT) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; DMSO was purchased from Sinopharm Chemical Reagent Co., Ltd.; TLR-2, TLR-4, IL-8, and TSLP primers were purchased from Shanghai Shengong Biotechnology Co., Ltd.; (3) CCK8 detection reagent was purchased from Japan Dojin Chemical Research Institute; Nile red staining kit was purchased from Shanghai Harling Biotechnology Co., Ltd.; mouse TNF-α ELISA kit and mouse IL-6 ELISA kit were purchased from Hangzhou Lianke Biotechnology Co., Ltd.; NO detection kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0029] 1.3 Solution preparation (1) DMEM complete medium for Hacat cells and RAW264.7 cells: 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. (2) Complete medium for SZ95 cells: Take 450 mL of MEM medium, add 50 mL of fetal bovine serum (FBS) and growth factors, mix thoroughly, and store in aliquots at 4°C. Preheat in a 37°C water bath before use. (3) Antibiotic stock solution: Dissolve gentamicin and criminicin powder in PBS to prepare a 10 mg / mL stock solution and store at -20°C away from light. (4) DHT stock solution: Dissolve the powder in DMSO to prepare a 100 mM stock solution and store at -20°C away from light. (5) DEX stock solution: Dissolve the powder in PBS to prepare a 50 mM stock solution and store at -20°C away from light. (6) LPS stock solution: Dissolve the powder in PBS to prepare a 1 mg / mL stock solution and store at -20°C away from light.

[0030] 1.4 Instruments Microplate reader (Thermo, USA), CO2 incubator (Thermo, USA), shaking incubator (Biostream, China), inverted microscope (Olympus, Japan), inverted fluorescence microscope (Leica, Germany), cell counter (Cellometer, USA), biological safety cabinet (Thermo, USA), high-speed low-temperature centrifuge (Thermo, USA), fluorescence quantitative PCR instrument (Thermo, USA), low-temperature refrigerator (Meiling, China), ultra-low temperature refrigerator (Thermo, USA), pipette (RAININ), ice maker (ZIEGRA).

[0031] 2. Raw materials to be tested The names of the cosmetic raw materials tested are shown in Table 1.

[0032] Table 1. serial number Sample name A Raw material A B Raw material B C Raw Material C D Raw material D 1. Establishment of in vitro antibacterial efficacy model 1. Bacterial Culture Resuscitate and stably culture Propionibacterium acnes strains and Staphylococcus aureus strains at 37°C in an anaerobic shaking incubator; 2. Dispense the sterilized Mueller-Hinton (MH) medium into sterile test tubes, each with 9 tubes as a group, set up a positive control group and a cosmetic raw material test substance group, of which 8 2mL test tubes are marked as test tubes 1-8, and 1 4mL test tube is marked as blank control tube 9; 3840μg / mL of the cosmetic raw material test substance is added to test tube No. 1, and the cosmetic raw material test substances in test tubes No. 2 to 7 are diluted in turn to 1920μg / mL, 960μg / mL, 480μg / mL, 240μg / mL, 120μg / mL, and 60μg / mL, and no cosmetic raw material test substance is added to tubes 8 and 9.

[0033] 3. Prepare the activated two strains to be tested with physiological saline to make bacterial suspensions respectively and inoculate them into test tubes 1 to 8, and do not add bacterial suspension to test tube 9; operate the two strains separately, and take out the observation culture medium after culturing for 18 to 24 hours, take 100 μL of each into a 96-well cell culture plate to measure the OD600nm absorbance, and calculate the relative antibacterial rate of the test substance.

[0034] 4. Relative antibacterial rate of the test substance The cell viability calculation formula is as follows: Relative inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100%; As: average absorbance of the well plate liquid. It should be noted that this refers to the liquid taken from test tubes 1 to 7 of the cosmetic raw material test group, the culture medium containing Propionibacterium acnes or Staphylococcus aureus, and the cosmetic raw material test substance; Ac: average absorbance of the liquid in the negative control well. It should be noted that this refers to the liquid taken out from test tube No. 8, which contains the culture medium of Propionibacterium acnes or Staphylococcus aureus and does not contain the cosmetic raw material test substance; Ab: average absorbance of blank control wells. It should be noted that this refers to the liquid taken out of test tube No. 9, i.e., the culture medium that does not contain Propionibacterium acnes or Staphylococcus aureus and the cosmetic raw material test substance.

[0035] 2. Establishment of in vitro keratinocyte anti-acne model 1. Cell Culture Hacat cells were cultured with complete culture medium at 37°C, 5% CO2 by volume and saturated humidity in a CO2 incubator 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 1:3.

[0036] 2. CCK8 Detection and Analysis of Cytotoxicity of Test Substances 2.1 Cell viability CCK8 detection Set up the cosmetic raw material test group, collect Hacat cells with good cell morphology and in the logarithmic growth phase, and use complete culture medium to prepare the concentration to 0.5~1.0×10 5 cell / mL, inoculated into 96-well cell culture plates, 100 μL per well, cultured in a CO2 incubator for 16-18 h, removed the original culture medium, added concentration gradient cosmetic raw material test substances prepared with fresh complete culture medium, set 6 concentrations for each test substance, the liquid raw material concentrations (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, 0.0316%, respectively, and the solid raw material concentrations were 1000 μg / mL, 316 μg / mL, 100 μg / mL, 31.6 μg / mL, 10 μg / mL, 3.16 μg / mL, respectively, 100 μL per well, set up a negative control group containing only cells and a blank control group, set up 3 replicates for each experimental well, added CCK8 detection solution after 18 h of incubation for 2-4 h, read the absorbance at a wavelength of 450 nm on an ELISA reader after shaking, and record the measurement results.

[0037] 2.2 Cell viability analysis and concentration determination The cell viability calculation formula is as follows: Cell survival rate = [(As1-Ab1) / (Ac1-Ab1)] × 100%; As1: average absorbance of the experimental well liquid. It should be noted that the experimental well liquid of the cosmetic raw material test group here contains cell culture medium, CCK8, and cosmetic raw material test substance; Ac1: average absorbance of the liquid in the negative control wells. It should be noted that the liquid in the negative control wells of the negative control group here contains cell culture medium and CCK8, but does not contain the cosmetic raw material test substance; Ab1: average absorbance of blank control wells. It should be noted that the blank control wells of the blank control group here do not contain culture medium and CCK8 of cells and cosmetic raw material test substances.

[0038] Calculation of 90% cell viability (CV90): LogCV90=((90-c)×Logb-(90-a)×Logd) / (ac); 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.

[0039] The cell viability of the test substance was calculated according to the above formula, and the concentration corresponding to CV90 or the concentration below CV90 was selected as the dosing concentration for evaluating the anti-acne efficacy.

[0040] 2.3 Preparation of Propionibacterium acnes bacterial solution Resuscitate and stably culture Propionibacterium acnes, pour the high-temperature sterilized agar medium into a sterile culture dish, wait for the culture medium to solidify, use an inoculation loop to streak the bacterial solution and then invert for culture, pick Propionibacterium acnes colonies in the culture medium and add 2-5wt% sebum mixture (purchased), culture at 37°C and 220 r / min for 3-7 days under shaking anaerobically, collect the supernatant after using a centrifuge, filter with a 0.22μm filter membrane to obtain the bacterial culture solution, and store in a -80°C refrigerator.

[0041] 2.4 Propionibacterium acnes secretions induce Hacat cell anti-acne model, and set up blank control group, induced model group, sample group and positive control group, among which, the blank control group was Hacat cells without adding any inducer or treatment agent; the induced model group was Hacat cells treated with Propionibacterium acnes secretions to simulate the pathogenesis of acne; the sample group was Hacat cells first treated with Propionibacterium acnes secretions, and then the cosmetic raw material test substances were added; the positive control group was Hacat cells treated with Propionibacterium acnes secretions, and then gentamicin and critamycin were added. After the administration of Propionibacterium acnes secretions in the induced model group, sample group and positive control group, the 24-well plate was placed in an incubator for 6 to 8 hours, and RNA extraction, reverse transcription and PCR amplification were performed on the cells in each group.

[0042] ΔCT = CT 目的基因 - CT 内参基因； ΔΔCT = ΔCT 样品组 - ΔCT 空白对照； Relative expression of target gene Fold Change = 2 -ΔΔCT。

[0043] 3. Establishment of in vitro sebaceous gland cell oil control model 1. Cell Culture SZ95 cells were cultured with complete culture medium at 37°C in a CO2 incubator with 5% volume percent 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.

[0044] 2. CCK8 Detection and Analysis of Cytotoxicity of Test Substances 2.1 Cell viability CCK8 detection The cosmetic raw material test group was set up, and SZ95 cells with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 2.0-5.0×10 5 cell / mL, inoculated into 96-well cell culture plates, 100 μL per well, cultured in a CO2 incubator for 16-18 h, removed the original culture medium, added concentration gradient test substances prepared with fresh complete culture medium, set 6 concentrations for each test substance, the liquid raw material concentrations (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, 0.0316%, respectively, and the solid raw material concentrations were 1000 μg / mL, 316 μg / mL, 100 μg / mL, 31.6 μg / mL, 10 μg / mL, 3.16 μg / mL, respectively, 100 μL per well, set up a negative control group containing only cells and a blank control group, set up 3 replicates for each experimental well, added CCK8 detection solution after 18 h of incubation for 2-4 h, and read the absorbance at a wavelength of 450 nm on an ELISA reader after shaking to record the measurement results.

[0045] 2.2 Cell viability analysis and concentration determination The cell viability calculation formula is as follows: Cell survival rate = [(As2-Ab2) / (Ac2-Ab2)] × 100%; As2: average absorbance of the test well liquid. It should be noted that the test well liquid of the cosmetic raw material test group here contains cell culture medium, CCK8, and cosmetic raw material test substance; Ac2: average absorbance of the liquid in the negative control wells. It should be noted that the liquid in the negative control wells of the negative control group here contains cell culture medium and CCK8, but does not contain the cosmetic raw material test substance; Ab2: average absorbance of blank control wells. It should be noted that the blank control wells of the blank control group here do not contain culture medium and CCK8 of cells and cosmetic raw material test substances.

[0046] Calculation of 90% cell viability (CV90): LogCV90=((90-c)×Logb-(90-a)×Logd) / (ac); 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.

[0047] The cell viability of the test substance was calculated according to the above formula, and the concentration corresponding to CV90 or the concentration below CV90 was selected as the administration concentration for evaluating the oil control efficacy.

[0048] 3. DHT induced SZ95 oil control model, set up blank control group, induced model group, sample group and positive control group, among which, the blank control group was SZ95 cells without any inducer or treatment agent; the induced model group was SZ95 cells treated with DHT to simulate the state of androgen-induced increased sebum secretion; the sample group was SZ95 cells treated with DHT first, and then the cosmetic raw material test substance was added; the positive control group was SZ95 cells treated with DHT and then DEX was added. After the administration of DHT in the induced model group, sample group and positive control group, the 24-well plate was placed in an incubator and cultured for 24-30 hours, and the cells in each group were stained with Nile red and photographed using a 20x objective lens of a fluorescence microscope. Five areas with similar plating rates were selected for each group, and one photo was taken for each area. ImageJ was used to count the cell lipid droplet secretion and calculate the lipid droplet inhibition rate.

[0049] Inhibition rate (%) = (1-T / C) × 100%; Where: T—average of the mean fluorescence intensity of Nile red in the sample group; C—Average of the mean fluorescence intensity of Nile red in the induced model group.

[0050] IV. Establishment of RAW264.7 macrophage anti-inflammatory and soothing effect model 1. Cell Culture RAW264.7 cells were cultured with complete culture medium at 37°C in a CO2 incubator with 5% CO2 and saturated humidity until the logarithmic growth phase.

[0051] 2. CCK8 Detection and Analysis of Cytotoxicity of Test Substances 2.1 Cell viability assay The cosmetic raw material test group was set up, and RAW264.7 cells with good cell morphology and in the logarithmic growth phase were collected and prepared with complete culture medium to a concentration of 1.0×10 5 cell / mL, inoculated into 96-well cell culture plates, 100 μL per well, cultured in a CO2 incubator for 16-18 h, removed the original culture medium, added concentration gradient test substances prepared with fresh complete culture medium, set 6 concentrations for each test substance, the liquid raw material concentrations (v / v) were 10.0%, 3.16%, 1.00%, 0.316%, 0.100%, 0.0316%, respectively, and the solid raw material concentrations were 1000 μg / mL, 316 μg / mL, 100 μg / mL, 31.6 μg / mL, 10 μg / mL, 3.16 μg / mL, respectively, 100 μL per well, set up a negative control group containing only cells and a blank control group, set up 3 replicates for each experimental well, added CCK8 detection solution after 18 h of incubation for 2-4 h, and read the absorbance at a wavelength of 450 nm on an ELISA reader after shaking to record the measurement results.

[0052] 2.2 Cell viability analysis and concentration determination The cell viability calculation formula is as follows: Cell survival rate = [(As3-Ab3) / (Ac3-Ab3)] × 100%; As3: average absorbance of the test well liquid. It should be noted that the test well liquid of the cosmetic raw material test group here contains cell culture medium, CCK8, and volume percentage concentration of the test substance; Ac3: average absorbance of the liquid in the negative control wells. It should be noted that the liquid in the negative control wells of the negative control group here contains cell culture medium and CCK8, but does not contain the test substance in volume percentage concentration; Ab3: average absorbance of blank control wells. It should be noted that the blank control wells of the blank control group here do not contain cells and culture medium and CCK8 with a volume percentage concentration of the test substance.

[0053] Calculation of 90% cell viability (CV90): LogCV90=((90-c)×Logb-(90-a)×Logd) / (ac); 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.

[0054] The cell viability of the test substance was calculated according to the above formula, and the concentration corresponding to CV90 or the concentration below CV90 was selected as the anti-inflammatory and soothing verification dosing concentration.

[0055] 3.1 Detection of NO, TNF-α, and IL-6 secretion in the supernatant of the LPS-induced RAW264.7 macrophage anti-inflammatory and soothing effect model: RAW264.7 cells with good cell morphology and in the logarithmic growth phase were collected and prepared with complete medium to a concentration of 3-5×10 5 cell / mL was inoculated in a 24-well cell culture plate, 1 mL per well, and cultured in a CO2 incubator for 16-18 h. The original culture medium was removed, and a blank control group, an induced model group and a sample group were set up. Complete culture medium was added to the blank control group and the induced model group, and the sample group was added with the test substance of the corresponding concentration of CV90 prepared by complete culture medium. The cells were cultured for 4 h at 37°C, 5% CO2 and saturated humidity. Except for the cell control group, LPS was added to each well to a final concentration of 1 μg / mL, and the cells were cultured for 20 h at 37°C, 5% CO2 and saturated humidity. The cell culture medium was collected, and the cells were centrifuged at 2000 rpm and 4°C for 10 min. The supernatant was collected, and the TNF-α, IL-1β and IL-6 contents of the supernatant were detected by ELISA kits, and the NO content of the supernatant was detected by NO detection kits. The operating steps were referred to the instructions of the kits.

[0056] 3.2 Data processing: Select data software to calculate the standard curve, substitute the OD value of the sample to be tested into the standard curve to calculate the content of NO, TNF-α, and IL-6, and calculate the relative expression with the LPS-induced model group as 100%. The calculation formula is as follows: Relative expression level = sample group content / induced model group content 5. Results Analysis GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. One-way ANOVA was used for statistical analysis among the groups. All statistical analyses were two-tailed, and p < 0.05 was considered to be significantly different.

[0057] 4.1 In vitro antimicrobial efficacy model evaluation results 4.1.1 Results of in vitro anti-Propionibacterium acnes activity of samples According to the in vitro antibacterial test results of raw material samples AD, the inhibition rate of each against Propionibacterium acnes was calculated and the antibacterial curve was drawn. Figure 1 .

[0058] 4.1.2 Results of in vitro anti-Staphylococcus aureus activity of samples According to the in vitro antibacterial test results of raw material samples AD, the inhibition rate of each against Propionibacterium acnes was calculated and the antibacterial curve was drawn. Figure 2 .

[0059] 4.1.3 Results of in vitro antimicrobial efficacy model evaluation of samples Table 2.

[0060] 4.2 Evaluation results of the in vitro keratinocyte anti-acne model 4.2.1 Hacat cytotoxicity test of samples According to the cell viability CCK8 test results of raw material samples AD, the corresponding raw material sample administration concentration when the Hacat cell viability 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 3 .

[0061] Table 3. serial number CV90 A 0.77mg / mL B 0.18mg / mL C 0.6mg / mL D 2.1% 4.2.2 Related gene inhibition Hacat cells were collected for RNA extraction, reverse transcription, and qPCR, and the relative expression levels of TLR2, TLR4, IL-8, and TSLP genes in the cells were detected and calculated. The test results are detailed in Figure 4 .

[0062] 4.3 Evaluation results of the sample in vitro sebaceous gland cell oil control model 4.3.1 Sample SZ95 cytotoxicity test According to the cell viability CCK8 test results of raw material samples AD, the corresponding raw material sample administration concentration when the SZ95 cell viability is 90% (CV90) is calculated, and the subsequent administration is incubated according to this concentration. The test results are detailed in Table 4 and Figure 5 .

[0063] Table 4. serial number CV90 A 0.31mg / mL B 0.1mg / mL C 0.26mg / mL D 1.9% 4.3.2 Relative lipid droplet inhibition rate of SZ95 sebaceous gland cells The lipid droplet secretion of SZ95 cells was observed and photographed using a fluorescence microscope, and the relative lipid droplet inhibition rate of the raw material sample AD was calculated. Figure 6 .

[0064] 4.4 Evaluation results of the anti-inflammatory and soothing effect model of sample RAW264.7 macrophages 4.4.1 Cytotoxicity test of RAW264.7 samples According to the cell viability CCK8 test results of sample AD, the corresponding raw material sample dosage concentration when the cell viability of the RAW264.7 macrophage model is 90% (CV90) is calculated, and the subsequent administration is incubated according to this concentration. The test results are detailed in Table 5 and Figure 7 .

[0065] Table 5. serial number CV90 A 1.1 mg / mL B 0.15 mg / mL C 0.36 mg / mL D 2% 4.4.2 NO, TNF-α, and IL-6 secretion in cell supernatant The supernatant of RAW264.7 cells was collected and the secretion of inflammatory factors NO, TNF-α, and IL-6 in the supernatant was detected and calculated according to the instructions of the NO detection kit and ELISA kit, and the relative secretion of the LPS-induced model group as the control was calculated. Figure 8 .

[0066] 4.4 Comprehensive evaluation results and analysis All samples were evaluated according to the method flow of the present invention. The four test substances detected in this example were based on Fig. 9 The screening process shown was tested, and evaluation and scoring were performed according to the scoring criteria in Tables 6-9. The total scores of the experiments under different models were summarized in Table 10. The anti-acne efficacy of the raw materials could be screened and ranked according to the total score results.

[0067] Table 6.

[0068] Table 7.

[0069] Table 8.

[0070] Table 9.

[0071] Summary of sample AD scores under each model: Table 10.

[0072] See also Fig. 9 The combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro of the present invention combines the test results of four in vitro models to score and screen the anti-acne efficacy of the target raw materials, and comprises the following steps: S1, the establishment and use of the model for evaluating the antimicrobial effect of raw material test substances, includes the following steps: S11, stable culture of Propionibacterium acnes and Staphylococcus aureus; the sterilized Mueller-Hinton (MH) medium was dispensed into sterile test tubes, 9 in a group, of which 1 4 mL tube was labeled as tube 1, and the other 9 2 mL tubes were labeled as tubes 2-9. 3840 μg / mL of the cosmetic raw material test substance was added to tube 1, and tubes 2-7 were diluted in turn by half, and no test substance was added to tubes 8-9.

[0073] S12, prepare the activated test strain into bacterial suspension with physiological saline and inoculate it into tubes 1 to 8. Operate on two strains separately. After culturing for 18 to 24 hours, take out the observation culture medium, take 100 μL of each well into a 96-well plate to measure the OD600nm absorbance, and calculate the relative inhibition rate of the test substance.

[0074] S13, collect the relative inhibition rate of the test substance against Propionibacterium acnes and Staphylococcus aureus at 480-1920 μg / mL. The inhibition rate <40% is considered as no inhibition, and 0 points are assigned; 40%≤inhibition rate <80% is considered as a certain inhibition, and 0.3 points are assigned; inhibition rate ≥80% is considered as a strong inhibition, and 1.0 points are assigned. When the total score of a test substance is >0.5 points, the test substance gets 1.0 points in the in vitro antibacterial model, and T1 is 1 point. The score of the test substance in the in vitro antibacterial model is calculated.

[0075] S2, the establishment and use of the in vitro keratinocyte anti-acne model for evaluating the raw material test object includes the following steps: S21, pick Propionibacterium acnes colonies in the culture medium and add 2-5wt% sebum mixture, culture anaerobically at 37℃ and 220 r / min for 3-7 days, collect the supernatant after centrifugation, filter with 0.22μm filter membrane to obtain bacterial culture solution, and store in a -80℃ refrigerator.

[0076] S22, the CCK8 method was used to screen and obtain the corresponding test substance dosage concentration when the Hacat cell survival rate was 90%; Hacat cells in the logarithmic growth phase were inoculated in a 12-well plate for culture, and a blank control group, an induced model group, a positive control group and a sample group were set up. The model was established using 5% of the bacterial culture solution prepared in S21, and the preset concentration of the test substance was added to each group for culture for 16h~24h, and then the cell RNA was collected to detect the expression levels of related genes TLR-2, TLR-4, IL-8, and TSLP.

[0077] S23, calculated the expression results of related genes of each group in S22, and used GraphPad Prism to draw graphs. One-way ANOVA statistical analysis was used to compare each group with the induced model group: for each factor, when p < 0.05 and the inhibition rate ≥ 15%, it indicated that it had a significant inhibitory effect compared with the induced model group; when a test substance had an effective inhibitory effect on at least one factor in the model, it would score 1.0, and T2 would score 1 point. The scores of the test substances in the in vitro keratinocyte anti-acne model were statistically analyzed.

[0078] S3, the establishment and use of the in vitro sebaceous gland cell oil control model for evaluating the raw material test object includes the following steps: S31, the CCK8 method was used to screen the corresponding test substance dosage concentration when the SZ95 cell survival rate was 90%; SZ95 cells in the logarithmic growth phase were inoculated in a 24-well plate, and a blank control group, an induced model group, a positive control group and a sample group were set up. Dihydrotestosterone DHT was used as an inducer, and the corresponding concentrations of the test substances were added to the other groups and cultured for 16h~24h, and then the Nile red staining method was used to detect the lipid droplet secretion level of each group.

[0079] S32, image J was used to count the lipid droplet secretion levels of each group in S31, and GraphPad Prism was used to draw graphs. One-way ANOVA statistical analysis was used to compare each group with the induced model group: when p < 0.05 and the inhibition rate ≥ 10%, the test substance was considered to have a significant inhibitory effect on lipid droplets, and scored 1.0 in this model, then T3 was 1 point, and the scores of the test substances in the SZ95 cell oil control model were counted.

[0080] S4, the establishment and use of a model for evaluating the anti-inflammatory and soothing effects of the raw material test subject RAW264.7 macrophages in vitro, including the following steps: S41. The CCK8 method was used to screen and obtain the corresponding test substance dosage concentration when the survival rate of raw264.7 cells was 90%. Raw264.7 cells in the logarithmic growth phase were inoculated in a 24-well plate for culture. A blank control group, an induced model group, a positive control group and a sample group were set up. Lipopolysaccharide LPS was used to model and the preset concentration of the test substance was added to each group for 18h~24h of culture. The cell supernatant was collected and the protein expression levels of NO, IL-6 and TNF-α were detected by ELISA.

[0081] S42, calculate the expression results of relevant genes of each group in S41, use GraphPad Prism to draw graphs, and use One-way ANOVA statistical analysis to compare each group with the induced model group: when p < 0.05 and the inhibition rate ≥ 10%, the test substance is considered to have a significant inhibitory effect on the factor and is assigned 0.3 points. When the total score of the test substance in this model is > 0.5 points, the test substance gets 1.0 points in the in vitro macrophage anti-inflammatory soothing model, and T4 is 1 point. The scores of the test substances in the in vitro macrophage anti-inflammatory soothing model are calculated.

[0082] S5, summarize the total score T of the raw material test substances in each model of S13, S23, S32, and S42. When T≥0.5, it is considered that the raw material test substance has a certain anti-acne effect.

[0083] In summary, the combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro of the present invention adopts multiple types of in vitro models, which basically covers the comprehensive evaluation of cosmetic anti-acne raw materials in combination with key nodes in the acne occurrence process, and has the advantages of being comprehensive, sensitive, rapid and cost-saving in the raw material screening stage of cosmetics.

[0084] 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, any changes or modifications to the above embodiments will fall within the scope of the claims of the present invention.

Claims

1. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro, characterized in that: The combination method comprises the following steps: Step S1: Establishing an in vitro antibacterial efficacy model for the cosmetic raw material to be evaluated and scoring it, where the score is recorded as T1; Step S2: establishing an in vitro keratinocyte anti-acne model for the cosmetic raw material to be evaluated and scoring it, where the score is recorded as T2; Step S3: establishing an in vitro sebaceous gland cell oil control model for the cosmetic raw material to be evaluated and scoring it, where the score is recorded as T3; Step S4: establishing a model for the anti-inflammatory and soothing effect of the RAW264.7 macrophages of the cosmetic raw material to be evaluated and scoring the model, wherein the score is recorded as T4; Step S5: Calculate the total score and give the in vitro anti-acne efficacy evaluation result of the cosmetic raw material. The total score calculation formula is as follows: ;in, a1 is the scoring weight coefficient of the in vitro antibacterial efficacy model, a2 is the scoring weight coefficient of the in vitro keratinocyte anti-acne model, a3 is the scoring weight coefficient of the in vitro sebaceous gland cell oil control model, and a4 is the scoring weight coefficient of the RAW264.7 macrophage anti-inflammatory and soothing effect model. Here, a1, a2, a3, and a4 are all 0.25; if the total score T is not less than the preset score, the in vitro anti-acne efficacy evaluation result of the cosmetic raw material is valid, and the preset score is 0.

5.

2. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 1, characterized in that: The step S1 comprises: Step S11: Stably culturing Propionibacterium acnes and Staphylococcus aureus; Step S12: setting test tubes for cosmetic raw material test substance groups and test tubes for positive control groups, wherein each group of test tubes for the cosmetic raw material test substance groups consists of test tubes No. 1 to No. 9, and the sterilized MH culture medium is divided into test tubes No. 1 to No. 9 of the cosmetic raw material test substance group, and 3840 μg / mL of the cosmetic raw material test substance is added to test tube No. 1 of the cosmetic raw material test substance group, and test tubes No. 2 to No. 7 are sequentially added with the cosmetic raw material test substance diluted by half, and test tubes No. 8 to No. 9 are not added with the cosmetic raw material test substance, then tube No. 8 is the negative control, and tube No. 9 is the blank control; in the positive control group, the cosmetic raw material test substances in test tubes No. 1 to No. 7 of the raw material test substance group are replaced with antibiotics; Step S13: The activated strains of Propionibacterium acnes and Staphylococcus aureus were prepared into bacterial suspensions with physiological saline, and added to test tubes 1 to 8 of different cosmetic raw material test groups, respectively. After culturing for 18 to 24 hours, 100 μL of liquid was taken from test tubes 1 to 9 of the cosmetic raw material test group to a 96-well cell culture plate to measure the OD600nm absorbance, and the relative antibacterial rate of the cosmetic raw material test at each concentration was calculated. The calculation formula of the relative antibacterial rate is as follows: Relative inhibition rate = [1-(As-Ab) / (Ac-Ab)] × 100%, where: As is the average absorbance of the liquid in the well plate, which involves the liquid taken out from test tubes 1 to 7 of the cosmetic raw material test group; Ac is the average absorbance of the liquid in the negative control well, which involves the liquid taken out from test tube 8; Ab is the average absorbance of the blank control well, which involves the liquid taken out from test tube 9; Step S14: Calculate the As value of the liquid taken out of each tube, and calculate the relative inhibition rate value of the corresponding test substance against Propionibacterium acnes and Staphylococcus aureus. For Propionibacterium acnes or Staphylococcus aureus, if the relative inhibition rate corresponding to any test tube from 2 to 4 is less than 40%, 0 points are assigned; if 40%≤the relative inhibition rate corresponding to any test tube from 2 to 4 is less than 80%, 0.3 points are assigned; if the relative inhibition rate corresponding to any test tube from 2 to 4 is ≥80%, 1 point is assigned; when the sum of the points assigned to the cosmetic raw material test substance in any test tube from 2 to 4 against Propionibacterium acnes and Staphylococcus aureus is greater than 0.5 points, T1 is 1 point.

3. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 2, characterized in that: The step S2 comprises: Step S21: Pick the Propionibacterium acnes colonies after stable culture in the culture medium and add 2-5wt% of sebum mixture by mass percentage, culture at 37°C and 220r / min for 3-7 days, collect the supernatant by centrifuge, filter with 0.22μm filter membrane to obtain Propionibacterium acnes culture fluid, and store in a -80°C refrigerator; Step S22: culturing Hacat cells and collecting Hacat cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating them in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using the CCK8 method to screen and obtain the corresponding dosage concentration of the cosmetic raw material test substance when the Hacat cell survival rate is 90%, taking the Hacat cells in the logarithmic growth phase and inoculating them in a 12-well cell culture plate for culture, setting up a blank control group, an induced model group, a positive control group and a sample group, using 5% by mass of the Propionibacterium acnes culture solution prepared in step S21 to model, and adding a preset concentration of the cosmetic raw material test substance to each group for culture for 16h~24h, and then collecting cell RNA to detect the expression levels of related genes TLR-2, TLR-4, IL-8, and TSLP; Step S23: Calculate the expression results of relevant genes of each group in step S22, use GraphPad Prism to draw a graph, and use One-way ANOVA statistical analysis to compare each group with the induced model group: for each gene, when p < 0.05 and the inhibition rate ≥ 15%, it indicates that it has a significant inhibitory effect compared with the induced model group; when a cosmetic raw material test substance has an inhibitory effect on at least 1 gene in the model, T2 is 1 point.

4. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 3, characterized in that: The step S3 comprises: Step S31: culturing SZ95 cells and collecting SZ95 cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating them in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using the CCK8 method to screen and obtain the corresponding dosage concentration of the cosmetic raw material test substance when the SZ95 cell survival rate is 90%; taking the logarithmic growth phase SZ95 cells and inoculating them in a 24-well cell culture plate, setting a blank control group, an induced model group, a positive control group and a sample group, using dihydrotestosterone DHT as an inducer, adding the preset concentration of the cosmetic raw material test substance to the remaining groups and culturing for 16h~24h, and then using the Nile red staining method to detect the lipid droplet secretion level of each group; Step S32: Image J was used to statistically analyze the lipid droplet secretion levels of each group in step S31, and GraphPad Prism was used to draw graphs. One-way ANOVA statistical analysis was used to compare each group with the induced model group: when p < 0.05 and the inhibition rate ≥ 10%, it was considered that the cosmetic raw material test substance had a significant inhibitory effect on lipid droplet secretion, and T3 was 1 point.

5. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 4, characterized in that: The step S4 comprises: Step S41: culturing raw264.7 cells and collecting raw264.7 cells with good cell morphology and in the logarithmic growth phase, culturing with complete medium and then inoculating in a 96-well cell culture plate, removing the original medium, adding a concentration gradient cosmetic raw material test substance configured with fresh complete medium; using CCK8 method to screen and obtain the corresponding test substance administration concentration when the raw264.7 cell survival rate is 90%; taking raw264.7 cells in the logarithmic growth phase and inoculating in a 24-well cell culture plate for culture, setting up a blank control group, an induced model group, a positive control group and a sample group, using lipopolysaccharide LPS to model and adding a preset concentration of the cosmetic raw material test substance to each group for culturing for 18h~24h, collecting the cell supernatant, and detecting the protein expression levels of NO gene, IL-6 gene and TNF-α gene by ELISA; Step S42: Calculate the expression results of NO gene, IL-6 gene and TNF-α gene of each group in step S41, and use GraphPadPrism to draw graphs. One-way ANOVA statistical analysis is used to compare each group with the induced model group: when p<0.05 and the inhibition rate is ≥10%, it is considered that the cosmetic raw material test substance has an inhibitory effect on the expression of NO gene, IL-6 gene and TNF-α gene, and is assigned 0.3 points. When the total score of the cosmetic raw material test substance in this model is greater than 0.5 points, the cosmetic raw material test substance gets 1.0 points in the in vitro macrophage anti-inflammatory soothing model, and T4 is 1 point.

6. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 5, characterized in that: The cosmetic raw materials include one or more of chemical products, biological products, and plant extracts.

7. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to any one of claims 3 to 5, characterized in that: The cosmetic raw material test substance can be uniformly dissolved in the culture medium, and when dimethyl sulfoxide or ethanol is used as the solvent, the volume percentage concentration of the solvent shall not exceed 0.5% of the total volume of the complete culture medium, and the volume ratio of the solvent in the positive control group and the sample group is the same.

8. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 5, characterized in that: The amount of bacterial suspension added in step S13 is (1.0-2.0)×10 6 CFU / mL; Hacat cells in step S22 were (1.0-4.0)×10 4 Cells were seeded in 96-well cell culture plates at a density of (2.0-5.0) × 10 5 cells / mL in a 12-well cell culture plate; in step S31, SZ95 cells were seeded at a density of 1.0×10 4 Cells were inoculated into 96-well cell culture plates at a density of (5.0-8.0)×10 4 cells / mL in a 24-well cell culture plate; in step S41, RAW264.7 cells were seeded at a density of 1.0×10 4 Cells were seeded in 96-well cell culture plates at a density of (2.0-5.0) × 10 5 The cells were seeded at a density of cells / mL in 24-well cell culture plates.

9. A combined method for evaluating the anti-acne efficacy of cosmetic raw materials in vitro according to claim 5, characterized in that: The test tubes of the positive control group in step S12 consist of test tubes 1 to 9, the positive drug in the positive control group is an antibiotic, and no antibiotic is added to test tubes 8-9; the positive control group in step S22 is DEX with a concentration of 10~50μM; the final concentration of DHT in S31 is 50~200μM, and the positive control group is DEX with a concentration of 10~50μM; the final concentration of LPS in S41 is 10~50μg / mL, and the positive control group is DEX with a concentration of 10~50μM, and 3 or more replicate wells are set for each cosmetic raw material test substance.

10. A combined method for evaluating the in vitro anti-acne efficacy of cosmetic raw materials according to claim 5, characterized in that: In step S31, the lipid droplet secretion amount is detected by a Nile red staining kit; in step S41, the inflammatory factor NO content is detected by a NO detection kit, and the inflammatory factor TNF-α and IL-6 content are detected by an ELISA kit.

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