TRPV1 receptor inhibitors containing oleic acid

Abstract

The invention provides an oleic acid-containing TRPV1 receptor inhibitor, and relates to an application of oleic acid in pain inhibition with a non-therapeutic purpose. The pain inhibiting effect means that burning hot stabbing pain of sensitive skin can be relieved. The invention also relates to an application of the long-chain monounsaturated fatty acid oleic acid in preparation of a skin external preparation for inhibiting pain.

Description

Technical Field

[0001] The present invention relates to the research field of the efficacy of cosmetic raw materials, and in particular to the ability of oleic acid to inhibit the activation of human TRPV1 receptors. The present invention can be used as a regulator of human TRPV1 receptor activity and has the efficacy of inhibiting pain. Background Art

[0002] Pain is a strong and unbearable discomfort caused by noxious stimulation, which is closely related to the activation of pain receptors on nerve fibers. There are a wide variety of sensory nerve endings on the human skin, mainly from dorsal root ganglia or trigeminal ganglia. Among them, there is a type of C nerve fiber that is thinner and unmyelinated, with slow nerve signal transmission, and mainly senses temperature, pain and itching. 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, and itching discomfort. At the same time, it can further cause the release of neuropeptides from 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.

[0003] The pain people feel is mainly related to TRPV1 receptors. For example, the discomfort of burning and tingling of sensitive skin is mainly related to the overactivation of TRPV1 receptors on the nerve endings of C nerve fibers on the skin. 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 temperature, low pH, and endogenous substances. It is a multimodal receptor. When TRPV1 receptors are 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 transmitting to the brain through nerve fibers and ganglia, producing a burning and tingling sensation. Therefore, research and development of substances or raw materials that inhibit the activation of TRPV1 receptors is an important research direction for alleviating discomfort such as burning and tingling of sensitive skin, and has important application value.

[0004] Oleic acid, also known as cis-9-octadecenoic acid, is a common long-chain monounsaturated fatty acid, belonging to n-9 (or ω-9, classified according to the position of the carbon-carbon double bond) unsaturated fatty acid. Oleic acid is also a natural fatty acid that exists in many animals and plants. It is widely used in the field of cosmetics and can be used as a moisturizer, emulsifier, skin conditioner, and penetration enhancer.

[0005] Since oleic acid has a certain ability to enhance the skin's ability to retain moisture, it can be added as a moisturizer in cosmetic formulas to make the skin's stratum corneum more lubricated and soft. Oleic acid can help mix oily ingredients and water-based ingredients together, so oleic acid can also be used as an emulsifier to make cosmetic formulas form more stable emulsions.

[0006] Oleic acid is also a physiologically active substance that can be used as a skin conditioner. For example, oleic acid has antioxidant properties, which can inhibit the formation of free radicals and reduce damage to the body; oleic acid has anti-inflammatory properties, which can inhibit inflammatory reactions, reduce inflammatory symptoms, and thus relieve pain; oleic acid also has anti-aging effects, which can promote the metabolism of skin cells, delay skin aging, and keep the skin soft and delicate.

[0007] In general, oleic acid has a variety of functions in skin care cosmetics, including moisturizing, emulsification, anti-oxidation, anti-inflammatory, promoting metabolism, etc. Therefore, it is widely used in various types of skin care cosmetics, such as creams, lotions, facial cleansers, etc.

[0008] At present, there are many patent applications involving the application of oleic acid in the field of cosmetics, and the application directions include promoter, edge activator, antioxidant, anti-aging, etc. The application of oleic acid in the field of cosmetics related to pain inhibition has not yet been found.

[0009] Our laboratory has previously established a stinging inhibition screening method based on human TRPV1 receptors, and tested and evaluated whether oleic acid in cosmetic raw materials has the ability to inhibit stinging. We unexpectedly discovered that oleic acid can be used as an important substance to regulate the activity of human TRPV1 receptors, and can be used in the cosmetic field as a skin conditioner for sensitive skin and other uncomfortable skin. SUMMARY OF THE INVENTION

[0011] In one aspect, the present invention provides the use of oleic acid for suppressing pain for non-therapeutic purposes.

[0012] In a preferred embodiment, the oleic acid is used at a concentration of at least 500 μM, more preferably 500-1000 μM.

[0013] In a preferred embodiment, the pain inhibition is achieved by inhibiting human TRPV1 receptor activation.

[0014] In a preferred embodiment, the pain suppression refers to relieving the burning and stinging pain of sensitive skin.

[0015] In another aspect, the present invention also relates to the use of oleic acid in the preparation of an external skin preparation for suppressing pain.

[0016] In a preferred embodiment, the content of oleic acid in the external skin preparation is 0.001-10% by weight.

[0017] In a preferred embodiment, the skin external preparation 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

[0018] Figure 1 The monounsaturated fatty acid oleic acid was shown to inhibit TRPV1 receptor activation. DETAILED DESCRIPTION

[0019] The present invention utilizes the stinging inhibition screening method based on human TRPV1 receptor established in the laboratory in the early stage to test and evaluate whether oleic acid in cosmetic raw materials has the ability to inhibit stinging.

[0020] The method for screening the inhibition of stinging pain based on human TRPV1 receptor comprises the following steps:

[0021] (a) A human TRPV1 receptor overexpressing cell line was inoculated into a culture plate, and a fluorescent indicator was added to load the cells;

[0022] (b) preparing a buffer containing an activator, a buffer containing an inhibitor, and a buffer containing a sample to be detected;

[0023] (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;

[0024] (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);

[0025] (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);

[0026] (f) detecting fluorescence value;

[0027] (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.

[0028] In a preferred embodiment, in step (a), 0.8×10 5 to 1.2×10 5In a preferred embodiment, the fluorescent indicator of step (a) is a green fluorescent calcium indicator. In a preferred embodiment, the buffer in step (b) is selected from KRH or PBS buffer. In a preferred embodiment, the activator is selected from dihydrocapsaicin or capsaicin. In a preferred embodiment, the inhibitor is selected from capsaicin or ruthenium red.

[0029] In a specific embodiment, the following groups are set up in the human TRPV1 receptor-based inhibition pain screening method: 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).

[0030] In some embodiments, step (e) is performed after incubation for 5-15 minutes after step (d). Since the incubation time of cells in the sample is 5-15 minutes, a cytotoxicity test is added to exclude the interference of cell death on the test.

[0031] In this method for screening pain inhibition based on human TRPV1 receptor, 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 sample tested has the effect of inhibiting pain. Therefore, this screening method only requires one detection to determine whether the sample tested has the effect of inhibiting pain. Therefore, a simple and rapid cytological evaluation method for screening pain inhibition substances is provided.

[0032] The present invention relates to the field of research on the efficacy of cosmetic raw materials. In fact, research has found that oleic acid has the efficacy of inhibiting stinging. Specifically, oleic acid has the ability to inhibit the activation of human TRPV1 receptors and can be used as a regulator of human TRPV1 receptor activity.

[0033] The beneficial effects of the present invention include: through a stinging inhibition screening method based on human TRPV1 receptor, it is found that oleic acid has the effect of inhibiting stinging, which can be used to guide and develop cosmetics suitable for sensitive skin with soothing effect, and has high research and application value.

[0034] Long-chain fatty acids

[0035] As used herein, the term "long chain fatty acids" (LCFA) is intended to mean fatty acids (FA) having a chain length greater than 14 carbon atoms, ie, a chain length of at least C14.

[0036] As used herein, the term "long chain monounsaturated fatty acids" (LCMUFA) is intended to mean monounsaturated fatty acids (MUFA) having a chain length greater than 16 carbon atoms.

[0037] In a preferred embodiment, the long chain monounsaturated fatty acid is oleic acid.

[0038] In some embodiments, the long chain monounsaturated fatty acid is used at a concentration of 500 μM to 1000 μM.

[0039] Skin topical products

[0040] The present invention also provides a skin topical agent capable of suppressing pain, which comprises a long-chain monounsaturated fatty acid oleic acid. In some embodiments, suppressing pain refers to suppressing skin stinging. For example, suppressing pain refers to relieving the burning and stinging pain of sensitive skin. In some embodiments, suppressing pain is achieved by suppressing human TRPV1 receptor activation.

[0041] The pain people feel is mainly related to TRPV1 receptors. For example, the discomfort of burning and tingling of sensitive skin is mainly related to the overactivation of TRPV1 receptors on the nerve endings of C nerve fibers on the skin. When TRPV1 receptors are 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, which are transmitted to the brain through nerve fibers and ganglia, producing burning and tingling sensations. The present invention studies and develops oleic acid, a substance with the effect of inhibiting the activation of TRPV1 receptors, which can relieve the discomfort of burning and tingling of sensitive skin and has important application value.

[0042] In some embodiments, the content of oleic acid in the external preparation for skin is 0.001-10 wt %, preferably 0.01-10 wt %.

[0043] The skin external preparation is a general concept for all ingredients usually used on the outside of the skin, for example, it can be a cosmetic composition. The cosmetic composition can be a basic cosmetic, a facial makeup cosmetic, a body cosmetic, a hair care cosmetic, etc., and there is no special restriction on its dosage form, which can be reasonably selected according to different purposes. The cosmetic composition also contains different media or matrix excipients allowed by the cosmetics level according to different dosage forms and purposes.

[0044] The skin external preparation is selected from: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

[0045] The skin topical preparation comprising oleic acid can be topically applied to human skin and / or hair. The skin topical preparation can also include a cosmetically acceptable topical carrier, which can be about 50 % by weight to about 99.99 % by weight of the skin topical preparation (such as about 80 % by weight to about 99 % by weight of the skin topical preparation). In a preferred embodiment of the present invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier can include one or more selected from wetting agents, emollients, grease, wetting agents and similar substances. In one embodiment, the cosmetically acceptable topical carrier includes a substrate such as a nonwoven fabric or a membrane material.

[0046] Skin topical agents can be prepared into a variety of product types, including but not limited to lotions, creams, gels, sticks, sprays, ointments, cleansing liquid lotions and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming products, facial masks and skin films, films and cosmetics such as foundation and mascara. These product types can contain several types of cosmetically acceptable topical carriers, including but not limited to solutions, suspensions, emulsions (e.g., microemulsions and nanoemulsions), gels, solids and liposomes.

[0047] Skin topical preparations containing oleic acid can be formulated as solutions. Solutions generally contain an aqueous solvent or an organic solvent (e.g., about 50% to about 99.99% or about 90% to about 99% of a cosmetically acceptable aqueous solvent or organic solvent). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerol, 1,2,4-butanetriol, sorbitol esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

[0048] Skin topical preparations can be formulated into solutions containing emollients. Such skin topical preparations preferably contain from about 2% to about 50% of one or more emollients. As used herein, "emollient" refers to a substance used to prevent or alleviate dryness, for example, by preventing the loss of skin moisture through the skin. Examples of emollients include, but are not limited to, vegetable oils, mineral oils, aliphatic esters, etc.

[0049] Lotions can be prepared from such solutions.Lotions typically contain from about 1% to about 20% (e.g., from about 5% to about 10%) of one or more emollients and from about 50% to about 90% (e.g., from about 60% to about 80%) water.

[0050] Another type of product that can be formulated from a solution is a cream. Creams typically contain from about 5% to about 50% (e.g., from about 10% to about 20%) of one or more emollients and from about 45% to about 85% (e.g., from about 50% to about 75%) water.

[0051] Although it is preferred that the skin external preparation containing oleic acid contains water, as an alternative, the skin external preparation can be anhydrous or an ointment containing no water but organic and / or silicone solvents, greases, lipids and waxes. The ointment can contain a simple base of animal or vegetable oil or semisolid hydrocarbon. The ointment can contain about 2% to about 10% of one or more emollients and about 0.1% to about 2% of one or more thickeners.

[0052] The topical agent for skin application can be formulated as an emulsion. If the topical carrier is an emulsion, about 1% to about 10% (e.g., about 2% to about 5%) of the topical carrier contains one or more emulsifiers. The emulsifier can be nonionic, anionic or cationic. Examples of suitable emulsifiers include those commonly identified as suitable emulsifiers in the field of personal care and cosmetic formulations.

[0053] Lotions and creams can be formulated as emulsions. Typically such lotions contain 0.5% to about 5% of one or more emulsifiers. Such creams typically contain about 1% to about 20% (e.g., about 5% to about 10%) of one or more emollients; about 20% to about 80% (e.g., 30% to about 70%) of water; and about 1% to about 10% (e.g., about 2% to about 5%) of one or more emulsifiers.

[0054] Oil-in-water and water-in-oil type single emulsion skin care preparations, such as lotions and creams, are well known in the cosmetics field and can be used in the present invention. Multiphase emulsion skin external preparations (such as water-in-oil-in-water type and oil-in-water-in-oil type) can also be used in the present invention. Generally, such single-phase emulsions or multiphase emulsions contain water, emollients and emulsifiers as their basic ingredients.

[0055] Skin topical preparations comprising oleic acid can also be formulated into gels (e.g., aqueous gels, alcoholic gels, alcohol / water gels or oily gels using suitable gelling agents). Suitable gelling agents for aqueous gels and / or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gelling agents typically contain between about 0.1% and 5% by weight of such gelling agents.

[0056] The external skin preparation containing oleic acid can also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing the powder).

[0057] In addition to the above-mentioned components, the external preparation for skin which can be used in the present invention may contain various other oil-soluble substances and / or water-soluble substances at levels conventionally established in its technical field for use in external preparations for skin for use on skin and hair.

[0058] The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin conditioners, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin external preparation and do not affect the effects of the oleic acid of the present invention.

[0059] In some embodiments of the skin topical preparation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl parahydroxybenzoate and phenoxyethanol. Based on the total weight of the composition, the amount of the preservative is about 0.5 to about 2 weight %, preferably about 0.5 to 1 weight %.

[0060] In one example of the skin external preparation of the present invention, one or more antioxidants can be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, hydroquinone, propyl gallate, nordihydroguaiaretic acid, vitamin E or vitamin E derivatives, vitamin C and its derivatives, calcium pantothenate, green tea extract and mixed polyphenols, and mixtures of the above-mentioned substances. The antioxidant used is about 0.02 to 0.5 weight % of the total weight of the composition, and more preferably from about 0.002 to 0.1 weight % of the amount used.

[0061] In one example of the skin external preparation of the present invention, one or more emollients can be used to act as lubricants through their ability to remain on the skin surface or in the stratum corneum to reduce peeling and improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, etc. Suitable examples of emollients include, but are not limited to, polypropylene glycol ("PPG")-15 stearyl ether, PPG-10 hexadecyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl alcohol ethylhexanoate, cetostearyl alcohol, glyceryl stearate, hydroxystearic acid octyl fat, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl alcohol ethylhexanoate, cetostearyl alcohol, glyceryl stearate and combinations thereof are preferred. When used, the emollient is based on about 0.1 to about 30 weight % of the total weight of the composition, preferably about 1 to about 30 weight % of the amount used.

[0062] In one example of the skin external preparation of the present invention, one or more moisturizing agents can be used. Moisturizing agents are also referred to as wetting agents, which help to improve the effect of emollients, reduce flaking, remove the scales of the stimulating composition, and improve the touch of the skin. Polyols can be used as moisturizing agents, including but not limited to glycerol, polyalkylene glycols, alkylene polyols and derivatives thereof, including butylene glycol, propylene glycol, dipropylene glycol, polyglycerol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol, and combinations thereof. When used, the amount of moisturizing agent is from about 0.1 to about 20% by weight, preferably from about 1 to about 15% by weight, based on the gross weight of the composition.

[0063] In an example of the skin external preparation of the present invention, one or more emulsifiers can be used. Emulsifiers can be used within the scope of effective stabilizing amount. Preferably, based on the total weight of the composition, the emulsifier is used in an amount of about 1.0 to about 10.0 weight %, more preferably about 3.0 to about 6.0 weight %. Any emulsifier compatible with the components in the composition can be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, stearyl alcohol, Steareth-2, Steareth-20, acrylic acid (ester) class / C10-30 alkyl alcohol acrylate cross-linked polymer, and combinations thereof.

[0064] In one example of the skin external preparation of the present invention, one or more pH regulators can be used. The pH regulator useful in the skin external preparation of the present invention includes tromethamine. When used, the amount of the pH regulator based on the total weight of the composition is about 0.1 to about 2 weight %, preferably about 0.1 to about 1 weight %.

[0065] In a specific embodiment of the present invention, the skin topical agent comprises acrylates / C10-30 alkyl acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine or a combination thereof.

[0066] The present invention is further described below in conjunction with specific embodiments and comparative examples. However, these embodiments are only used to illustrate the present invention and do not constitute a limitation of the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0067] The reagents, consumables, instruments and purchasing manufacturers used in the following examples are as follows:

[0068] 1. Cells, consumables and reagents

[0069] Human neuroblastoma cell line hTRPV1-SH-SY5Y overexpressing human TRPV1 receptor; 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.

[0070] 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.

[0071] Sodium chloride, potassium chloride, magnesium sulfate heptahydrate, calcium chloride, potassium dihydrogen phosphate, glucose, and DMSO were purchased from Sinopharm Chemical Reagent Co., Ltd.; HEPES was purchased from Sigma, USA.

[0072] Oleic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0073] 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.

[0074] 2. Instruments

[0075] Fluorescence microplate reader Fluoroskan Ascent FL and microplate reader Multiskan Mk3 were purchased from Thermo Corporation, USA, and fluorescence microscope DMi8 was purchased from Leica Corporation, USA.

[0076] Example 1: Test on the inhibition of TRPV1 receptor activation by long-chain monounsaturated fatty acid oleic acid

[0077] 1. Experimental Principle

[0078] The pain inhibition evaluation method based on the human TRPV1 receptor overexpression cell line is used to evaluate whether the tested substance has the ability / efficacy to inhibit pain. TRPV1 receptor is a non-selective ion channel. When TRPV1 receptor is activated by substances such as dihydrocapsaicin, extracellular calcium ions will enter the cell through the TRPV1 receptor. Fluo-4, AM is an 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 in the form of 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, fluorescence microplate readers, etc. can be used to detect changes in intracellular calcium ion concentration. When the TRPV1 receptor is activated, extracellular calcium ions enter the cell and bind to the Fluo-4 that is retained in the cell after loading, and then produce green fluorescence under the stimulation of the excitation light. When the TRPV1 receptor antagonist binds to the TRPV1 receptor and inhibits its activity, the activity of dihydrocapsaicin to activate 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 human TRPV1 receptor, and then determine whether the detected substance has the effect of inhibiting stinging.

[0079] 2. Experimental Methods

[0080] 1. Cytotoxicity Test

[0081] Before evaluating the inhibition of stinging, the CCK-8 kit is required to evaluate the toxicity of the sample to the cells to avoid interference of the sample's cytotoxicity with the stinging inhibition evaluation test. Since the cells need to be exposed to the sample for 5-15 minutes in subsequent tests, it is necessary to test the cell viability of the cells after 15 minutes of exposure to the sample.

[0082] 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%.

[0083] 2.2 Sample preparation: First prepare 200 mM oleic acid sample stock solution with DMSO, then dilute the sample with KRH buffer. The highest sample concentration is 1000 μM, the dilution ratio is 1:2 or 1:3.16, and the dilution gradient is 5-7.

[0084] 2.3 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.

[0085] 2.4 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.

[0086] 2.5 Statistical analysis: After the test, the data was imported into Excel to calculate and analyze the cell viability.

[0087] 2. Suppress stinging evaluation

[0088] 2.1 Cell inoculation

[0089] 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%.

[0090] 2.2 Prepare samples to be tested

[0091] First, prepare a 200mM stock solution of oleic acid sample with DMSO, and then dilute it with KRH buffer in a proportional gradient, with a dilution ratio of 1:2, 7 concentration gradients, the highest concentration is 1000μM, and the other concentrations are 500μM, 250μM, 125μM, 62.5μM, 31.25μM, and 15.625μM. In addition, prepare 180nM dihydrocapsaicin, a TRPV1 receptor activator sample, and KRH buffer containing 10μM inhibitor capsaicin. Note: Dihydrocapsaicin and capsaicin need to be dissolved in DMSO to prepare the stock solution, and then diluted with KRH buffer.

[0092] 2.3 Loading cells with green fluorescent calcium indicator

[0093] 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.

[0094] 2.4 Add the test sample and KRH buffer containing the inhibitor capsaicin

[0095] 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.

[0096] 2.5 Adding the activator dihydrocapsaicin

[0097] 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.

[0098] 2.6 Fluorescence microplate reader data collection

[0099] Immediately measure the fluorescence value of each well using a fluorescence microplate reader.

[0100] 2.7 Data Analysis

[0101] The data collected by the fluorescence microplate reader were imported into an Excel table, and the average value of KRH rent (blank control group) in 3 wells was subtracted from the value of each well, and the value of the DHC group (negative control group) (after deducting the value of KRH buffer) 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.

[0102] 3. Experimental Results

[0103] 1. The information of long-chain monounsaturated fatty acid oleic acid and cytotoxicity results are shown in the following table:

[0104] The results of CCK-8 cytotoxicity test showed that oleic acid had no cytotoxicity within the test range of 1000μM.

[0105] Serial number name Carbon chain type Cytotoxicity results 1 Oleic acid 18∶1(n-9) No cytotoxicity within the tested range of 1000 μM

[0106] 2. Long-chain monounsaturated fatty acid oleic acid inhibits stinging test results

[0107] The long-chain monounsaturated fatty acid oleic acid was evaluated using a pain inhibition evaluation method based on a human TRPV1 receptor overexpressing cell line. The results are shown below.

[0108] exist Figure 1 In 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), and the others are different concentrations of oleic acid in the fatty acid sample to be tested. Compared with the KRH group (blank control group), the p value <0.01 in 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 in 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 in 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 "**".

[0109] from Figure 1 The results show that human TRPV1 receptors can be activated by dihydrocapsaicin and inhibited by capsaicin, and the difference is significant, indicating that the experimental method is reliable. The sample test results are as follows: the relative fluorescence intensity of the tested sample oleic acid is -53.32% at a concentration of 1000μM, and the relative fluorescence intensity is 39.74% at a concentration of 500μM, which means that oleic acid has a significant inhibitory effect on the activation of human TRPV1 receptors by dihydrocapsaicin at high concentrations.

[0110] In summary, the results showed that the long-chain monounsaturated fatty acid oleic acid showed an inhibitory effect on the activation of human TRPV1 receptor by dihydrocapsaicin and had human TRPV1 receptor antagonist activity.

Claims

1. Application of oleic acid in suppressing pain for non-therapeutic purposes.

2. The use according to claim 1, wherein: The oleic acid is used at a concentration of at least 500 μM.

3. The use according to claim 2, wherein: The oleic acid is used at a concentration of 500-1000 μM.

4. The use according to claim 1, wherein: The pain suppression is achieved by inhibiting the activation of human TRPV1 receptor.

5. The use according to claim 1, wherein: The pain suppression refers to relieving the burning and stinging pain of sensitive skin.

6. Use of oleic acid in the preparation of an external skin preparation for suppressing pain.

7. The use according to claim 6, wherein: The content of oleic acid in the external skin preparation is 0.001-10% by weight.

8. The use according to claim 7, wherein: The skin external preparation is 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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