A method for evaluating anti-dandruff efficacy based on zinc enzyme homeostasis

Abstract

The invention discloses a method for evaluating the anti-dandruff efficacy based on zinc enzyme homeostasis, comprising: performing cytotoxicity detection on a test object; culturing cells and dividing them into groups, adding TPEN to a model control group, adding TPEN and a test object of a safe concentration to an experimental group, removing the culture medium after incubation of each group, adding a total RNA extraction reagent to cells collected by centrifugation and storing them; performing gene expression detection on the cells collected from each group; constructing a zinc-deficient skin model and performing detection; the invention constructs a scalp desquamation laboratory model based on the principle of zinc enzyme imbalance, uses cells as an experimental system, has high throughput and high sensitivity, can quickly screen functional raw materials, and shortens the formulation development time; the skin model used in the invention is closer to the actual human body situation, and is suitable for any raw material, mixture, formulation and product testing; the invention has the advantages of comprehensive detection parameters and high specificity through detecting specific gene expression and tissue morphology observation, and can evaluate the anti-dandruff efficacy of the test object as a whole.

Description

Technical Field

[0001] The invention relates to an anti-dandruff efficacy evaluation method, in particular to an anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis. Background Art

[0002] Zinc is one of the essential trace elements for the human body. It has anti-inflammatory, antioxidant, immune regulation, antibacterial and antiviral effects, and participates in many aspects of collective cellular metabolism.

[0003] Zinc homeostasis is dynamically regulated by zinc transporters (ZnTs), Zrt- and irt-like proteins (zips), and zinc storage proteins metallothioneins (MTs). The broad zinc transporter family includes two transmembrane protein families involved in zinc transport: the zinc transporter family (ZnTs) and the zinc-regulated transporter and iron-regulated transporter like protein family (ZIPs). The main function of the ZnTs family is to expel excess zinc ions from the cell or transfer them into vesicles for storage, thereby reducing the intracellular zinc concentration. The main function of the ZIPs family is to take up zinc ions. Ion channels have been proven to play an important role in barrier repair. Zinc ions can act as neurotransmitters to produce physiological activities through ion channels, and have the function of regulating and controlling voltage-gated ion channels and ligand-gated pore-type ion channels. In addition, zinc can be retained in metallothionein reserves or transferred in metal transporters of ZIPs and ZnTs family transporters.

[0004] Zinc is involved in the formation of a variety of zinc-containing metalloenzymes. It is known that zinc is a cofactor for more than 200 enzymes and metalloproteins, such as carbonic anhydrase, alkaline phosphatase, carboxypeptidase, thymidine nucleotidase, DNA and RNA polymerase, etc. When zinc transporters mutate or become disordered, it will cause zinc enzyme dysfunction, which manifests in the following aspects:

[0005] (1) Inflammatory response: Zinc participates in histone deacetylation and gene transcription involving zinc finger transcription factors. A decrease in intracellular zinc ion concentration will affect the function of macrophages, neutrophils, and natural killer cells, as well as complement activity, leading to the production of inflammatory mediators.

[0006] (2) Reduced antioxidant activity: Zinc exerts its antioxidant effect through two direct or indirect pathways. In the first pathway, Zn2+ ions bind to the active center of antioxidant enzymes, such as superoxide dismutase, as auxiliary metals in the structural center, zinc and copper (SOD1 type), thereby stimulating the production of proteins, such as sulfhydryl-rich metallothioneins (MT), such as the high chemical affinity of zinc sulfate. Studies have found that zinc protects the skin from UV radiation-induced immunosuppression by inducing MT production. In the second pathway, zinc can enhance the activity of antioxidant enzymes, such as glutathione catalase (GSH) or superoxide dismutase (SOD), and reduce the induced oxidation potential by reducing the activity of inducible nitrate synthase (iNOS) and NADPH oxidase. In addition, zinc also has an indirect antioxidant effect. Zinc has a protective effect on skin fibroblasts exposed to UVA and UVB radiation, reducing cytotoxicity and lipid peroxidation. Due to the imbalance of zinc homeostasis, the intracellular zinc ion concentration decreases, which in turn triggers a decrease in antioxidant enzyme activity.

[0007] Skin barrier function damage caused by zinc enzyme imbalance occurs on the scalp in the form of physiological desquamation. Among them, kallikrein-related peptidases (KLKs) play an important role in physiological desquamation as zinc metalloenzymes. Kininase-related peptidases (KLKs) are involved in various events such as skin inflammation, wound healing, itching, antibacterial activity and viral susceptibility. Kininase-related peptidase 5 (KLK5) and kininase-related peptidase 7 (KLK7) are mainly responsible for physiological desquamation. Kininase-related peptidase 5 (KLK5) can cleave all three corneodesmosin constituent proteins: desmoglein 1 (DSG1), desmocollin 1 (DSC1) and corneodesmosin (CDSN). KLK7 can cleave DSC1 and CDSN. Zinc ions can inhibit the activity of KLK5, KLK7 and kininase-related peptidase 8 (KLK8).

[0008] At present, the main test methods for evaluating the anti-dandruff efficacy of hair products include human test methods and in vitro test samples for antibacterial or bactericidal properties. The human test method includes clinical evaluation by researchers before and after use, self-evaluation by patients, and detection of Malassezia on the scalp. Although the evaluation results of the human test method are direct and accurate, it has the defects of long test cycle and insufficient detection throughput. In addition, it is mainly based on finished product testing, and cannot detect cosmetic raw materials. The screening effect of raw materials in the early stage of research and development is not ideal, and the detection method does not match the market demand. Most in vitro test methods test the antibacterial or bactericidal effects of samples. Many methods only target the aspect of microecological imbalance in the mechanism of scalp desquamation, and there is no solution for desquamation caused by scalp barrier damage caused by zinc deficiency or zinc enzyme imbalance. Summary of the invention

[0009] The purpose of the present invention is to provide an anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis, which is simple to operate, highly standardized, has intuitive experimental results, accurately evaluates the anti-dandruff effect, has a short experimental cycle, and can detect cosmetic raw materials. The method can be used to evaluate the repair effect of the test substance on scalp barrier damage.

[0010] The above-mentioned object of the present invention is achieved by the following technical scheme: a method for evaluating the anti-dandruff efficacy based on zinc enzyme homeostasis, characterized by comprising the following steps:

[0011] S1. Conduct cytotoxicity test on the test substance to determine the safe concentration of the test substance;

[0012] S2. Cell culture was performed and divided into a negative control group, a model control group and an experimental group. The negative control group was not treated with any treatment. The model control group was added with TPEN to construct a zinc-deficient cell model. The experimental group was added with TPEN and a safe concentration of the test substance during the cell culture process. After incubation, the culture medium was removed from each group, and the cells collected by centrifugation were added with a total RNA extraction reagent and then stored.

[0013] S3, performing gene expression detection on the cells collected from each group, if the relative gene expression of the model control group is significantly downregulated compared with the relative gene expression of the negative control group, it means that the zinc-deficient cell model is completed, otherwise, it goes to step S2; when the zinc-deficient cell model is completed, if the relative gene expression of the experimental group is significantly upregulated compared with the relative gene expression of the model control group, it is preliminarily determined that the test substance has the effect of removing dandruff;

[0014] S4. Construct a zinc-deficient skin model and conduct tests, including:

[0015] (1) Pre-incubating a skin model, wherein the skin model is a three-dimensional simulated human skin having barrier function and metabolic function;

[0016] ⑵The pre-incubated skin models were divided into a negative control group, a model control group and an experimental group. The negative control group was not treated with any treatment, the model control group was washed with a buffer solution, and the surface of the skin model in the experimental group was treated with different concentrations of the test substance;

[0017] ⑶ The negative control group, the model control group and the experimental group were re-incubated, wherein the re-incubation of the experimental group was performed after the test substance on the skin model was cleaned, and TPEN was added to the culture medium of the model control group and the experimental group;

[0018] (4) After the re-incubation, the cell activity of each group was tested, and the cell activity was required to be greater than or equal to 50%;

[0019] (5) Gene expression test was performed on each group that met the cell activity requirements. If the relative gene expression of the model control group was significantly downregulated compared with the relative gene expression of the negative control group, it indicated that the zinc-deficient skin model was successfully constructed. In this case, if the relative gene expression of the experimental group was significantly upregulated compared with the relative gene expression of the model control group, it indicated that the test substance had the effect of removing dandruff;

[0020] (6) Perform tissue morphological sections on each group. If the desquamation of the skin model tissue in the model control group is significantly higher than that in the negative control group, it means that the experimental model is successfully constructed. In this case, if the desquamation of the skin model tissue in the test substance group is significantly lower than that in the model control group, it is finally determined that the test substance has an anti-dandruff effect.

[0021] The TPEN of the present invention is a zinc ion chelating agent tetrapyridylmethylethylenediamine.

[0022] The present invention constructs a scalp desquamation laboratory model based on the principle of zinc enzyme imbalance, uses cells as the experimental system, has high throughput and high sensitivity, can quickly screen effective raw materials, and shorten the formulation development time; the skin model used in the present invention is relatively stable in technology and highly standardized, is a three-dimensional simulated human skin with barrier function and metabolic function, can directly contact the sample, is suitable for the testing of any raw materials, mixtures, formulas and products, and is not affected by physical properties such as sample solubility and color, and is closer to the actual situation of the human body; the present invention detects specific gene expressions of cellular antioxidant enzymes, cellular glycoproteins and skin barrier kininase, and observes tissue morphology, has the advantages of comprehensive detection parameters and high specificity, and can comprehensively evaluate the desquamation efficacy of the test object.

[0023] In the step S1 of the present invention, the cell activity of the blank control group is taken as 100%, the relative cell activity of each group is calculated, and the test substance concentration at which the relative cell activity is greater than or equal to 90% is selected as the appropriate test substance concentration.

[0024] In step S4⑵ of the present invention, each group sets up at least three parallel models, and the experimental group sets up at least three parallel models corresponding to each concentration of the test substance, and performs cell activity test, gene expression test and tissue morphology section respectively.

[0025] In the step S4⑵ of the present invention, the treatment dosage of the test substance is 10-50 μL, and the treatment time is 15-60 min.

[0026] In the steps S2 and S4 of the present invention, the concentration of TPEN added is 2 to 8 μmol / L.

[0027] The cells used in steps S1 and S2 of the present invention are immortalized keratinocytes (HaCaT), primary isolated keratinocytes, and the like.

[0028] In the step S2 of the present invention, the genes related to antioxidant enzymes and cell glycoproteins in the cells are SOD1, CAT, CDH1, VCAN or FBN.

[0029] In the step S4⑸ of the present invention, the gene expression of the zinc deficiency skin model is detected as barrier damage target genes such as KLK5 and KLK7.

[0030] In the step S4⑴ of the present invention, pre-incubation is carried out in an incubator environment with a temperature of 37.5±0.5°C, a humidity of 90±8%, and 5±1% CO2 for 12-24 hours.

[0031] In the step S4(1) of the present invention, the skin model adopts a commercial three-dimensional skin model or a non-commercial skin model, and the commercial three-dimensional skin model is an epidermal model, a full-thickness skin model or a melanin-containing skin model; the non-commercial skin model is a three-dimensional reconstructed epidermal model constructed in vitro by primary keratinocytes of children's foreskin tissue, and the foreskin tissue is derived from boys aged 5-11, and keratinocytes and fibroblasts are separated and cultured, and the constructed cells should be within 3 generations; the keratinocytes are inoculated in an insert culture dish, and the bottom of the culture dish is a scaffold material with biological activity or an artificial dermis containing fibroblasts. The keratinocytes are grown into a multi-layer three-dimensional epidermis or a full-thickness skin model after 12-14 days of weather-liquid interface culture, and the model is required to meet the structural characteristics and functional characteristics of the in vitro reconstructed three-dimensional skin model, that is, a layered keratin structure and a barrier function.

[0032] Compared with the prior art, the present invention has the following significant effects:

[0033] (1) The present invention constructs a scalp desquamation laboratory model based on the principle of zinc enzyme imbalance, uses cells as the experimental system, has high throughput and high sensitivity, can quickly screen functional raw materials, and shorten the formulation development time.

[0034] (2) The skin model used in the present invention is relatively stable in technology and highly standardized. It is a three-dimensional simulated human skin with barrier function and metabolic function. It can directly contact the sample and is suitable for the testing of any raw materials, mixtures, formulas and products. It is not affected by the physical properties of the sample such as solubility and color, and is closer to the actual situation of the human body.

[0035] ⑶ The present invention has the advantages of comprehensive detection parameters and high specificity through the detection of specific gene expressions such as cellular antioxidant enzymes, cellular glycoproteins and skin barrier kininase, and tissue morphological observation, and can comprehensively evaluate the anti-dandruff effect of the test substance. DETAILED DESCRIPTION

[0036] Example 1

[0037] This embodiment is used to evaluate the anti-dandruff efficacy of a certain shampoo, comprising the following steps:

[0038] S1. Conduct cytotoxicity test on the test substance to determine the safe concentration of the test substance;

[0039] The cell activity of the blank control group was taken as 100%, and the relative cell activity of each group was calculated. The test substance concentration with a relative cell activity greater than or equal to 90% was selected as the appropriate test substance concentration, that is, 0.16 mg / mL was selected for subsequent experiments.

[0040] S2. Cell culture was performed and divided into a negative control group, a model control group and an experimental group. The negative control group did not receive any treatment, TPEN was added to the model control group to construct a zinc-deficient cell model, and TPEN and 0.16 mg / mL of the test substance were added to the experimental group during the cell culture process. After incubation, the culture medium was removed from each group, and the cells collected by centrifugation were added with a total RNA extraction reagent and then stored;

[0041] Specifically, the experiment was designed with a negative control group, a model control group, and an experimental group, each group having at least 3 parallel samples. The negative control group did not receive any treatment, and the model control group was added with 4 μmol / L zinc ion chelator tetrapyridylmethylethylenediamine (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine, TPEN) to construct a zinc-deficient cell model. The experimental group was added with 4 μmol / L TPEN and 0.16 mg / mL of the test substance during cell culture and incubated for 24 hours. After the incubation, the culture medium was removed, and the cells were washed with PBS buffer 3 times. The cells collected by centrifugation were added with 1 mL of Trizol and stored at -80°C for subsequent experiments.

[0042] S3. Gene expression detection was performed on the cells collected from each group by RT-qPCR method, and the relative expression of the tested genes was calculated by 2(-ΔΔCt) method. If the relative expression of the gene in the model control group is significantly downregulated compared with the relative expression of the gene in the negative control group, it means that the zinc-deficient cell model is completed. Otherwise, it goes to step S2; when the zinc-deficient cell model is completed, if the relative expression of the gene in the experimental group is significantly upregulated compared with the relative expression of the gene in the model control group, it is preliminarily determined that the test substance has the effect of removing dandruff;

[0043] The cytotoxicity results of a certain shampoo exposed to keratinocytes, the relative activity of each group of cells (Mean±SD) are shown in the following table:

[0044] Group(mg / mL) Viability(%) Control 100.00±8.64 50.00 1.88±0.21 15.82 46.69±4.62 5.01 55.53±4.31 1.58 78.57±8.45 0.50 82.93±3.84 0.16 92.03±4.10 0.05 98.91±7.83 0.02 99.96±5.37

[0045] (Table 1)

[0046] Primer design is shown in the table below:

[0047] name sequence SOD1-F 5'-GGTGTGGCGATGTGTCT-3'- SOD1-R 5'-TCCACCTTTGCCCAAGTCA-3' FBN-F 5'-ArCCAACCACGTGCATCAGT-3' FBN-R 5'-AGAGCGGGTATCAACACAGC-3' 18s-F 5'-CCTGGAIACCGCAGCTAGGA-3' 18s-R 5'-GCGGCGCAATACGAATGCCCC-3'

[0048] (Table 2)

[0049] The results of SOD1 and FBN gene expression detection in zinc-deficient cell model are shown in the following table: Relative expression of SOD1 mRNA in each group (Mean±SD)

[0050]

[0051] *Indicates that the difference is statistically significant (p<0.05) compared with the model control group (M):

[0052] # indicates that the difference was statistically significant compared with the negative control group (NC) (p < 0.05).

[0053] (Table 3)

[0054] See the table below: Relative expression of FBN mRNA in each group (Mean±SD)

[0055]

[0056] *Indicates that the difference is statistically significant (p < 0.05) compared with the model control group (M);

[0057] # indicates that the difference was statistically significant compared with the negative control group (NC) (P<0.05).

[0058] (Table 4)

[0059] S4. Construct a zinc-deficient skin model and conduct tests, including:

[0060] (1) Pre-incubating a skin model, wherein the skin model is a three-dimensional simulated human skin having barrier function and metabolic function;

[0061] 1. Preliminary preparation of zinc-deficient skin model:

[0062] The commercially available epidermal skin model was incubated in a 12-well culture dish of the same size, and 2 mL of the skin model supporting culture medium was added to the dish in advance;

[0063] Incubate in an incubator at 37.5±0.5℃, 90±8% humidity and 5±1% CO2 for 12-24h and set aside;

[0064] 2. Preparation of test substances:

[0065] A certain amount of shampoo was weighed into a centrifuge tube, and then a certain volume of sterile PBS phosphate buffer saline was added to prepare a uniform solution of 0.16 mg / mL.

[0066] ⑵ The pre-incubated skin model was divided into a negative control group, a model control group and an experimental group, and three parallel models were set up in each experimental group. Among them, the negative control group did not receive any treatment, the model control group was rinsed with buffer solution, and the surface of the skin model in the experimental group was treated with 0.16 mg / mL shampoo for 15 minutes and then the tested shampoo was rinsed off with buffer solution;

[0067] (3) The negative control group, model control group, and experimental group were re-incubated. The re-incubation of the experimental group was performed after the test substance on the skin model was cleaned, and the culture medium of the model control group and the experimental group was 2 mL of culture medium containing 4 μmol / L TPEN, while the culture medium of the negative control group did not contain 4 μmol / L TPEN;

[0068] (4) After 42±1 h of incubation, one of the three parallel skin models was used for cell activity testing, and the other two groups were used for gene expression testing and tissue morphology sectioning;

[0069] (5) After the re-incubation, each group was tested for cell activity, and the cell activity was required to be greater than or equal to 50%;

[0070] 3. Zinc deficiency skin model activity test:

[0071] After the test reaches the end point, take another 12-well plate and add 0.3mg / mL MTT test culture medium to each well, 2mL / well. Transfer the epidermal model of one of the three parallel skin models to the culture well containing MTT test culture medium. Incubate in an incubator at 37℃ for 3h. Since active cells can metabolize MTT, the epidermis turns blue due to the formation of formazan crystals in the cells.

[0072] Formazan Extraction:

[0073] Prepare 2 mL EP tubes with caps and label the groups;

[0074] Remove the culture basket and blot the tissue dry with absorbent paper;

[0075] Remove the epidermal units using a hole punch;

[0076] Use flat-tipped forceps to gently remove and separate the epidermal tissue from the collagen matrix and place the epidermis together with the supporting collagen in the EP tube;

[0077] Add 500 μL of acidic isopropanol to each tube;

[0078] Close the lid tightly to prevent evaporation, and mix thoroughly to ensure that all biological materials are immersed in the solvent;

[0079] If you plan to extract on the same day, read the sample on a microplate reader and store at room temperature away from light for 4 hours. During storage, vortex each tube once to assist extraction. You can also store the sample in a 4°C refrigerator away from light overnight or read the sample on the 5th day of the experiment.

[0080] Remove the MTT solution from the culture wells of the test culture plate and discard the culture plate as required;

[0081] After the formazan extraction is completed, vortex to mix. If visible suspended cells or tissue fragments are observed, they should be removed by centrifugation (500r / min) to avoid interference with the absorbance value;

[0082] Pipette 2 200 μL samples from each EP tube into a 96-well plate, i.e., 2 × 200 μL samples / 96 wells (2 wells for each tissue);

[0083] Acidified isopropanol was used as blank control, and the OD value of the 96-well plate was read at 570 nm.

[0084] 4. Gene expression test of zinc deficiency skin model:

[0085] Gene expression tests were performed on each group that met the cell activity requirements. If the relative gene expression of the model control group was significantly lower than that of the negative control group, it indicated that the zinc-deficient skin model was successfully constructed. In this case, if the relative gene expression of the experimental group was significantly higher than that of the model control group, it indicated that the test substance had an anti-dandruff effect.

[0086] Gene expression detection in zinc deficiency skin model (KLK5, KLK7):

[0087] When the test reaches the end point, one of the three parallel skin models is removed from the fixed support, and the cell layer and the support layer are simply peeled off, and then both are placed in the prepared TRIzol reagent to separate and obtain total ribonucleic acid (RNA); 1 μg of total RNA is added to a total volume of 20 μl liquid containing random primers, and reverse transcription of the relevant RNA is performed according to the reverse transcription instrument operating instructions; then, PCR amplification is performed using oligonucleotide primers, and finally the relative expression of genes is detected by a quantitative polymerase chain reaction (PCR) instrument;

[0088] 5. Histomorphological sections of zinc-deficient skin model:

[0089] Histomorphological sections were performed on each group. If the desquamation of the skin model tissue in the model control group was significantly higher than that in the negative control group, it indicated that the experimental model was successfully constructed. In this case, if the desquamation of the skin model tissue in the test substance group was significantly lower than that in the model control group, it was finally determined that the test substance had an anti-dandruff effect.

[0090] When the test reached the end point, one of the three parallel skin models was removed from the fixed scaffold, and the cell layer and the scaffold layer were simply peeled off, fixed in 4% paraformaldehyde, and stained with hematoxylin-eosin (HE), and then the skin tissue was observed under an optical microscope. Six areas were randomly selected under a 200x microscope, and the desquamation of each area was recorded.

[0091] The results of tissue activity test of a certain shampoo exposed to zinc-deficient skin model are shown in the table below. The relative tissue activity of each group (Mean±SD):

[0092] Group OD value Tissue activity Blank control 0.0313±0.0004 -- Negative control 1.1742±0.0103 100.00％±1.13％ Model comparison 1.0558±0.0023 89.91％±0.20％ Sample group to be tested 1.0879±0.0190 92.64％±1.62％

[0093] (Table 5)

[0094] Primer design is shown in the table below:

[0095] name sequence KLK5-F 5'-CAAATCCCCCACCCCCTAC-3' KLK5-R 5'-GGCGAGAACATGCTCTGTGA-3' KLK7-F 5'-GTCGGAACTTGGATCACATCAG-3' KLK7-R 5'-AATCTTGTCACCCTGGGCTTC-3' 18s-F 5'-CCTGGATACCGCAGCTAGGA-3' 18s-R 5'-GCGGCGCAATACGAATGCCCC-3'

[0096] (Table 6)

[0097] The results of KLK5 gene expression detection in zinc-deficient skin model are shown in the table below:

[0098]

[0099]

[0100] (Table 7)

[0101] The relative expression of KLK7 mRNA in each group (Mean±SD) is shown in the following table:

[0102]

[0103] (Table 8)

[0104] Example 2

[0105] This embodiment is used to evaluate the anti-dandruff efficacy of a hair product raw material, and comprises the following steps:

[0106] S1. Conduct cytotoxicity test on the test substance to determine the safe concentration of the test substance;

[0107] The cell activity of the blank control group was taken as 100%, and the relative cell activity of each group was calculated. The test substance concentration with a relative cell activity greater than or equal to 90% was selected as the appropriate test substance concentration, that is, 10% concentration was selected for subsequent experiments.

[0108] S2. Cell culture was performed and divided into a negative control group, a model control group and an experimental group. The negative control group was not treated with any treatment. The model control group was added with TPEN to construct a zinc-deficient cell model. The experimental group was added with TPEN and 10% of the test substance during the cell culture process. After incubation, the culture medium was removed from each group, and the cells collected by centrifugation were added with a total RNA extraction reagent and then stored.

[0109] Specifically, the experiment was designed with a negative control group, a model control group, and an experimental group, each group having at least 3 parallel samples. The negative control group did not receive any treatment, and the model control group was added with 4 μmol / L zinc ion chelator tetrapyridylmethylethylenediamine (N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine, TPEN) to construct a zinc-deficient cell model. The experimental group was added with 4 μmol / L TPEN and 10% of the test substance during cell culture and incubated for 24 hours. After the incubation, the culture medium was removed, and the cells were washed with PBS buffer 3 times. After centrifugation, 1 mL of Trizol was added to the cells and stored at -80°C for subsequent experiments.

[0110] S3. Gene expression detection was performed on the cells collected from each group by RT-qPCR method, and the relative expression of the tested genes was calculated by 2(-ΔΔCt) method. If the relative expression of the gene in the model control group is significantly downregulated compared with the relative expression of the gene in the negative control group, it means that the zinc-deficient cell model is completed. Otherwise, it goes to step S2; when the zinc-deficient cell model is completed, if the relative expression of the gene in the experimental group is significantly upregulated compared with the relative expression of the gene in the model control group, it is preliminarily determined that the test substance has the effect of removing dandruff;

[0111] The cytotoxicity results of a hair product raw material exposed to keratinocytes, the relative activity of each group of cells (Mean±SD) are shown in the following table:

[0112] Group(%) Viability(%) Control 100±5.12 20.00 23±0.47 10.00 91.85±6.01 5.00 92.46±4.86 2.50 94.79±4.99 1.25 96.22±4.75 0.63 96.4±3.81 0.31 97.73±4.16 0.16 100.36±8.77

[0113] (Table 9)

[0114] Primer design is shown in the table below:

[0115] name sequence SOD1-F 5'-GGTGTGGCCGATGTGTCT-3' SOD1-R 5'-TCCACCTTTGCCCAAGTCA-3' FBN-F 5'-ATCCAACCACGTGCArCAGT-3' FBN-R 5'-AGAGCGGGTATCAACACAGC-3' 18s-F 5'-CCTGGAIACCGCAGCTAGGA-3' 18s-R 5'-GCGGCGCAATACGAATGCCCC-3'

[0116] (Table 10)

[0117] The results of SOD1 and FBN gene expression detection in zinc-deficient cell model are shown in the following table: Relative expression of SOD1 mRNA in each group (Mean±SD)

[0118]

[0119]

[0120] *Indicates that the difference is statistically significant (p<0.05) compared with the model control group (M);

[0121] # indicates that the difference was statistically significant compared with the negative control group (NC) (p<0.05).

[0122] (Table 11)

[0123] See the table below: Relative expression of FBN mRNA in each group (Mean±SD)

[0124]

[0125] *Indicates that the difference is statistically significant (p<0.05) compared with the model control group (M);

[0126] # indicates that the difference was statistically significant compared with the negative control group (NC) (p<0.05).

[0127] (Table 12)

[0128] S4. Construct a zinc-deficient skin model and conduct tests, including:

[0129] (1) Pre-incubating a skin model, wherein the skin model is a three-dimensional simulated human skin having barrier function and metabolic function;

[0130] 1. Preliminary preparation of zinc-deficient skin model

[0131] The commercially available epidermal skin model was incubated in a 12-well culture dish of the same size, and 2 mL of the skin model supporting culture medium was added to the dish in advance;

[0132] Incubate in an incubator at 37.5±0.5℃, 90±8% humidity and 5±1% CO2 for 12-24h and set aside;

[0133] 2. Preparation of test substances

[0134] The product to be tested is a hair product raw material soluble in water. A certain amount of a hair product raw material is weighed into a centrifuge tube, and then a certain volume of sterile PBS phosphate buffer saline is added to prepare a 10% uniform solution.

[0135] ⑵ The pre-incubated skin model was divided into a negative control group, a model control group and an experimental group, wherein the negative control group was not treated with any treatment, the model control group was washed with a buffer solution, and the surface of the skin model of the experimental group was treated with 10% of the test substance;

[0136] (4) The negative control group, model control group and experimental group were re-incubated, wherein the re-incubation of the experimental group was performed after the test substance on the skin model was cleaned, and TPEN was added to the culture medium of both the model control group and the experimental group;

[0137] Zinc deficiency skin model and test substance exposure:

[0138] The test substance was directly treated on the surface of the skin model. The test substance of the negative control group and the model control group was PBS phosphate buffer, and the test substance of the experimental group was 10% uniform solution. The test substance dose was 10 μL, and the treatment time was 15 minutes.

[0139] Then, the test substance on the surface of the skin model was cleaned, and 2 mL of new culture medium containing 4 μmol / LTPEN was added to the skin model culture medium, and the skin model was incubated for 42 ± 1 h. Different test concentrations were set for each test substance, and 3 parallel models were set for each concentration;

[0140] After 42±1h incubation, one of the three parallel skin models was used for cell activity test, and the other two groups were used for gene expression test and tissue morphology section; the experiment set up negative control group, model control group and experimental group;

[0141] (5) After the re-incubation, the cell activity of each group was tested, and the cell activity was required to be greater than or equal to 50%;

[0142] Zinc deficiency skin model activity test:

[0143] After the test reaches the end point, take another 12-well plate and add 0.3mg / mL MTT test culture medium to each well, 2mL / well. Transfer the epidermal model of one of the three parallel skin models to the culture well containing MTT test culture medium. Incubate in an incubator at 37℃ for 3h. Since active cells can metabolize MTT, the epidermis turns blue due to the formation of formazan crystals in the cells.

[0144] Formazan Extraction:

[0145] Prepare 2 mL EP tubes with caps and label the groups;

[0146] Remove the culture basket and blot the tissue dry with absorbent paper;

[0147] Remove the epidermal units using a hole punch;

[0148] Use flat-tipped forceps to gently remove and separate the epidermal tissue from the collagen matrix and place the epidermis together with the supporting collagen in the EP tube;

[0149] Add 500 μL of acidic isopropanol to each tube;

[0150] Close the lid tightly to prevent evaporation, and mix thoroughly to ensure that all biological materials are immersed in the solvent;

[0151] If you plan to extract on the same day, read the sample on a microplate reader and store at room temperature away from light for 4 hours. During storage, vortex each tube once to assist extraction. You can also store the sample in a 4°C refrigerator away from light overnight or read the sample on the 5th day of the experiment.

[0152] Remove the MTT solution from the culture wells of the test culture plate and discard the culture plate as required;

[0153] After the formazan extraction is completed, vortex to mix. If visible suspended cells or tissue fragments are observed, they should be removed by centrifugation (500r / min) to avoid interference with the absorbance value;

[0154] Pipette 2 200 μL samples from each EP tube into a 96-well plate, i.e., 2 × 200 μL samples / 96 wells (2 wells for each tissue);

[0155] Acidified isopropanol was used as blank control, and the OD value of the 96-well plate was read at 570 nm.

[0156] (5) Gene expression test was performed on each group that met the cell activity requirements. If the relative gene expression of the model control group was significantly downregulated compared with the relative gene expression of the negative control group, it indicated that the zinc-deficient skin model was successfully constructed. In this case, if the relative gene expression of the experimental group was significantly upregulated compared with the relative gene expression of the model control group, it indicated that the test substance had the effect of removing dandruff;

[0157] Gene expression detection in zinc deficiency skin model (KLK5)

[0158] When the test reaches the end point, one of the three parallel skin models is removed from the fixed support, and the cell layer and the support layer are simply peeled off, and then both are placed in the prepared TRIzol reagent to separate and obtain total ribonucleic acid (RNA); 1 μg of total RNA is added to a total volume of 20 μl liquid containing random primers, and reverse transcription of the relevant RNA is performed according to the reverse transcription instrument operating instructions; then, PCR amplification is performed using oligonucleotide primers, and finally the relative expression of genes is detected by a quantitative polymerase chain reaction (PCR) instrument;

[0159] (6) Perform tissue morphological sections on each group. If the desquamation of the skin model tissue in the model control group is significantly higher than that in the negative control group, it means that the experimental model is successfully constructed. In this case, if the desquamation of the skin model tissue in the test substance group is significantly lower than that in the model control group, it is finally determined that the test substance has an anti-dandruff effect.

[0160] When the test reached the end point, one of the three parallel skin models was removed from the fixed scaffold, and the cell layer and the scaffold layer were simply peeled off, fixed in 4% paraformaldehyde, and stained with hematoxylin-eosin (HE), and then the skin tissue was observed under an optical microscope. Six areas were randomly selected under a 200x microscope, and the desquamation of each area was recorded.

[0161] The results of the tissue activity test of a hair product raw material exposed to a zinc-deficient skin model are shown in the table below. The relative activity of each group of tissues (Mean±SD):

[0162]

[0163]

[0164] (Table 13)

[0165] Primer design is shown in the table below:

[0166] name sequence KLK5-F 5'-CAAATCCCCCACCCCCTAC-3' KLK5-R 5'-GGCGAGAACATGCTCTGTGA-3' KLK7-F 5'-GTCGGAACTTGGATCACATCAG-3' KLK7-R 5'-AATCTTGTCACCCTGGGCTTC-3' 18s-F 5'-CCTGGATACCGCAGCTAGGA-3' 18s-R 5'-GCGGCGCAATACGAATGCCCC-3'

[0167] (Table 14)

[0168] The results of KLK5 gene expression detection in zinc-deficient skin model are shown in the table below:

[0169]

[0170] (Table 15)

[0171] Relative expression of KLK7 mRNA in zinc-deficient skin model (M e an±SD), see the table below:

[0172]

[0173] (Table 16)

[0174] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Although the present invention only lists some cosmetic raw materials and products. However, other non-single ingredient systems mentioned in the present invention, such as personal care products (shampoo, conditioner, hair dye, skin care, etc.), medicines, plant extracts (crude extracts, refined extracts and mixtures thereof), etc., can also be evaluated for anti-dandruff efficacy using the method described in the present invention. They are not listed one by one here.

Claims

1. A method for evaluating the anti-dandruff efficacy based on zinc enzyme homeostasis, characterized in that The specific steps include: S1. Conduct cytotoxicity test on the test substance to determine the safe concentration of the test substance; S2. Cell culture was performed and divided into a negative control group, a model control group and an experimental group. The negative control group was not treated with any treatment. The model control group was added with TPEN to construct a zinc-deficient cell model. The experimental group was added with TPEN and a safe concentration of the test substance during the cell culture process. After incubation, the culture medium was removed from each group, and the cells collected by centrifugation were added with a total RNA extraction reagent and then stored. S3, performing SOD1 and FBN gene expression detection on the cells collected from each group, if the relative gene expression of the model control group is significantly downregulated compared with the relative gene expression of the negative control group, it means that the zinc-deficient cell model is completed, otherwise, proceed to step S2; when the zinc-deficient cell model is completed, if the relative gene expression of the experimental group is significantly upregulated compared with the relative gene expression of the model control group, it is preliminarily determined that the test substance has an anti-dandruff effect; S4. Construct a zinc-deficient skin model and conduct tests, including: (1) Pre-incubating a skin model, wherein the skin model is a three-dimensional simulated human skin having barrier function and metabolic function; ⑵The pre-incubated skin models were divided into a negative control group, a model control group and an experimental group. The negative control group was not treated with any treatment, the model control group was washed with a buffer solution, and the surface of the skin model in the experimental group was treated with different concentrations of the test substance; ⑶ The negative control group, the model control group and the experimental group were re-incubated, wherein the re-incubation of the experimental group was performed after the test substance on the skin model was cleaned, and TPEN was added to the culture medium of the model control group and the experimental group; (4) After the re-incubation, the cell activity of each group was tested, and the cell activity was required to be greater than or equal to 50%; (5) Perform KLK5 and KLK7 gene expression tests on each group that meets the cell activity requirements. If the relative gene expression of the model control group is significantly downregulated compared with the relative gene expression of the negative control group, it means that the zinc-deficient skin model is successfully constructed. In this case, if the relative gene expression of the experimental group is significantly upregulated compared with the relative gene expression of the model control group, it means that the test substance has an anti-dandruff effect; (6) Perform tissue morphological sections on each group. If the desquamation of the skin model tissue in the model control group is significantly higher than that in the negative control group, it means that the experimental model is successfully constructed. In this case, if the desquamation of the skin model tissue in the test substance group is significantly lower than that in the model control group, it is finally determined that the test substance has an anti-dandruff effect; The concentration of TPEN in steps S2 and S4 is 2-8 μmol / L; The cells used in the steps S1 and S2 are immortalized keratinocytes or primary isolated keratinocytes.

2. The anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis according to claim 1, characterized in that: In step S1, the cell activity of the blank control group is taken as 100%, the relative cell activity of each group is calculated, and the test substance concentration at which the relative cell activity is greater than or equal to 90% is selected as the appropriate test substance concentration.

3. The anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis according to claim 2, characterized in that: In step S4⑵, at least three parallel models are set up for each group, and at least three parallel models are set up for each concentration of the test substance in the experimental group, and cell activity test, gene expression test and tissue morphology section are performed respectively.

4. The anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis according to claim 3, characterized in that: In step S4⑵, the treatment dose of the test substance is 10-50 μL, and the treatment time is 15-60 min.

5. The anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis according to claim 1, characterized in that: In step S4(1), pre-incubation is performed in an incubator environment with a temperature of 37.5±0.5°C, a humidity of 90±8%, and 5±1% CO2 for 12-24 hours.

6. The anti-dandruff efficacy evaluation method based on zinc enzyme homeostasis according to claim 5, characterized in that: In the step S4(1), the skin model adopts a commercial three-dimensional skin model; The commercial three-dimensional skin model is an epidermis model, a full-thickness skin model or a melanin-containing skin model.

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