Raw material or product pain risk evaluation method based on human TRPV1 receptor combined inflammatory factor

Abstract

The invention relates to a method for evaluating the pain risk of a raw material or a product of a combined inflammatory factor based on a human TRPV1 receptor, which comprises the following steps of: (a) providing keratinocytes with the human TRPV1 receptor, and establishing a detection method based on the TRPV1 receptor; (b) adding the inflammatory factor and a to-be-detected raw material or product into a detection system, and exposing the cells to the inflammatory factor and the to-be-detected raw material or product; and (c) analyzing and judging the activation condition of the TRPV1 receptor by observing and recording the change of the fluorescence intensity in the cells so as to judge the generation of the skin pain effect.

Description

Technical Field

[0001] The present invention relates to the field of safety evaluation of cosmetics and their ingredients, and in particular to a method for evaluating the pain risk of raw materials or products based on human TRPV1 receptor combined with inflammatory factors. Background Art

[0002] Cosmetics have become a necessity in people's daily lives. They play a great role in beautifying the face and skin and promoting physical and mental health. Generally, cosmetics contain water, oil and other active ingredients. Microorganisms will grow in a certain environment. In addition, during the use and storage of cosmetics, the products may also be contaminated by microorganisms, which may bring hidden dangers to the products. In order to ensure the safety of cosmetics, it is necessary to add preservatives, but preservatives may also cause adverse skin reactions.

[0003] There are many types of preservatives, the main ones are the following: 1) Aromatic alcohols: phenoxyethanol, benzyl alcohol; 2) Parabens: propylparaben, butylparaben; 3) Formaldehyde releasers: imidazolidinyl urea, DMDM ​​hydantoin, methylchloroisothiazolinone and methylisothiazolinone; 4) Propynyl butylcarbamate; 5) Benzoic acid, sorbic acid and its sodium salt; In addition, there are some types without preservative claims, such as 6) 1,2-pentanediol, 1,2-hexanediol, phenylethyl alcohol; 7) Ketones: p-hydroxyacetophenone; 8) Medium and long carbon chain polar amphiphilic substances: ethylhexylglycerin, glyceryl caprate, sorbitan caprylate, glyceryl undecylenate; 9) Organic acids: levulinic acid, anisic acid, caprylhydroxamic acid, etc.

[0004] Phenoxyethanol has a wide range of antibacterial activity, and is effective against various Gram-negative and Gram-positive bacteria and yeasts, and has a weak inhibitory effect on the inherent flora of the skin, that is, it has a weak impact on the inherent microecological system of the skin. As parabens and formaldehyde-releasing preservatives are gradually replaced, aromatic alcohol phenoxyethanol has become one of the most widely used cosmetic preservatives. According to the requirements of the Cosmetic Safety Technical Specifications (2015 Edition), the maximum allowable concentration of phenoxyethanol when used in cosmetics is 1%. However, based on feedback from some consumers or the actual application of product formulators, product formulas containing about 0.5% phenoxyethanol will cause a stinging reaction, especially in some people with sensitive skin or imperfect barrier function.

[0005] In order to verify the pain-inducing phenomenon of phenoxyethanol, the evaluation method based on the human TRPV1 receptor overexpression cell line disclosed in the applicant's previous patent application CN 113667697A was adopted. TRPV1 receptor (transient receptor potential vanilloid type 1, transient receptor potential vanilloid subtype 1, also known as capsaicin receptor) plays an important role in the generation and transmission of pain in mammals. TRPV1 receptors are mainly distributed in small to medium-sized neurons in dorsal root ganglia and trigeminal ganglia, especially small-diameter unmyelinated sensory nerve fibers. Based on the mechanism of action of human TRPV1 receptors in pain generation and transmission, the applicant independently constructed a neural cell model that overexpresses human TRPV1 receptors. "Overexpression" mainly refers to the integration of TRPV1 receptor genes into the genome of SH-SY5Y cells by means of lentiviral infection, amplifying the expression signal of TRPV1 receptors by about 26 times, and establishing a pain evaluation method based on this. However, on this detection platform, no signal activating TRPV1 receptors was detected at 0.1%-1% phenoxyethanol.

[0006] The fact that phenoxyethanol’s pain-inducing effect was not detected in the human TRPV1 receptor overexpressing cell line basically ruled out the sensitivity of the detection method, so it was considered that it might be produced through the combined mediation of certain factors. Based on the common characteristics of sensitive skin and skin with imperfect barriers, both may have chronic or weak skin inflammation, so we tried to add the combined exposure of inflammatory factors to the development of the pain-inducing detection method of preservatives represented by phenoxyethanol.

[0007] Interleukin, abbreviated as interleukin, refers to lymphokines that interact between white blood cells or immune cells. It belongs to the same category as blood cell growth factor, which is a cytokine. The two coordinate and interact with each other to complete the hematopoietic and immunoregulatory functions. Interleukin plays an important role in transmitting information, activating and regulating immune cells, mediating T and B cell activation, proliferation and differentiation, and inflammatory response. Interleukin-1 (IL-1, also known as lymphocyte stimulating factor) is mainly produced by activated monocytes and macrophages. There are two forms, IL-1α and IL-1β. The main biological functions are: 1. Local low concentration - immune regulation: synergistically stimulate APC and T cell activation, promote B cell proliferation and secretion of antibodies; 2. Large-scale production - endocrine effect: induce acute phase protein synthesis in the liver; cause fever and malignant lesions.

[0008] Therefore, in order to more accurately evaluate the safety and pain-causing properties of cosmetics and their ingredients, the present application provides a method for evaluating the pain-causing risks of raw materials or products based on the human TRPV1 receptor in combination with inflammatory factors (such as interleukin-1). SUMMARY OF THE INVENTION

[0010] In one aspect, the present invention provides a method for assessing the pain risk of raw materials or products of combined inflammatory factors based on human TRPV1 receptor, comprising the following steps:

[0011] (a) providing keratinocytes with human TRPV1 receptors and establishing a detection method based on TRPV1 receptors;

[0012] (b) adding inflammatory factors and raw materials or products to be detected into the detection system, so that the cells are exposed to the inflammatory factors and the raw materials or products to be detected;

[0013] (c) By observing and recording the changes in intracellular fluorescence intensity, the activation of TRPV1 receptors is analyzed and determined, thereby determining the skin pain effect.

[0014] In a preferred embodiment, the inflammatory factor is selected from IL-1α or IL-1β. In a preferred embodiment, the inflammatory factor is used at a concentration of at least 12.5 ng / mL.

[0015] In a preferred embodiment, the raw material or product to be detected is a preservative or a raw material or product with a preservative function, wherein the preservative is phenoxyethanol, and the raw material with a preservative function is selected from ethylhexylglycerin and p-hydroxyacetophenone.

[0016] In a preferred embodiment, after the TRPV1 receptor is activated, extracellular calcium ions enter the cell through the TRPV1 receptor, bind to the fluorescent probe pre-loaded in the cell, and generate fluorescence under the excitation of excitation light, wherein the probe is a calcium ion probe.

[0017] The evaluation method of the present invention is applied to the field of cosmetics and is used to evaluate the pain risk of cosmetic raw materials or cosmetic final products.

[0018] On the other hand, the present invention provides a method for screening ingredients that have the effect of alleviating the pain-causing effect of raw materials in cosmetics or cosmetics, comprising: using the evaluation method of the present invention to evaluate the pain risk and select raw materials that can alleviate the pain risk.

[0019] In a preferred embodiment, the cosmetic is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The calcium flux test results of Example 1 are shown.

[0021] Figure 2 A bar graph of the calcium flux test results of Example 1 is shown.

[0022] Figure 3A bar graph showing the results of the calcium flux test of IL-1α in Example 2 is shown.

[0023] Figure 4 A bar graph showing the results of the calcium flux test of IL-1β in Example 2 is shown.

[0024] Figure 5 A bar graph showing the calcium flux test results of IL-1α+phenoxyethanol in Example 3.

[0025] Figure 6 The calcium flux test results of IL-1β+phenoxyethanol in Example 3 are shown.

[0026] Figure 7 A bar graph showing the calcium flux test results of IL-1β+phenoxyethanol in Example 3.

[0027] Figure 8 A bar graph showing the results of the calcium flux test of IL-1β+ethylhexylglycerol / parahydroxyacetophenone in Example 4 is shown. DETAILED DESCRIPTION

[0028] In order to better observe the activation effect of combined exposure to interleukins and preservatives (eg, phenoxyethanol) on TRPV1 receptors, normal human keratinocytes (which have a normal number of TRPV1 receptors on their surface) were selected, rather than the SH-SY5Y cell line overexpressing TRPV1 receptors.

[0029] The detection index is the signal intensity of intracellular calcium ions after the sample acts on it. The principle is that the TRPV1 receptor is a non-selective ion channel. When the TRPV1 receptor is activated, extracellular calcium ions will enter the cell through the TRPV1 receptor and combine with the fluorescent probe pre-loaded in the cell, which will produce green fluorescence under the excitation of the excitation light. Therefore, by observing and recording the changes in the fluorescence intensity in the cell, the activation of the TRPV1 receptor can be analyzed and determined.

[0030] The present invention uses a calcium ion probe to observe and record changes in intracellular fluorescence intensity, thereby analyzing and determining the activation of the TRPV1 receptor. The probes that can be used in the evaluation method of the present invention include, but are not limited to: Fluo-3 AM, Fluo-4 AM, Rhod-2 AM, Calbryte 520, Calbryte 590, and Calbryte 630. These calcium ion probes provide powerful fluorescence measurement tools, greatly improving intracellular retention and loading efficiency, which is conducive to determining the activation of the TRPV1 receptor.

[0031] The evaluation method of the present invention is mainly divided into 4 steps for verification and analysis:

[0032] 1. Establish a detection method based on TRPV1 receptor and verify it with positive control;

[0033] 2. Expose IL-1α and IL-1β to the detection system respectively to observe their effects on TRPV1 receptor;

[0034] 3. Add IL-1α and IL-1β and different concentrations of phenoxyethanol into the detection system to observe whether phenoxyethanol can activate TRPV1 receptors and thus produce skin pain effects.

[0035] 4. Explore whether the IL-1 combined exposure model is also applicable to the prediction of the pain-inducing effects of other preservatives.

[0036] Therefore, the present invention provides a method for assessing the pain risk of raw materials or products based on human TRPV1 receptor combined with inflammatory factors, the key point of which is the need to introduce combined exposure to inflammatory factors (e.g., IL-1β). The model cells used in the present invention are keratinocytes, and the activation of TRPV1 receptors is analyzed and determined by observing and recording the changes in intracellular fluorescence intensity.

[0037] Evaluation of the pain risk of raw materials or products

[0038] Necessary ingredients are often added to cosmetics. For example, in order to ensure the safety of cosmetics, it is necessary to add preservatives. However, these added ingredients (e.g., preservatives) may also cause adverse skin reactions (e.g., irritation and pain).

[0039] As for preservatives, they mainly include: 1) aromatic alcohols: phenoxyethanol, benzyl alcohol; 2) parabens: propylparaben, butylparaben; 3) formaldehyde releasers: imidazolidinyl urea, DMDM ​​hydantoin, methylchloroisothiazolinone and methylisothiazolinone; 4) propynyl butylcarbamate; 5) benzoic acid, sorbic acid and its sodium salt; in addition, there are some types without preservative claims, such as 6) 1,2-pentanediol, 1,2-hexanediol, phenylethyl alcohol; 7) ketones: p-hydroxyacetophenone; 8) medium and long carbon chain polar amphiphilic substances: ethylhexylglycerin, glyceryl caprate, sorbitan caprylate, glyceryl undecylenate; 9) organic acids: levulinic acid, anisic acid, caprylhydroxamic acid, etc.

[0040] Starting from the problem of detecting and quantifying the pain-inducing effects of preservatives (e.g., phenoxyethanol), the applicant found that the evaluation method based on human TRPV1 receptor overexpression cell line in previous studies (CN 113667697A) could not detect the activation of TRPV1 receptor by phenoxyethanol.

[0041] However, the inventors unexpectedly discovered that combined exposure to interleukins (eg, IL-1α and IL-1β) and preservatives (eg, phenoxyethanol) can activate the TRPV1 receptor.

[0042] Based on the widespread use of preservatives (e.g., phenoxyethanol) in products and the universal use of preservatives in products, this study established an innovative method for detecting the skin nociceptive effects of preservatives (e.g., phenoxyethanol).

[0043] At the same time, this application confirms through further testing that this method can also be extended to the pain risk assessment of some raw materials that have no preservative claims but have certain preservative effects.

[0044] In addition, from a further application perspective, it can also be extended to the study of the efficacy of screening different raw materials in alleviating the pain-inducing effects of preservatives, as well as the pain risk assessment of cosmetic final products.

[0045] 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.

[0046] Example 1: Establishment of a detection method based on TRPV1 receptor

[0047] 1. Test system

[0048] The cells used in this test were keratinocytes, batch number: Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0049] 2. Main reagents

[0050] KC2500 culture medium (Guangdong Boxi Biotechnology), PBS (Solarbio), fetal bovine serum (FBS, Lanzhou Rongye), trans-4-tert-butyl cyclohexanol (TCI), capsaicin CAP (Vokay), Fluo-3AM calcium ion fluorescent probe.

[0051] 3. Main equipment

[0052] CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500).

[0053] 4. Sample information

[0054] Capsaicin CAP: TRPV1 receptor activator; trans-4-tert-butylcyclohexanol: TRPV1 receptor inhibitor.

[0055]

[0056] 5. Calcium flow test

[0057] Test method:

[0058] 1) Cell seeding: 1.8×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0059] 2) Solution preparation: According to the test groups, use PBS to prepare the working solutions of the test substances.

[0060] 3) Probe loading: Aspirate the keratinocyte culture medium, wash 3 times with PBS, add 0.5mL probe loading working solution to each well, and incubate in a 37℃ incubator for 45min. After the incubation, aspirate the probe working solution and wash 3 times with PBS. The probe working solution can also be called "probe solution". The probe is prepared with PBS and the concentration is 2μM.

[0061] 4) Stimulation and drug administration: 0.5 mL of PBS solution was added to each well of the BC group, 0.05 μM capsaicin CAP was added to each well of the NC group, and 0.05 μM capsaicin CAP and 3.9 μg / mL trans-4-tert-butyl cyclohexanol were added to each well of the PC group. After drug administration, the cells were incubated in an incubator for 1 h.

[0062] 5) Calcium flow detection: After incubation, observe and take pictures using a fluorescence microscope to collect cell morphology under a 200× field of view. The photography time is controlled within 30 minutes.

[0063] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0064] 6. Test results

[0065] Figure 1 Calcium flux test results are shown. Figure 2 A bar graph of calcium flux test results is shown.

[0066] Table 1 below summarizes the calcium flux test results.

[0067] Table 1

[0068] Group Relative IOD average SD P-value BC 1.00 0.11 / NC 2.06 0.27 0.000## PC 1.11 0.22 0.000**

[0069] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0070] 7. Conclusion

[0071] Compared with the BC group, the cellular calcium ion signal intensity of the NC group increased significantly, indicating that the TRPV1 receptor activator (capsaicin CAP) effectively activated the TRPV1 receptor; compared with the NC group, the cellular calcium ion signal intensity of the PC group decreased significantly, indicating that the TRPV1 receptor inhibitor (trans-4-tert-butyl cyclohexanol) can effectively inhibit TRPV1 receptor activation. Both prove that this detection system is effective and suitable for the detection of TRPV1 receptors.

[0072] Example 2: Effects of IL-1α and IL-1β exposure on TRPV1 receptor

[0073] 1. Test system

[0074] The cells used in this test were keratinocytes, batch number: Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0075] 2. Main reagents

[0076] KC2500 culture medium (Guangdong Boxi Biotechnology), PBS (Solarbio), fetal bovine serum (FBS, Lanzhou Rongye), trans-4-tert-butyl cyclohexanol (TCI), IL-1α, IL-1β (Solarbio), Fluo-3 AM calcium ion fluorescent probe.

[0077] 3. Main equipment

[0078] CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500).

[0079] 4. Sample information

[0080] Interleukins IL-1α and IL-1β are predicted to activate TRPV1 receptors; trans-4-tert-butylcyclohexanol: a TRPV1 receptor inhibitor.

[0081]

[0082]

[0083] 5. Calcium flow test

[0084] Test method:

[0085] 1) Cell seeding: 1.8×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0086] 2) Solution preparation: According to the test groups, use PBS to prepare the working solutions of the test substances.

[0087] 3) Probe loading: Aspirate the keratinocyte culture medium, wash 3 times with PBS, add 0.5mL probe loading working solution to each well, and incubate in a 37℃ incubator for 45min. After the incubation, aspirate the probe working solution and wash 3 times with PBS. The probe working solution can also be called "probe solution". The probe is prepared with PBS and the concentration is 2μM.

[0088] 4) Drug administration:

[0089] IL-1α group: 0.5 mL PBS was added to each well of the BC group; 0.5 mL PBS solution containing 12.5 ng / mL (denoted as: NC-12.5 ng / mL), 25 ng / mL (denoted as: NC-25 ng / mL), and 50 ng / mL (denoted as: NC-50 ng / mL) IL-1α was added to each well of the NC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1α + 3.9 μg / mL trans-4-tert-butylcyclohexanol (denoted as: PC-NC-12.5 ng / mL), 25 ng / mL IL-1α + 3.9 μg / mL trans-4-tert-butylcyclohexanol (denoted as: PC-NC-25 ng / mL), and 50 ng / mL IL-1α was added to each well of the PC group. After administration, a PBS solution of IL-1α+3.9 μg / mL trans-4-tert-butylcyclohexanol (denoted as: PC-NC-50 ng / mL) was incubated in an incubator for 1 h.

[0090] IL-1β group: 0.5 mL PBS was added to each well of the BC group; 0.5 mL PBS solution containing 12.5 ng / mL (denoted as: NC-12.5 ng / mL) and 250 ng / mL (denoted as: NC-250 ng / mL) IL-1β was added to each well of the NC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β+3.9 μg / mL trans-4-tert-butylcyclohexanol (denoted as: PC-NC-12.5 ng / mL) and 250 ng / mL IL-1β+3.9 μg / mL trans-4-tert-butylcyclohexanol (denoted as: PC-NC-250 ng / mL) was added to each well of the PC group. After the administration, the cells were incubated in the incubator for 1 h.

[0091] 5) Calcium flow detection: After incubation, observe and take pictures using a fluorescence microscope to collect cell morphology under a 200× field of view. The photography time is controlled within 30 minutes.

[0092] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0093] 6. Test results

[0094] Figure 3 A bar graph showing the results of the calcium flux assay for IL-1α.

[0095] Table 2 below summarizes the calcium flux assay results for IL-1α.

[0096] Table 2

[0097]

[0098] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0099] The results showed that compared with the BC group, the cellular calcium ion signal intensity of the NC-12.5ng / mL, NC-25ng / mL, and NC-50ng / mL groups increased significantly, and the relationship was dose-response. Compared with the NC-12.5ng / mL group, the cellular calcium ion signal intensity of the PC-NC-12.5ng / mL group decreased significantly. Compared with the NC-25ng / mL group, the cellular calcium ion signal intensity of the PC-NC-25ng / mL group decreased significantly. Compared with the NC-50ng / mL group, the cellular calcium ion signal intensity of the PC-NC-50ng / mL group decreased significantly. It can be seen that IL-1α can activate TRPV1 receptors, and at the same time, TRPV1 receptor inhibitors (trans-4-tert-butylcyclohexanol) can effectively inhibit the activation of TRPV1 receptors by IL-1α.

[0100] Figure 4 A bar graph showing the results of the calcium flux assay for IL-1β.

[0101] Table 3 below summarizes the calcium flux assay results for IL-1β.

[0102] Table 3

[0103]

[0104] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0105] The results showed that compared with the BC group, the cellular calcium ion signal intensity of the NC-12.5ng / mL and NC-250ng / mL groups increased significantly, and the relationship was dose-response. Compared with the NC-12.5ng / mL group, the cellular calcium ion signal intensity of the PC-NC-12.5ng / mL group decreased significantly. Compared with the NC-250ng / mL group, the cellular calcium ion signal intensity of the PC-NC-250ng / mL group decreased significantly. It can be seen that IL-1β can activate TRPV1 receptors, and at the same time, TRPV1 receptor inhibitors (trans-4-tert-butyl cyclohexanol) can effectively inhibit the activation of TRPV1 receptors by IL-1β.

[0106] 7. Conclusion

[0107] Both IL-1α and IL-1β can activate TRPV1 receptors, and TRPV1 receptor inhibitors can effectively inhibit receptor activation. IL-1α and IL-1β have similar activation effects on TRPV1 receptors.

[0108] Example 3: Effects of combined exposure of IL-1α and IL-1β with different concentrations of phenoxyethanol on TRPV1 receptors

[0109] 1. Test system

[0110] The cells used in this test were keratinocytes, batch number: Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0111] 2. Main reagents

[0112] KC2500 culture medium (Guangdong Boxi Biotechnology), PBS (Solarbio), fetal bovine serum (FBS, Lanzhou Rongye), trans-4-tert-butyl cyclohexanol (TCI), IL-1α, IL-1β (Solarbio), phenoxyethanol (Weimaikong Biotechnology (Shanghai) Co., Ltd.), and Fluo-3 AM calcium ion fluorescent probe.

[0113] 3. Main equipment

[0114] CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500)

[0115] 4. Sample information

[0116] Interleukins IL-1α and IL-1β, which can activate TRPV1 receptors; trans-4-tert-butylcyclohexanol: a TRPV1 receptor inhibitor; phenoxyethanol, predicted to activate TRPV1 receptors through combined exposure.

[0117]

[0118]

[0119] 5. Calcium flow test

[0120] Test method:

[0121] 1) Cell seeding: 1.8×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0122] 2) Solution preparation: According to the test groups, use PBS to prepare the working solutions of the test substances.

[0123] 3) Probe loading: Aspirate the keratinocyte culture medium, wash 3 times with PBS, add 0.5mL probe loading working solution to each well, and incubate in a 37℃ incubator for 45min. After the incubation, aspirate the probe working solution and wash 3 times with PBS. The probe working solution can also be called "probe solution". The probe is prepared with PBS and the concentration is 2μM.

[0124] 4) Drug administration:

[0125] IL-1α group: 0.5 mL PBS was added to each well of the BC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1α was added to each well of the NC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1α + 3.9 μg / mL trans-4-tert-butylcyclohexanol was added to each well of the PC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1α + 0.1% phenoxyethanol (denoted as phenoxyethanol-0.1%), 12.5 ng / mL IL-1α + 0.2% phenoxyethanol (denoted as phenoxyethanol-0.2%), 12.5 ng / mL IL-1α + 0.4% phenoxyethanol (denoted as phenoxyethanol-0.4%), and 12.5 ng / mL IL-1α + 0.8% phenoxyethanol (denoted as phenoxyethanol-0.8%) was added to each well of the sample group, respectively. After the administration, the cells were incubated in the incubator for 1 h.

[0126] IL-1β group: 0.5 mL PBS was added to each well of the BC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β was added to each well of the NC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β + 3.9 μg / mL trans-4-tert-butylcyclohexanol was added to each well of the PC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β + 0.1% phenoxyethanol (denoted as phenoxyethanol-0.1%), 12.5 ng / mL IL-1β + 0.2% phenoxyethanol (denoted as phenoxyethanol-0.2%), 12.5 ng / mL IL-1β + 0.4% phenoxyethanol (denoted as phenoxyethanol-0.4%), and 12.5 ng / mL IL-1β + 0.8% phenoxyethanol (denoted as phenoxyethanol-0.8%) was added to each well of the sample group, respectively. After the administration, the cells were incubated in the incubator for 1 h.

[0127] 5) Calcium flow detection: After incubation, observe and take pictures using a fluorescence microscope to collect cell morphology under a 200× field of view. The photography time is controlled within 30 minutes.

[0128] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0129] 6. Test results

[0130] Figure 5 A bar graph showing the calcium flux test results for IL-1α+phenoxyethanol.

[0131] Table 4 below shows the calcium flux test results of IL-1α+phenoxyethanol.

[0132] Table 4

[0133] Experimental Grouping Relative IOD average SD P-value BC 1.00 0.19 / NC (IL-1α) 2.34 0.22 0.000## PC 0.89 0.12 0.000** Phenoxyethanol - 0.1% 0.90 0.11 0.000** Phenoxyethanol - 0.2% 1.61 0.31 0.000** Phenoxyethanol - 0.4% 1.74 0.21 0.000** Phenoxyethanol - 0.8% 2.08 0.17 0.013*

[0134] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0135] The results showed that compared with the BC group, the intensity of the cell calcium ion signal in the NC group increased significantly, indicating that the experimental stimulation conditions of IL-1α were effective. Compared with the NC group, the intensity of the cell calcium ion signal in the PC group decreased significantly, indicating that the positive control test was effective. Compared with the NC group, the intensity of the cell calcium ion signal in the phenoxyethanol-0.1%, phenoxyethanol-0.2%, phenoxyethanol-0.4%, and phenoxyethanol-0.8% groups decreased significantly. This shows that IL-1α can activate TRPV1 receptors, and no activation effect of phenoxyethanol on TRPV1 receptors was observed when IL-1α and phenoxyethanol were jointly exposed.

[0136] Figure 6 The results of the calcium flux assay for IL-1β+phenoxyethanol are shown. Figure 7 A bar graph showing the calcium flux test results for IL-1β+phenoxyethanol.

[0137] Table 5 below shows the calcium flux test results of IL-1β+phenoxyethanol.

[0138] Table 5

[0139] Experimental Grouping Relative IOD average SD P-value BC 1.00 0.13 / NC (IL-1β) 1.64 0.12 0.000## PC 0.94 0.13 0.000** Phenoxyethanol - 0.1% 1.53 0.17 0.133 Phenoxyethanol - 0.2% 1.78 0.16 0.047* Phenoxyethanol - 0.4% 4.96 0.66 0.000** Phenoxyethanol - 0.8% 15.78 1.53 0.000**

[0140] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0141] The results showed that compared with the BC group, the intensity of the cell calcium ion signal in the NC group increased significantly, indicating that the IL-1β experimental stimulation conditions were effective. Compared with the NC group, the intensity of the cell calcium ion signal in the PC group decreased significantly, indicating that the positive control test was effective. Compared with the NC group, there was no difference in the intensity of the cell calcium ion signal in the phenoxyethanol-0.1% group, and the intensity of the cell calcium ion signal in the phenoxyethanol-0.2%, phenoxyethanol-0.4%, and phenoxyethanol-0.8% groups increased significantly. It can be seen that IL-1β can activate TRPV1 receptors. The combined exposure method of IL-1β and phenoxyethanol effectively detected the activation effect of phenoxyethanol on TRPV1 receptors, and as the concentration of phenoxyethanol increased, the activation effect also increased in a concentration-dependent manner.

[0142] 7. Conclusion

[0143] Both IL-1α and IL-1β can activate TRPV1 receptors, but the effects exhibited when co-exposed with phenoxyethanol are different. No activation effect of phenoxyethanol on TRPV1 receptors was observed when IL-1α and phenoxyethanol were co-exposed, but it was unexpectedly found that the activation effect of phenoxyethanol on TRPV1 receptors was effectively detected when IL-1β and phenoxyethanol were co-exposed, and as the concentration of phenoxyethanol increased, the activation effect also increased in a concentration-dependent manner, indicating that the pain-inducing phenomenon of phenoxyethanol on the skin can be quantitatively detected through this model. At the same time, starting from a phenoxyethanol concentration of about 0.4%, the intensity of the cellular calcium ion signal increased sharply compared with the NC group (IL-1β activation group) and the 0.2% phenoxyethanol group, which is consistent with the feedback from consumers or the perception of formulators when using phenoxyethanol at a concentration of about 0.5%, which may cause skin pain, indicating that this detection model has high practical applicability in subsequent practice.

[0144] Example 4: Applicability of the IL-1 co-exposure model to the detection of pain-inducing effects of other preservatives

[0145] 1. Test system

[0146] The cells used in this test were keratinocytes, batch number: Ep220707, provided by Guangdong Boxi Biotechnology Co., Ltd.

[0147] 2. Main reagents

[0148] KC2500 culture medium (Guangdong Boxi Biotechnology), PBS (Solarbio), fetal bovine serum (FBS, Lanzhou Rongye), trans-4-tert-butyl cyclohexanol (Tianjin Ai Pharmaceutical Co., Ltd.), ethylhexylglycerol (Ashland LLC), p-hydroxyacetophenone (Dezhixin Flavors and Fragrances (Nantong) Co., Ltd.), Fluo-3 AM calcium ion fluorescent probe.

[0149] 3. Main equipment

[0150] CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), fluorescence microscope (Leica, DM2500)

[0151] 4. Sample information

[0152] Interleukin IL-1β can activate TRPV1 receptors; trans-4-tert-butyl cyclohexanol: TRPV1 receptor inhibitor; ethylhexylglycerin (a raw material with no preservative claims and a certain preservative effect), parahydroxyacetophenone (a raw material with no preservative claims and a certain preservative effect).

[0153]

[0154] 5. Calcium flow test

[0155] Test method:

[0156] 1) Cell seeding: 1.8×10 5 Keratinocytes were seeded into 6-well plates at a seeding density of 100 cells / well and incubated in an incubator (37°C, 5% CO 2 ) and incubate overnight.

[0157] 2) Solution preparation: According to the test groups, use PBS to prepare the working solutions of the test substances.

[0158] 3) Probe loading: Aspirate the keratinocyte culture medium, wash 3 times with PBS, add 0.5mL probe loading working solution to each well, and incubate in a 37℃ incubator for 45min. After the incubation, aspirate the probe working solution and wash 3 times with PBS. The probe working solution can also be called "probe solution". The probe is prepared with PBS and the concentration is 2μM.

[0159] 4) Drug administration:

[0160] 0.5 mL PBS was added to each well of the BC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β was added to each well of the NC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β + 3.9 μg / mL trans-4-tert-butylcyclohexanol was added to each well of the PC group; 0.5 mL PBS solution containing 12.5 ng / mL IL-1β + 0.05% ethylhexylglycerol (denoted as Jahwa-2-0.05%), 12.5 ng / mL IL-1β + 0.1% ethylhexylglycerol (denoted as Jahwa-2-0.1%), 12.5 ng / mL IL-1β + 0.1% p-hydroxyacetophenone (denoted as Jahwa-3-0.1%), 12.5 ng / mL IL-1β + 0.2% p-hydroxyacetophenone (denoted as Jahwa-3-0.2%), and 12.5 ng / mL After administration, the PBS solution of IL-1β+0.4% p-hydroxyacetophenone (referred to as Jahwa-3-0.4%) was incubated in an incubator for 1 hour.

[0161] 5) Calcium flow detection: After incubation, observe and take pictures using a fluorescence microscope to collect cell morphology under a 200× field of view. The photography time is controlled within 30 minutes.

[0162] 6) Statistical analysis of results: GraphPad Prism was used for plotting, and the results were expressed as Mean ± SD. The t-test was used for statistical analysis for comparison between groups. All statistical analyses were two-tailed. P < 0.05 was considered to be significantly different, and P < 0.01 was considered to be extremely significantly different.

[0163] 6. Test results

[0164] Figure 8 A bar graph showing the results of the calcium flux assay for IL-1β+ethylhexylglycerol / parahydroxyacetophenone.

[0165] Table 6 below shows the results of the calcium flux test for IL-1β+ethylhexylglycerol / parahydroxyacetophenone.

[0166] Table 6

[0167] Experimental Grouping Relative IOD average SD P-value BC 1.00 0.11 / NC (IL-1α) 9.63 0.72 0.000## PC 1.96 0.21 0.000** Jahwa - 2 - 0.05% 4.86 0.65 0.001** Jahwa - 2 - 0.1% 23.13 2.22 0.001** Jahwa - 3 - 0.1% 0.94 0.12 0.000** Jahwa - 3 - 0.2% 1.34 0.17 0.000** Jahwa - 3 - 0.4% 2.98 0.43 0.000**

[0168] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P value < 0.05 was indicated by #, and P value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P value < 0.05 was indicated by *, and P value < 0.01 was indicated by **.

[0169] The results showed that compared with the BC group, the cellular calcium signal intensity of the NC group increased significantly. Compared with the NC group, the cellular calcium signal intensity of the PC group decreased significantly. Compared with the NC group, the cellular calcium signal intensity of the Jahwa-2-0.1% group increased significantly, and the cellular calcium signal intensity of the Jahwa-2-0.05%, Jahwa-3-0.1%, Jahwa-3-0.2%, and Jahwa-3-0.4% groups decreased significantly. It can be seen that the combined exposure of IL-1β and ethylhexylglycerol, starting from a concentration of ethylhexylglycerol above about 0.1%, will cause a significant increase in the cellular calcium signal intensity and activate TRPV1 signaling. The combined exposure of IL-1β and parahydroxyacetophenone did not cause an increase in the cellular calcium signal intensity within the concentration range of 0.1%-0.4%.

[0170] 7. Conclusion

[0171] In addition to phenoxyethanol, the pain risk assessment method of human TRPV1 receptor combined with inflammatory factor IL-1β was used to test two raw materials that have no preservative claims but have certain preservative effects: ethylhexylglycerol and p-hydroxyacetophenone. Ethylhexylglycerol showed a certain effect similar to phenoxyethanol in activating TRPV1 receptors, suggesting that it may have skin pain risks starting from a concentration of 0.1%; no similar phenomenon was observed with p-hydroxyacetophenone, which is speculated to be milder. This is a further promotion and application of the pain risk assessment method of human TRPV1 receptor combined with inflammatory factor IL-1β.

Claims

1. A method for assessing the pain risk of raw materials or products of combined inflammatory factors based on human TRPV1 receptor, comprising the following steps: (a) providing keratinocytes with human TRPV1 receptors and establishing a detection method based on TRPV1 receptors; (b) adding inflammatory factors and raw materials or products to be detected into the detection system, so that the cells are exposed to the inflammatory factors and the raw materials or products to be detected; (c) By observing and recording the changes in intracellular fluorescence intensity, the activation of TRPV1 receptors is analyzed and determined, thereby determining the skin pain effect.

2. The method of claim 1, wherein: The inflammatory factor is selected from IL-1α or IL-1β.

3. The method of claim 1, wherein: The raw material or product to be tested is a preservative or a raw material or product with preservative function.

4. The method of claim 3, wherein: The preservative is phenoxyethanol, and the raw material with preservative function is selected from ethylhexylglycerin and p-hydroxyacetophenone.

5. The method of claim 1, wherein: After the TRPV1 receptor is activated, extracellular calcium ions enter the cell through the TRPV1 receptor, bind to the fluorescent probe pre-loaded in the cell, and produce fluorescence under the excitation of the excitation light.

6. The method of claim 5, wherein: The probe is a calcium ion probe.

7. The method of claim 1, wherein: The inflammatory factor is used at a concentration of at least 12.5 ng / mL.

8. The method of claim 1, wherein: The method is applied in the field of cosmetics to evaluate the pain risk of cosmetic raw materials or cosmetic final products.

9. A method for screening ingredients that have an effect of relieving the pain-causing effect of raw materials or cosmetics in cosmetics, comprising: The pain risk is assessed by the method described in any one of claims 1 to 8, and raw materials that can alleviate the pain risk are selected.

10. The method of claim 9, wherein: The cosmetics are selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

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