Cytological evaluation method for screening stabbing-inhibiting substances

Abstract

The cytological evaluation method for screening the substances for inhibiting stabbing comprises the following steps: (a) inoculating a human TRPV1 receptor overexpression cell strain into a culture plate, and adding a fluorescent indicator to load cells; (b) preparing a buffer solution containing an activator, a buffer solution containing an inhibitor and a buffer solution containing a sample to be detected; (c) dividing the cell inoculation pores into a blank control group, a negative control group, a positive control group and a sample group; (d) adding a buffer solution into the blank control group and the negative control group, adding a buffer solution containing an inhibitor into the positive control group, and adding a buffer solution containing a sample to be detected into the sample group; (e) continuously adding a buffer solution into the blank control group, and adding a buffer solution containing an activating agent into the negative control group, the positive control group and the sample group; (f) detecting a fluorescence value; and (g) taking the detected fluorescence value of the negative control group as 100%, and if the fluorescence value of the sample group is lower than 100%, determining that the detected sample has the effect of inhibiting the stabbing pain.

Description

Technical Field

[0001] The present invention relates to the field of efficacy evaluation of cosmetics and their ingredients, and in particular to a method for screening cosmetic raw materials or active substances with stinging inhibition efficacy based on a human TRPV1 receptor overexpressing cell line. Background Art

[0002] Pain is a strong and unbearable discomfort caused by harmful stimulation. This discomfort reminds people to avoid dangerous environments and stay away from dangerous factors. However, some pain, such as the tingling sensation of sensitive skin or pain from some diseases such as cancer, will have a certain degree of impact or serious impact on people's lives, but they cannot be escaped or relieved like avoiding dangerous external environments or removing external factors that cause pain. Analgesia or other treatment methods are needed to improve and cure.

[0003] Sensitive skin is a syndrome defined by the reaction of unpleasant sensations (stinging, burning, pain, itching and tingling) to stimuli that normally should not cause such sensations. These unpleasant sensations cannot be explained by the lesions caused by any skin disease. The skin may appear normal or be accompanied by erythema. Sensitive skin can affect all parts of the body, especially the face.

[0004] At present, the incidence of sensitive skin is high worldwide. A global epidemiological survey on sensitive skin shows that people with sensitive skin account for more than half of the surveyed population, of which 60-70% of women and 50-60% of men have sensitive skin, which is higher for women than for men. A domestic epidemiological study surveyed communities in the three major cities of Beijing, Shanghai and Guangzhou, and the results showed that the average proportion of people with sensitive skin in the three cities was 13%, 15.9% for women and 8.6% for men, which is significantly lower than that in Europe and the United States.

[0005] The causes of sensitive skin are very complex. Current research suggests that there are four factors related to sensitive skin, including impaired skin barrier function, skin nerve hyperreactivity, skin immune inflammatory response, and skin vascular hyperreactivity. These factors often promote and interfere with each other, causing sensitive skin problems to fall into a vicious cycle. Among these four factors, skin nerve hyperreactivity is closely related to the discomfort of sensitive skin, such as burning, stinging, and itching.

[0006] There are a wide variety of sensory nerve endings on the human skin, with different structures and shapes, mainly from dorsal root ganglia or trigeminal ganglia. These nerve endings of various shapes can detect pain, temperature changes, pressure or vibration and other environmental changes inside and outside the body, so as to make the body seek benefits and avoid harm. Among them, there is a type of C nerve fiber that is thinner, unmyelinated, and has slow nerve signal transmission. It mainly senses temperature, pain and itching, which is closely related to the discomfort of sensitive skin. There are many receptors related to the perception of temperature, pain and itching distributed on the free nerve endings of C nerve fibers, such as TRPV1, TRPA1, TRPM8, etc.; after these receptors are activated, the nerve fibers can generate action potentials, which are transmitted to the brain through the ganglia and spinal cord, producing burning, tingling, itching discomfort. At the same time, it can further cause the release of neuropeptides from the nerve fiber endings, such as substance P, calcitonin gene-related polypeptide CGRP, etc., causing neurogenic inflammation, leading to delayed skin barrier repair, tissue edema, redness, etc.

[0007] It is currently believed that the burning and tingling discomfort of sensitive skin is mainly related to the overactivation of TRPV1 receptors on the nerve endings of C nerve fibers. TRPV1 receptor, whose Chinese name is transient receptor potential vanilloid subtype 1, also known as capsaicin receptor, is a non-selective cation channel composed of four subunits. TRPV1 receptor can be activated by a variety of factors such as chemicals such as capsaicin, damaging high temperatures, low pH values, and endogenous substances. It is a multimodal receptor. When TRPV1 receptor is activated by these exogenous factors, calcium ions outside the cell membrane can be released into the cytoplasm, causing depolarization of the cell membrane, generating action potentials, and transmitted to the brain through nerve fibers and ganglia, producing a burning and tingling sensation. Therefore, research and development of cytological methods for screening substances with inhibitory effects on tingling, as well as research and screening of substances or raw materials with inhibitory effects on TRPV1 receptor activation, are important ways and research directions to alleviate the burning and tingling discomfort of sensitive skin.

[0008] Methods for studying TRPV1 receptor activators and antagonists include cellular methods, animal experiments, and human clinical tests. Cellular methods include cell lines that overexpress TRPV1 receptors, and cell patch clamp technology combined with electrophysiology, which are used to study the regulation of TRPV1 receptor activity by activators and antagonists.

[0009] Chinese invention patent application CN106537149A discloses a method for screening compounds for regulating TRPA1, TRPV1 and / or TRPA1V1, the method using a cell line stably co-expressing TRPA1, TRPV1, and / or TRPA1V1, and studying compounds regulating TRPA1, TRPV1 and / or TRPA1V1 by measuring the baseline ion channel activity of the cell line.

[0010] Chinese invention patent application CN112626163A discloses a cytological method for evaluating sensitive skin discomfort. This method uses a HaCaT cell line that stably expresses the TRPV1 receptor, a calcium influx-fluorescent probe Fluo-8, and a FlexStation instrument for quantitative detection to evaluate the efficacy of plant extracts in relieving skin discomfort.

[0011] The disclosed method in the above patent application uses the human embryonic kidney cell line HEK and the human keratinocyte cell line HaCaT, both of which are non-neural cell lines. The neural cells mainly related to skin discomfort do not belong to the same type of cells and have obvious heterogeneity.

[0012] The inventor's previous Chinese invention patent application CN113667697A (and PCT patent WO2021227811A1) disclosed an evaluation method based on a human TRPV1 receptor overexpressing cell line, in which a neural cell model overexpressing human TRPV1 receptors was constructed, and a pain evaluation method was established based on this neural cell model, which can be used to evaluate whether cosmetics and their ingredients have a risk of causing pain to human eyes. The method uses the human neuroblastoma cell line SH-SY5Y, which does not have background TRPV1 receptor expression, eliminating the interference of endogenous TRPV1 receptors in the test.

[0013] The present invention adopts the neural cell model overexpressing human TRPV1 receptors in the previous invention patent CN113667697A. By redesigning and improving the original evaluation method for screening substances with stinging activity and performing multiple rounds of adjustment and optimization, a cytological evaluation method that can be used to screen raw materials and chemical substances with stinging inhibition effects is established, and the known TRPV1 antagonist trans-4-tert-butyl cyclohexanol and 6 commercially available cosmetic raw materials are evaluated for stinging inhibition. SUMMARY OF THE INVENTION

[0015] In one aspect, the present invention provides a cytological evaluation method for screening substances that inhibit stinging pain, comprising the following steps:

[0016] (a) A human TRPV1 receptor overexpressing cell line was inoculated into a culture plate, and a fluorescent indicator was added to load the cells;

[0017] (b) preparing a buffer containing an activator, a buffer containing an inhibitor, and a buffer containing a sample to be detected;

[0018] (c) dividing the cell inoculation wells on the culture plate in step (a) into a blank control group, a negative control group, a positive control group and a sample group;

[0019] (d) adding a buffer to the blank control group and the negative control group in step (c), adding the buffer containing the inhibitor prepared in step (b) to the positive control group in step (c), and adding the buffer containing the sample to be tested prepared in step (b) to the sample group in step (c);

[0020] (e) continuing to add buffer to the blank control group in step (d), and adding the buffer containing the activator prepared in step (b) to the negative control group, the positive control group and the sample group in step (d);

[0021] (f) detecting fluorescence value;

[0022] (g) The fluorescence value of the negative control group in step (e) is taken as 100%. If the fluorescence value of the sample group is lower than 100%, the sample tested has an inhibitory effect on pain.

[0023] In a preferred embodiment, in step (a), 0.8×10 5 to 1.2×10 5 The cells were seeded into the culture plate at a density of 10 cells / well.

[0024] In a preferred embodiment, the fluorescent indicator in step (a) is a green fluorescent calcium indicator.

[0025] In a preferred embodiment, the buffer in step (b) is selected from: KRH or PBS buffer.

[0026] In a preferred embodiment, the sample to be detected is a pure chemical, such as trans-4-tert-butyl cyclohexanol. In a preferred embodiment, the sample to be detected is a mixture, such as SymSitive 1609, SymCare W, DCR 516, Calmnerv CR, NEUROFENSE or PACIFEEL.

[0027] In a preferred embodiment, the sample to be tested in step (b) is tested at a dose less than or equal to the maximum non-toxic dose. In a preferred embodiment, the sample to be tested is prepared according to the mass volume fraction, and the sample concentration gradient is diluted according to 1.25-10 times, preferably, according to 1.5-5 times, and more preferably, according to 2-3.16 times.

[0028] In a preferred embodiment, the activator is selected from: dihydrocapsaicin or capsaicin.

[0029] In a preferred embodiment, the inhibitor is selected from: capsaicin or ruthenium red.

[0030] In a preferred embodiment, the cytological method for screening substances with stinging-inhibiting efficacy is evaluated by the inhibitory effect on the activation of TRPV1 receptors.

[0031] In a preferred embodiment, the inhibitory effect on the activation of TRPV1 receptors demonstrates a soothing effect on burning and stinging of sensitive skin. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Sting Inhibition Evaluation Method The known TRPV1 antagonist trans-4-tert-butylcyclohexanol was evaluated.

[0033] Figure 2 The stinging inhibition evaluation method was used to evaluate 6 commercially available cosmetic raw materials. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art and researchers to which the present invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, those described herein are preferred methods and materials. For purposes of the present invention, the following terms are defined.

[0035] Activators are compounds that bind to receptors and activate them, producing physiological responses. The activators referred to in the present invention are substances that bind to TRPV1 receptors and change the conformation of the receptors, opening the pores in the middle of the receptors, and then causing monovalent and divalent cations (mainly calcium ions) to enter the cells. The activators applicable to the present invention are mainly vanillamide derivatives, including capsaicin, dihydrocapsaicin and other substances with high ability to activate TRPV1 receptors.

[0036] An inhibitor is a substance that can compete with a certain receptor, resulting in the inhibition of the receptor action. The inhibitor referred to in the present invention is a substance that competitively binds to the TRPV1 receptor, causing the pore in the middle of the receptor to close and cannot be opened by the activator, so that monovalent and divalent cations (mainly calcium ions) cannot enter the cell. The inhibitors applicable to the present invention are mainly capsaicin, ruthenium red and other substances.

[0037] According to the present invention, the term "cosmetics" refers to chemical industrial products or fine chemical products that are spread on any part of the human body surface, such as skin, hair, nails, lips and teeth, etc., by smearing, spraying or other similar methods, in order to achieve the purpose of cleaning, maintenance, beautification, modification and change of appearance, or to correct human body odor and maintain a good state.

[0038] According to the present invention, the term "cosmetic raw materials" refers to the raw materials used in the cosmetic production process, including cosmetic base raw materials, such as oily raw materials, surfactants, solvents, powdery raw materials, high molecular polymers and other additives; cosmetic auxiliary raw materials, such as flavors, colorants, pearlescent agents, preservatives, bactericides, antioxidants and chelating agents; and special-purpose raw materials, such as depilatories, hair dyes, perming agents, astringents, antiperspirants, deodorants, anti-freckle agents, sunscreens, etc.

[0039] Based on previous research (CN113667697A), the present invention establishes a cytological evaluation method that can be used to screen raw materials and chemical substances with stinging-inhibiting effects, and evaluates the stinging-inhibiting effects of a known TRPV1 antagonist, trans-4-tert-butyl cyclohexanol, and 6 commercially available cosmetic raw materials.

[0040] The present invention actually provides an evaluation method for screening substances with stinging inhibitory effects, specifically a cytological evaluation method for screening cosmetic raw materials or active substances with stinging inhibitory effects based on human TRPV1 receptor overexpressing cell lines.

[0041] The beneficial effects of the present invention are as follows: a cytological evaluation method for inhibiting pain based on a human TRPV1 receptor overexpressing cell line is established, which can be used to screen cosmetic raw materials, chemical substances or mixtures, and plant extracts, etc., with TRPV1 receptor antagonism, and raw materials that can relieve the burning pain and discomfort of sensitive skin can be obtained, guiding the design and development of cosmetics for sensitive skin, and having high research and application value.

[0042] The present invention provides a cytological method for screening substances with stinging-inhibiting efficacy, comprising the following steps:

[0043] (a) A human TRPV1 receptor overexpressing cell line was inoculated into a culture plate, and a fluorescent indicator was added to load the cells;

[0044] (b) preparing a buffer containing an activator, a buffer containing an inhibitor, and a buffer containing a sample to be detected;

[0045] (c) dividing the cell inoculation wells on the culture plate in step (a) into a blank control group, a negative control group, a positive control group and a sample group;

[0046] (d) adding a buffer to the blank control group and the negative control group in step (c), adding the buffer containing the inhibitor prepared in step (b) to the positive control group in step (c), and adding the buffer containing the sample to be tested prepared in step (b) to the sample group in step (c);

[0047] (e) continuing to add buffer to the blank control group in step (d), and adding the buffer containing the activator prepared in step (b) to the negative control group, the positive control group and the sample group in step (d);

[0048] (f) detecting fluorescence value;

[0049] (g) The fluorescence value of the negative control group in step (e) is taken as 100%. If the fluorescence value of the sample group is lower than 100%, the sample tested has an inhibitory effect on pain.

[0050] In a specific embodiment, the method of the present invention sets up the following groups: a blank group (KRH group), a negative control group (dihydrocapsaicin DHC), a positive control group (inhibitor capsaicin CZ + dihydrocapsaicin DHC) and a sample group (sample + dihydrocapsaicin DHC).

[0051] In some embodiments, in the method of the present invention, the incubation is carried out for 5-15 minutes after step (d), and then step (e) is performed. Since the incubation time of cells in the sample in the method of the present invention is 5-15 minutes, a cytotoxicity test is added to eliminate the interference of cell death on the test.

[0052] In the cytological evaluation method of the present invention, after step (e), detection is performed. If the fluorescence value of the sample group is lower than the fluorescence value of the negative control group, the detected sample has the effect of inhibiting stinging. Therefore, the method of the present invention only needs to be detected once to determine whether the detected sample has the effect of inhibiting stinging. Therefore, the present invention provides a simple and rapid cytological evaluation method for screening substances that inhibit stinging.

[0053] The cytological method for screening substances with stinging-inhibiting effects of the present invention is evaluated by the inhibitory effect on the activation of TRPV1 receptors. Such inhibitory effect on the activation of TRPV1 receptors indicates a soothing effect on burning and stinging of sensitive skin.

[0054] In the screening method of the present invention, the fluorescence value of the negative control group (after deducting the value of the blank control well) is taken as 100%, and the fluorescence value of the sample group is compared with it to obtain the relative fluorescence intensity percentage.

[0055] The screening method of the present invention also includes the calcium channel inhibitor capsaicin (CZ) as a positive control group. In the screening method of the present invention, the fluorescence value of the positive control group (CZ+DHC group) is less than 100% (for example, the relative fluorescence intensity is -23.56%), indicating that capsaicin (CZ) can strongly inhibit the activation of TRPV1 receptors by dihydrocapsaicin.

[0056] In the screening method of the present invention, TRPV1 receptor can be activated by dihydrocapsaicin (DHC) and inhibited by capsaicin (CZ), indicating that the experimental method is reliable. Therefore, the present invention provides a simple and reliable cytological evaluation method for screening substances with anti-stinging efficacy.

[0057] When the effective substance to be tested is trans-4-tert-butyl cyclohexanol, the concentration range of 0.0025-0.04% shows the effect of inhibiting the activation of TRPV1 receptor. The lower the relative fluorescence intensity value, the higher the degree of inhibition of TRPV1 receptor.

[0058] When the active substance to be tested is SymSitive 1609, it exhibits an inhibitory effect on the activation of TRPV1 receptor by dihydrocapsaicin within a concentration range of 0.0025-0.04%.

[0059] When the active substance to be tested is SymCare W, it exhibits an effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin within a concentration range of 0.005-0.02%.

[0060] When the active substance to be tested is DCR 516, it exhibits an inhibitory effect on the activation of TRPV1 receptor by dihydrocapsaicin within a concentration range of 0.25-1.0%.

[0061] When the effective substance to be tested is Calmnerv CR, it has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin within the concentration range of 0.01-0.316%.

[0062] When the effective substance to be tested is NEUROFENSE, it has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin within the concentration range of 0.01-1.0%.

[0063] When the effective substance to be tested is PACIFEEL, it has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin within the concentration range of 0.01-1.0%.

[0064] The present invention is further described below in conjunction with specific embodiments and comparative examples. However, these embodiments and comparative examples are only used to illustrate the present invention and do not constitute a limitation of the scope of the present invention. The experimental methods for which specific conditions are not specified in the following embodiments and comparative examples are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0065] The prepared concentrations of trans-4-tert-butyl cyclohexanol and the six raw materials in Examples 1 and 2 are mass / volume percentage concentrations (w / v) during testing.

[0066] The reagents, consumables, instruments and purchasing manufacturers used in the following examples are as follows:

[0067] 1. Cells, consumables and reagents

[0068] The human neuroblastoma cell line hTRPV1-SH-SY5Y overexpressing human TRPV1 receptor was obtained based on the method of CN 113667697A; T75 cell culture flasks and black-edged transparent-bottom 96-well plates were purchased from Corning, USA. Fetal bovine serum, cell culture medium high-glucose DMEM, antibiotic mixture (PSN), and 0.25% trypsin-EDTA were purchased from ThermoFisher, USA.

[0069] Dihydrocapsaicin (DHC) and green fluorescent calcium indicator Fluo-4, AM were purchased from ThermoFisher, USA; calcium channel inhibitor Capsazepine (CZ) was purchased from Merck, Germany. CCK-8 kit was purchased from Dojindo, Japan.

[0070] Sodium chloride, potassium chloride, magnesium sulfate heptahydrate, calcium chloride, potassium dihydrogen phosphate, glucose, and DMSO were purchased from Sinopharm Chemical Reagent Co., Ltd.; trans-4-tert-butyl cyclohexanol and HEPES were purchased from Sigma, USA.

[0071] KRH buffer (Krebs-Ringer HEPES buffer) formula: 125mM NaCl, 5mM KCl, 1.2mM MgSO4×7H2O, 2mM CaCl2, 1.2mM KH2PO4×2H2O, 25mM HEPES [free acid], 6mM glucose; adjust the pH to 7.4 with 1M NaOH.

[0072] 2. Instruments

[0073] Fluorescence microplate reader Fluoroskan Ascent FL and microplate reader Multiskan Mk3 were purchased from Thermo Company, USA.

[0074] Example 1: Evaluation of known TRPV1 antagonist trans-4-tert-butyl cyclohexanol based on human TRPV1 receptor overexpressing cell line

[0075] 1. Experimental Principle

[0076] Fluo-4, AM is the acetyl methyl ester derivative of Fluo-4. It is a fluorescent dye that can penetrate the cell membrane and is often used to detect intracellular calcium ion concentration. After Fluo-4, AM penetrates the cell membrane and enters the cell, it is cleaved by the esterase in the cell to form Fluo-4, which is then retained in the cell. Fluo-4 is almost non-fluorescent when it exists as a free ligand, but when it binds to intracellular calcium ions, it can produce strong fluorescence with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516nm. Fluorescence microscopes, fluorescent microplate readers, etc. can be used to detect changes in intracellular calcium ion concentration. The TRPV1 receptor is a non-selective ion channel. When the TRPV1 receptor is activated by substances such as dihydrocapsaicin, extracellular calcium ions will enter the cell through the TRPV1 receptor, and after binding to the Fluo-4 retained in the cell after loading, green fluorescence will be produced under the excitation of the excitation light. When the TRPV1 receptor antagonist binds to the TRPV1 receptor and inhibits its activity, the activity of dihydrocapsaicin in activating the TRPV1 receptor is inhibited, the ion channel will no longer open, and calcium ions cannot enter the cell, so the fluorescence produced after activation is no longer observed. Therefore, by observing and recording the changes in the fluorescence intensity in the cell, it is possible to analyze and determine the inhibition of the TRPV1 receptor, and then determine whether the detected substance has the effect of inhibiting stinging pain.

[0077] 2. Experimental Methods

[0078] 1. Seeding cells

[0079] The cells of the human TRPV1 receptor overexpressing cell line were inoculated in a 96-well plate with a black border and a transparent bottom. For each test object, 30 wells (well plate numbers B2-D11) were inoculated, with 1×10 5 100 μL 1×PBS buffer was added to the peripheral wells of the 96-well plate to reduce the effect of liquid evaporation on cell proliferation. The cells were cultured in a cell culture incubator for about 48 hours (culture conditions: 37° C., 5% CO2) until the cell confluence reached more than 95%.

[0080] 2. Prepare samples to be tested

[0081] Prepare the mother solution of trans-4-tert-butyl cyclohexanol with KRH buffer at a concentration of 0.04%. Then prepare 2-fold isoproportionate dilution solutions with KRH buffer, 7 concentration gradients, from low to high concentrations of 0.00063%, 0.00125%, 0.00250%, 0.00500%, 0.01000%, 0.02000%, 0.04000%. Also prepare the activator sample 180nM dihydrocapsaicin (prepared with KRH buffer) and KRH buffer containing 10μM inhibitor capsaicin.

[0082] 3. Loading cells with green fluorescent calcium indicator

[0083] Take out the 96-well plate, remove the old culture medium, add 50 μL of loading DMEM medium containing 2 μM Fluo-4, AM to each well, and incubate at 37°C for 25-35 minutes.

[0084] 4. Add the test sample and KRH buffer containing the inhibitor capsaicin

[0085] Pour out the loading medium, rinse once with 100 μL KRH buffer, then add 100 μL KRH buffer to each well of the KRH group (blank control group) and DHC group (negative control group), add 100 μL KRH buffer containing the inhibitor capsaicin to the wells of the CZ+DHC group (positive control group), add 100 μL of samples of different dilution concentrations to each well of the sample group, put in a cell culture incubator, and incubate at 37°C for 5-15 minutes.

[0086] 5. Add the activator dihydrocapsaicin

[0087] 20 μL of KRH buffer was added to each well of the KRH group (blank control group), and 20 μL of KRH buffer containing 180 nM dihydrocapsaicin was added to each well of the DHC group (negative control group), CZ+DHC group (positive control group) and sample group.

[0088] 5. Fluorescence microplate reader to collect data

[0089] Immediately measure the fluorescence value of each well using a fluorescence microplate reader.

[0090] 6. Data Analysis

[0091] The data collected by the fluorescence microplate reader were imported into an Excel table, and the average value of the 3-well KRH group (blank control group) was subtracted from the value of each well, and the value of the DHC group (negative control group) (after deducting the value of the KRH buffer) was taken as 100%, and the values ​​of the CZ+DHC group (positive control group) and the sample group with different concentrations were compared with it to obtain the relative fluorescence intensity percentage. The differences in the values ​​between the DHC group (negative control group) and the KRH (blank control group), the CZ+DHC group (positive control group) and the DHC group (negative control group), and the sample group and the DHC group (negative control group) were subjected to a t test, and each group was analyzed independently.

[0092] 3. Experimental Results

[0093] The results of the pain inhibition evaluation method based on human TRPV1 receptor overexpression cell lines showed that the known TRPV1 antagonist trans-4-tert-butyl cyclohexanol has the effect of inhibiting the activation of TRPV1 receptors by dihydrocapsaicin.

[0094] exist Figure 1In the figure, KRH represents KRH buffer control (blank control group), DHC represents dihydrocapsaicin control group (negative control group), CZ+DHC represents the group with TRPV1 receptor inhibitor capsaicin (CZ) and dihydrocapsaicin (positive control group), 0.00063%, 0.00125%, 0.00250%, 0.00500%, 0.01000%, 0.02000%, 0.04000% represent the mass concentration of trans-4-tert-butylcyclohexanol in the tested sample. Compared with the KRH group (blank control group), the p value <0.01 of the DHC group (negative control group) indicates that the difference between the two groups is highly statistically significant, which is indicated by "&&". Compared with the DHC group (negative control group), the p value <0.01 of the CZ+DHC group (positive control group) indicates that the difference between the two groups is highly statistically significant, which is indicated by "##". When the sample group was compared with the DHC group (negative control group), a p value of <0.05 indicated that the difference between the two groups was statistically significant, indicated by "*", and a p value of <0.01 indicated that the difference between the two groups was highly statistically significant, indicated by "**".

[0095] from Figure 1 The results showed that the DHC group (negative control group) showed that dihydrocapsaicin activated the TRPV1 receptor, with a relative fluorescence intensity of 100% compared to the KRH group, while the relative fluorescence intensity of the CZ+DHC group (positive control group) was -23.56%, indicating that capsaicin can strongly inhibit the activation of the TRPV1 receptor by dihydrocapsaicin. In the test group, trans-4-tert-butyl cyclohexanol showed that it had an inhibitory effect on the activation of the TRPV1 receptor within the range of 0.00250%-0.04000% by mass volume, and the lower the relative fluorescence intensity value, the higher the degree of inhibition of the TRPV1 receptor. The relative fluorescence intensity of trans-4-tert-butyl cyclohexanol was 61.06% at a concentration of 0.00250%, 14.51% at a concentration of 0.00500%, 17.53% at a concentration of 0.01000%, -0.43% at a concentration of 0.02000%, and 12.21% at a concentration of 0.04000%.

[0096] Example 2: Evaluation of 6 commercially available cosmetic raw materials based on the stinging inhibition evaluation method of human TRPV1 receptor overexpressing cell lines

[0097] 1. Experimental Principle

[0098] Fluo-4, AM is the acetyl methyl ester derivative of Fluo-4. It is a fluorescent dye that can penetrate the cell membrane and is often used to detect intracellular calcium ion concentration. After Fluo-4, AM penetrates the cell membrane and enters the cell, it is cleaved by the esterase in the cell to form Fluo-4, which is then retained in the cell. Fluo-4 is almost non-fluorescent when it exists as a free ligand, but when it binds to intracellular calcium ions, it can produce strong fluorescence with a maximum excitation wavelength of 494nm and a maximum emission wavelength of 516nm. Fluorescence microscopes, fluorescent microplate readers, etc. can be used to detect changes in intracellular calcium ion concentration. TRPV1 receptors are non-selective ion channels. When TRPV1 receptors are activated by substances such as dihydrocapsaicin, extracellular calcium ions enter the cell through the TRPV1 receptors, and after binding to Fluo-4 retained in the cell after loading, green fluorescence is produced under the excitation of excitation light. When the TRPV1 receptor antagonist binds to the TRPV1 receptor and inhibits its activity, the activity of dihydrocapsaicin in activating the TRPV1 receptor is inhibited, the ion channel will no longer open, and calcium ions cannot enter the cell, so the fluorescence produced after activation is no longer observed. Therefore, by observing and recording the changes in the fluorescence intensity in the cell, it is possible to analyze and determine the inhibition of the TRPV1 receptor, and then determine whether the detected substance has the effect of inhibiting stinging pain.

[0099] 2. Experimental Methods

[0100] 1. Cytotoxicity Test

[0101] Before evaluating the inhibition of stinging pain, a CCK-8 kit is used to evaluate the toxic effect of the sample on the cells to avoid interference of the cytotoxicity of the sample with the stinging pain inhibition evaluation test. As described in Example 1, the exposure time of the cells in the sample is 5-15 minutes, so it is necessary to detect the cell viability of the cells after exposure to the sample for 15 minutes.

[0102] 1.1 Inoculation of cells: Inoculate the cells of human TRPV1 receptor overexpressing cell line in 96-well plates, with 1×10 5 100 μL 1×PBS buffer was added to the peripheral wells of the 96-well plate, and the cells were cultured in a cell culture incubator for about 48 hours (culture conditions: 37° C., 5% CO 2 ) until the cell confluence reached more than 95%.

[0103] 1.2 Prepare samples: dilute the samples with KRH buffer, the highest sample concentration is 10%, the dilution ratio is 3.16, and the dilution gradient is 5-6. Some samples are highly toxic and need to be reduced in concentration for a second round of cytotoxicity testing.

[0104] 1.3 Cell incubation: Calculate the number of wells that need to be tested for toxicity, prepare the volume of CCK-8 working solution, and at least 1 blank well. Remove the old culture medium in the 96-well transparent plate, add CCK-8 working solution with a spray gun, 100 μL per well, and then place in a 37-degree incubator for 1-2 hours.

[0105] 1.4 Cell viability detection: After incubation, take out the transparent plate, remove the plate cover, and measure the OD value on an ELISA reader at a wavelength of 450 nm.

[0106] 1.5 Statistical analysis: After the test, the data was imported into Excel to calculate and analyze the cell viability.

[0107] 2. Suppress stinging evaluation

[0108] 2.1 Cell inoculation

[0109] The cells of the human TRPV1 receptor overexpressing cell line were seeded in a 96-well plate with a black border and a transparent bottom. For each test object, 30 wells were seeded, with 1×10 5 Cells, one 96-well plate can detect 2 substances (well plate number B2-D11 or E2-G11). Add 100μL 1×PBS buffer to the peripheral wells of the 96-well plate to reduce the effect of liquid evaporation on cell proliferation. Culture in a cell culture incubator for about 48 hours (culture conditions 37℃, 5% CO2) to make the cell confluence reach more than 95%.

[0110] 2.2 Prepare samples to be tested

[0111] Prepare commercial cosmetic raw materials with KRH buffer. Then prepare 2-fold or 3.16-fold isoproportional dilution solutions with 7 concentration gradients, measured by mass fraction, with KRH buffer. Also prepare a TRPV1 receptor activator sample of 180nM dihydrocapsaicin (prepared with KRH buffer) and a KRH buffer containing 10μM inhibitor capsaicin.

[0112] 2.3 Loading cells with green fluorescent calcium indicator

[0113] Take out the 96-well plate, remove the old culture medium, add 50 μL of loading DMEM medium containing 2 μM Fluo-4, AM to each well, and incubate at 37°C for 25-35 minutes.

[0114] 2.4 Add the test sample and KRH buffer containing the inhibitor capsaicin

[0115] Pour out the loading medium, rinse once with 100 μL KRH buffer, then add 100 μL KRH buffer to each well of the KRH group (blank control group) and DHC group (negative control group), add 100 μL KRH buffer containing the inhibitor capsaicin to the wells of the CZ+DHC group (positive control group), add 100 μL of samples of different dilution concentrations to each well of the sample group, put in a cell culture incubator, and incubate at 37°C for 5-15 minutes.

[0116] 2.5 Adding the activator dihydrocapsaicin

[0117] 20 μL of KRH buffer was added to each well of the KRH group (blank control group), and 20 μL of KRH buffer containing 180 nM dihydrocapsaicin was added to each well of the DHC group (negative control group), CZ+DHC group (positive control group) and sample group.

[0118] 2.6 Fluorescence microplate reader data collection

[0119] Immediately measure the fluorescence value of each well using a fluorescence microplate reader.

[0120] 2.7 Data Analysis

[0121] The data collected by the fluorescence microplate reader were imported into an Excel table, and the average value of the KRH buffer solution (blank control group) in 3 wells was subtracted from the value of each well. The value of the dihydrocapsaicin well (negative control group) (after deducting the value of the KRH buffer solution) was taken as 100%, and the values ​​of the wells of the CZ+DHC group (positive control group) and the sample group with different concentrations were compared with it to obtain the relative fluorescence intensity percentage. The differences in the values ​​between the DHC group (negative control group) and the KRH group (blank control group), the CZ+DHC group (positive control group) and the DHC group (negative control group), and the sample group and the DHC group (negative control group) were subjected to a t test, and each group was analyzed independently.

[0122] 3. Experimental Results

[0123] 1. Information and cytotoxicity results of 6 commercially available cosmetic raw materials are shown in the following table:

[0124] The results of CCK-8 cytotoxicity test showed that SymSitive 1609 had no cytotoxicity in the concentration range of ≤0.04%, SymCare W had no cytotoxicity in the concentration range of ≤0.02%, DCR 516 had no cytotoxicity in the test range of 10%, Calmnerv CR had no cytotoxicity in the concentration range of ≤0.316%, NEUROFENSE had no cytotoxicity in the test range of 10%, and Calmnerv CR had no cytotoxicity in the concentration range of ≤1.00%. In the subsequent sting inhibition evaluation test, the sample concentration shall not be higher than the above concentration.

[0125] Serial number name Raw material suppliers Cytotoxicity results 1 SymSitive 1609 Symrise ≤0.04％ 2 SymCare W Symrise ≤0.02％ 3 DCR 516 Xi'an Green Sky 10% of the tested area is non-toxic 4 Calmnerv CR Shanghai Jiakai ≤0.316％ 5 NEUROFENSE Sliab 10% of the tested area is non-toxic 6 PACIFEEL sederma ≤1.00％

[0126] 2. Suppression of sting test results

[0127] Six commercially available cosmetic raw materials were evaluated using a pain inhibition evaluation method based on a human TRPV1 receptor overexpressing cell line. The results showed that SymSitive 1609, SymCare W and DCR 516 had the effect of inhibiting the activation of TRPV1 receptors by dihydrocapsaicin; while Calmnerv CR, NEUROFENSE and PACIFEEL did not show the effect of inhibiting the activation of TRPV1 receptors by dihydrocapsaicin.

[0128] exist Figure 2 In the table, KRH represents KRH buffer control (blank control group), DHC represents dihydrocapsaicin control group (negative control group), CZ+DHC represents the group with TRPV1 receptor inhibitor capsaicin (CZ) and dihydrocapsaicin (positive control group), and the others are different concentrations of 6 commercially available cosmetic raw materials. Compared with the KRH group (blank control group), the p value <0.01 of the DHC group (negative control group) indicates that the difference between the two groups is highly statistically significant, indicated by "&&". Compared with the DHC group (negative control group), the p value <0.01 of the CZ+DHC group (positive control group) indicates that the difference between the two groups is highly statistically significant, indicated by "##". Compared with the DHC group (negative control group), the p value <0.05 of the sample group indicates that the difference between the two groups is statistically significant, indicated by "*", and the p value <0.01 indicates that the difference between the two groups is highly statistically significant, indicated by "**".

[0129] from Figure 2 The results showed that TRPV1 receptors could be activated by dihydrocapsaicin and inhibited by capsaicin, indicating that the experimental method was reliable. The tested sample SymSitive 1609 has the effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.0025-0.04%; the tested sample SymCare W has the effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.005%-0.02%; the tested sample DCR 516 has the effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.25%-1.0%; the tested sample Calmnerv CR has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.01%-0.316%; the tested sample NEUROFENSE has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.01%-1.0%; the tested sample PACIFEEL has no effect of inhibiting the activation of TRPV1 receptor by dihydrocapsaicin in the range of 0.01%-1.0%.

[0130] It should be noted here that since the factors affecting stinging pain are very complex, and this method is based on the detection of the inhibitory effect (antagonistic effect) of the detection substance on the activation of human TRPV1 receptors to evaluate the potential soothing effect of commercially available cosmetic raw materials on burning and stinging of sensitive skin. The sample evaluation results do not have the effect of inhibiting the activation of TRPV1 receptors by dihydrocapsaicin, which does not mean that the sample cannot achieve the purpose of soothing skin burning and stinging through other effects. For example, the sample can reduce the expression of TRPV1 receptors and reduce skin stinging by regulating inflammatory factors.

Claims

1. A cytological evaluation method for screening substances that inhibit stinging pain, comprising the following steps: (a) A human TRPV1 receptor overexpressing cell line was inoculated into a culture plate, and a fluorescent indicator was added to load the cells; (b) preparing a buffer containing an activator, a buffer containing an inhibitor, and a buffer containing a sample to be detected; (c) dividing the cell inoculation wells on the culture plate in step (a) into a blank control group, a negative control group, a positive control group and a sample group; (d) adding a buffer to the blank control group and the negative control group in step (c), adding the buffer containing the inhibitor prepared in step (b) to the positive control group in step (c), and adding the buffer containing the sample to be tested prepared in step (b) to the sample group in step (c); (e) continuing to add buffer to the blank control group in step (d), and adding the buffer containing the activator prepared in step (b) to the negative control group, the positive control group and the sample group in step (d); (f) detecting fluorescence value; (g) The fluorescence value of the negative control group in step (e) is taken as 100%. If the fluorescence value of the sample group is lower than 100%, the sample tested has an inhibitory effect on pain.

2. The method of claim 1, wherein: In the step (a), 0.8×10 5 to 1.2×10 5 The cells were seeded into the culture plate at a density of 10 cells / well.

3. The method of claim 1, wherein: The fluorescent indicator in step (a) is a green fluorescent calcium indicator.

4. The method of claim 1, wherein: The buffer in step (b) is selected from: KRH or PBS buffer.

5. The method of claim 1, wherein: The sample to be detected is a pure chemical or a mixture.

6. The method of claim 5, wherein: The pure chemical is trans-4-tert-butyl cyclohexanol, and the mixture is selected from: SymSitive 1609, SymCare W, DCR 516, Calmnerv CR, NEUROFENSE or PACIFEEL.

7. The method of claim 1, wherein: In the step (b), the sample to be tested is tested at a dose less than or equal to the maximum non-toxic dose, the sample to be tested is prepared according to the mass volume fraction, and the prepared sample concentration gradient is diluted geometrically by 1.25-10 times, preferably, by 1.5-5 times, and more preferably, by 2-3.16 times.

8. The method of claim 1, wherein the activator is selected from the group consisting of dihydrocapsaicin and capsaicin.

9. The method of claim 1, wherein the inhibitor is selected from the group consisting of capsaicin and ruthenium red.

10. The method according to claim 1, wherein the cytological method for screening substances with anti-stinging efficacy is evaluated by the inhibitory effect on the activation of TRPV1 receptor.

11. The method of claim 10, wherein the inhibitory effect on activation of TRPV1 receptors demonstrates a soothing effect on burning and stinging of sensitive skin.

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