Application of long-chain fatty acid with unsaturation greater than or equal to 3 in pain inhibition

Abstract

The present invention relates to the use of long-chain fatty acids with unsaturation > = 3 for pain inhibition for non-therapeutic purposes. The pain inhibiting effect means that burning hot stabbing pain of sensitive skin can be relieved. The invention also relates to the application of the long-chain fatty acid with the unsaturation degree being greater than or equal to 3 in preparing a skin external preparation for inhibiting pain.

Description

Technical Field

[0001] The present invention relates to the field of research on the efficacy of cosmetic raw materials, and in particular to the ability of long-chain fatty acids with a degree of unsaturation of ≥3 to inhibit the activation of human TRPV1 receptors. The present invention can be used as a regulator of 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 discomfort such as burning, tingling, and itching. 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] In the process of studying the activation of TRPV1 receptors, it was unexpectedly discovered that fatty acids can be used as important substances to regulate the activity of human TRPV1 receptors and have certain physiological significance. Fatty acids are a class of compounds composed of three chemical elements (carbon, hydrogen, and oxygen). They are the main components involved in the formation of neutral fats, phospholipids, and glycolipids in organisms and play an important role in life activities. At the same time, fatty acids are widely used in the fields of food, health products, and cosmetics.

[0005] There are many types of naturally occurring fatty acids. Depending on whether the fatty acids contain carbon-carbon double bonds, they can be divided into saturated fatty acids (SFA) without carbon-carbon double bonds and unsaturated fatty acids with carbon-carbon double bonds. Unsaturated fatty acids can be further divided into monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) according to whether they contain one or two or more carbon-carbon double bonds.

[0006] According to the synthesis of animals or humans, fatty acids can be divided into non-essential fatty acids (NEFA) and essential fatty acids (EFA). Most fatty acids can be synthesized by animals or humans themselves and do not need to be directly ingested from food. These fatty acids are called non-essential fatty acids. Non-essential fatty acids are mainly saturated fatty acids and monounsaturated fatty acids, such as oleic acid. Animals or humans cannot synthesize polyunsaturated fatty acids containing 2 or more double bonds and must obtain them from plants. These fatty acids are called essential fatty acids, such as α-linolenic acid and linoleic acid.

[0007] According to the position of double bonds on the carbon chain of unsaturated fatty acids, they can also be divided into n-3, n-6, n-9 and other series. If the first double bond appears on the third carbon atom from the carbon atom farthest from the carboxyl group in the straight-chain fatty acid, this fatty acid is called the n-3 series; if the first double bond appears on the sixth carbon atom, it is called the n-6 series; and so on.

[0008] Depending on the length of the carbon chain, fatty acids can be divided into short-chain fatty acids (containing 2 to 4 carbon atoms), medium-chain fatty acids (containing 6 to 12 carbon atoms) and long-chain fatty acids (containing more than 14 carbon atoms).

[0009] The long-chain fatty acids with one or two unsaturations are myristic acid (cis-9-tetradecenoic acid, 14:1 (n-5)), sapiens acid (cis-6-hexadecenoic acid, 16:1 (n-10)), arachidonic acid (cis-7-hexadecenoic acid, 16:1 (n-9)), palmitoleic acid (cis-9-hexadecenoic acid, 16:1 (n-7)), petroselinic acid (cis-6-octadecenoic acid, 18:1 (n-12)), oleic acid (cis- -9-octadecenoic acid, 18:1(n-9)), gondoic acid (cis-11-eicosenoic acid, 20:1(n-9)), erucic acid (cis-13-eicosenoic acid, 22:1(n-9)), neuraminic acid (cis-15-eicosadienoic acid, 24:1(n-9)), linoleic acid (cis-9,12-octadecadienoic acid, 18:2(n-6)), cis-11,14-eicosadienoic acid (20:2(n-6)), etc.

[0010] The long-chain fatty acids with unsaturation ≥3 are γ-linolenic acid (cis-9,12,15-octadecatrienoic acid, 18:3 (n-6)), dihomo-γ-linolenic acid (cis-8,11,14-eicosatrienoic acid, 20:3 (n-6)), arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid, 20:4 (n-6)), 4,7,10,13,16-docosapentaenoic acid (22:5 (n-6)), adrenic acid (7,10,13,16-docosatetraenoic acid, 22:4 (n-6)), 4,7,10,13,16,19,22-octadecanoic acid (28:7 (n-6)), 4,7,10,13,16,19,22,25,28-triadecanoic acid (34:9 (n-6)). -6)), α-linolenic acid (cis-9,12,15-octadecatrienoic acid, 18:3(n-3)), 11,14,17-eicosatetraenoic acid (20:3(n-3)), stearic acid (6,9,12,15-octadecatrienoic acid, 18:4(n-3)), 3,6,9,12,15-octadecatrienoic acid (18:5(n-3)), 8,11,14,17-eicosatetraenoic acid (20:4(n-3)), EPA (5,8,11,14,17-eicosapentaenoic acid, 20:5(n-3)), 7,10,13,16,19-docosapentaenoic acid (22:5(n-3)), DHA (4,7,10,13,16,19-docosahexaenoic acid, 22:6(n-3)), etc.

[0011] Fatty acids not only have important application value in nutrition, but also have wide applications in the field of cosmetics. There are many fatty acids or fatty acid-related derivatives in the catalog of used cosmetic raw materials. The characteristics of fatty acids are closely related to the chain length and degree of unsaturation. According to their different characteristics, some fatty acids can be used as surfactants and emulsifiers, while other fatty acids, especially unsaturated fatty acids, have important skin conditioning functions, such as increasing the fluidity of cell membranes, strengthening and repairing the natural barrier function of cell membranes, reducing transdermal water loss, improving skin moisturizing status, protecting against harmful environmental substances, increasing beneficial anti-inflammatory and anti-inflammatory ingredients, and alleviating skin diseases. In addition to being excellent moisturizers, skin conditioners, protective agents and anti-inflammatory ingredients, unsaturated fatty acids can also be used as carriers and penetration enhancers for transporting substances to increase the skin's absorption of other active substances. At present, there are many patents that discuss the application of fatty acids in the above-mentioned functions.

[0012] Chinese invention patent application CN115282068A discloses an organic gel carrier, a preparation method thereof and cosmetics. The carrier uses at least one of fatty acids, fatty acid esters and oils, as well as hydroxystearic acid, higher fatty alcohols and surfactants, and can embed different types of unstable or water-insoluble active ingredients therein, thereby serving as an organic gel carrier for the active ingredients, and promoting the penetration of the active ingredients into the skin and transporting them to the deep layers of the skin epidermis to fully exert their biological effects.

[0013] Chinese invention patent application CN115175739A discloses a personal care composition and methods of preparing and using the same, wherein the personal care composition comprises a complex comprising a plurality of short-chain fatty acids for increasing the firmness and / or elasticity of a patient's skin.

[0014] Chinese invention patent application CN113397992A discloses a sunscreen composition and an oil-in-water emulsified cosmetic having the sunscreen composition. The sunscreen composition contains a certain proportion of higher fatty acids, silicone and zinc oxide powder, which can improve the dispersion performance of zinc oxide and maintain its fluidity, and improve the stability of high-content zinc oxide in the oil-in-water system. While maintaining skin moisture, it can also improve the dryness of high-content zinc oxide powder on the skin surface, giving the skin a soft and smooth use experience.

[0015] Chinese invention patent application CN113350262A provides a moisturizer suitable for children with atopic dermatitis, which can assist in the treatment of children with atopic dermatitis, reduce the dosage of therapeutic hormones, and effectively relieve the irritation caused by therapeutic hormones. The formula contains a certain proportion of plant sterols, fatty acids, and ceramides, which can reduce TEWL and keep the skin's moisture content stable.

[0016] Chinese invention patent application CN111557874A discloses a bionic, camellia extract-containing composition for repairing the skin barrier, which contains a certain proportion of fatty acids, combined with squalene substances, fatty acids, triglycerides, and cholesterol analogs, and is used for repairing the sebum membrane.

[0017] At present, there are no patents or research related to the use of fatty acids in the field of cosmetics to inhibit pain. Therefore, this application uses the pain inhibition screening method based on human TRPV1 receptor established in the laboratory in the early stage to test and evaluate for the first time whether various long-chain fatty acids in cosmetic raw materials have the ability to inhibit pain. SUMMARY OF THE INVENTION

[0019] In one aspect, the present invention provides the use of a long-chain fatty acid with a degree of unsaturation ≥ 3 for suppressing pain for non-therapeutic purposes, wherein the long-chain fatty acid has a chain length of 14 or more carbon atoms.

[0020] In a preferred embodiment, the long chain fatty acid has a chain length of 18-20 carbon atoms. In a preferred embodiment, the long chain fatty acid with a degree of unsaturation ≥ 3 is a LCFA n3 or LCFA n6 with a length of C18-C20 having 3-5 double bonds. In a preferred embodiment, the long chain fatty acid is selected from: α-linolenic acid, arachidonic acid, eicosapentaenoic acid or a combination thereof.

[0021] In a preferred embodiment, the long chain fatty acid is used at a concentration of at least 30 μM.

[0022] In a preferred embodiment, the pain inhibition is achieved by inhibiting human TRPV1 receptor activation.

[0023] In a preferred embodiment, the pain suppression refers to relieving the burning and stinging pain of sensitive skin.

[0024] On the other hand, the present invention also provides the use of a long-chain fatty acid with an unsaturation degree ≥ 3 in the preparation of an external skin preparation for inhibiting pain, wherein the long-chain fatty acid has a chain length of 14 or more carbon atoms.

[0025] In a preferred embodiment, the content of the long-chain fatty acid in the external skin preparation is 0.001-10% by weight.

[0026] 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

[0027] Figure 1 Long-chain fatty acids with a degree of unsaturation ≥3 inhibit human TRPV1 receptor activation.

[0028] Figure 2 Long-chain fatty acids with 1 and 2 degrees of unsaturation inhibit human TRPV1 receptor activation. DETAILED DESCRIPTION

[0029] 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 various long-chain fatty acids in cosmetic raw materials have the ability to inhibit stinging.

[0030] The method for screening the inhibition of stinging pain based on human TRPV1 receptor comprises the following steps:

[0031] (a) A human TRPV1 receptor overexpressing cell line was inoculated into a culture plate, and a fluorescent indicator was added to load the cells;

[0032] (b) preparing a buffer containing an activator, a buffer containing an inhibitor, and a buffer containing a sample to be detected;

[0033] (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;

[0034] (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);

[0035] (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);

[0036] (f) detecting fluorescence value;

[0037] (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.

[0038] In a preferred embodiment, in step (a), 0.8×10 5 to 1.2×10 5 In 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.

[0039] 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).

[0040] 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.

[0041] 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.

[0042] The present invention relates to the field of research on the efficacy of cosmetic raw materials. In fact, the research has found that a series of unsaturated fatty acids have the efficacy of inhibiting stinging, specifically a series of long-chain fatty acids with an unsaturation degree of ≥3 have a strong ability to inhibit the activation of human TRPV1 receptors and can be used as regulators of human TRPV1 receptor activity.

[0043] The beneficial effects of the present invention include: screening and obtaining a variety of long-chain fatty acid cosmetic raw materials with a degree of unsaturation of ≥3 and a certain ability to inhibit stinging, which can be used to guide and develop cosmetics suitable for sensitive skin with soothing effects, and have high research and application value.

[0044] Long-chain fatty acids

[0045] 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.

[0046] As used herein, the term "long chain polyunsaturated fatty acids" (LCPUFAs) is intended to mean polyunsaturated fatty acids (PUFAs) having a chain length greater than 16 carbon atoms.

[0047] 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.

[0048] As used herein, the term "LCn3" is intended to mean a polyunsaturated n-3 fatty acid having a chain length greater than 16 carbon atoms, but it is understood that LCn3 represents a subclass of LCPUFA. Similarly, the term "LCn6" is intended to mean a polyunsaturated n-6 ​​fatty acid having a chain length greater than 16 carbon atoms.

[0049] Thus, the chain length of the LCFA used herein is C14-C28, such as C14-C26, preferably C14-24. In some embodiments, the chain length of the LCFA used herein is C16-C28, such as C16-C26, preferably C16-24. In some embodiments, the chain length of the LCFA used herein is C18-C28, such as C18-C26, preferably C18-24. In some embodiments, the chain length of the LCFA used herein is C18-C20.

[0050] In a preferred embodiment, the present invention uses a long-chain fatty acid with a degree of unsaturation of ≥ 3. In some embodiments, the long-chain fatty acid with a degree of unsaturation of ≥ 3 is selected from: γ-linolenic acid (cis-9,12,15-octadecatrienoic acid, 18:3 (n-6)), dihomo-γ-linolenic acid (cis-8,11,14-eicosatrienoic acid, 20:3 (n-6)), arachidonic acid (cis-5,8,11,14-eicosatetraenoic acid, 20:4 (n-6)), 4,7,1 0,13,16-docosapentaenoic acid (22:5(n-6)), adrenoic acid (7,10,13,16-docosapentaenoic acid, 22:4(n-6)), 4,7,10,13,16,19,22-octadecanoic acid (28:7(n-6)), 4,7,10,13,16,19,22,25,28-triadecanoic acid (34: 9 (n-6)), α-linolenic acid (cis-9,12,15-octadecatrienoic acid, 18:3 (n-3)), 11,14,17-tricosenoic acid (20:3 (n-3)), stearic acid (6,9,12,15-octadecatetraenoic acid, 18:4 (n-3)), 3,6,9,12,15-octadecatrienoic acid (18:5 (n-3)), 8,11, 14,17-eicosatetraenoic acid (20:4(n-3)), EPA (5,8,11,14,17-eicosapentaenoic acid, 20:5(n-3)), 7,10,13,16,19-docosapentaenoic acid (22:5(n-3)), DHA (4,7,10,13,16,19-docosahexaenoic acid, 22:6(n-3)) or a combination thereof.

[0051] In some embodiments, the long chain fatty acid with a degree of unsaturation of ≥3 is a long chain fatty acid with 3-5 double bonds. In some embodiments, the long chain fatty acid with a degree of unsaturation of ≥3 is VLCn3 or VLCn6. In a preferred embodiment, the long chain fatty acid with a degree of unsaturation of ≥3 is a LCFA n3 or LCFA n6 with a length of C18-C20 with 3-5 double bonds.

[0052] In a preferred embodiment, the long-chain fatty acid with a degree of unsaturation ≥ 3 is selected from: α-linolenic acid, arachidonic acid, eicosapentaenoic acid, or a combination thereof.

[0053] In some embodiments, the long-chain fatty acid is used at a concentration of at least 30 μM. In some embodiments, the long-chain fatty acid is used at a concentration of 30 μM-1000 μM. In some embodiments, the long-chain fatty acid is used at a concentration of 500 μM-1000 μM. In some embodiments, the long-chain fatty acid is used at a concentration of 200 μM-500 μM. In some embodiments, the long-chain fatty acid is used at a concentration of 50 μM-200 μM. In some embodiments, the long-chain fatty acid is used at a concentration of 50 μM-150 μM.

[0054] Skin topical products

[0055] The present invention also provides a skin topical agent capable of suppressing pain, comprising a long-chain fatty acid with a degree of unsaturation of ≥ 3. In some embodiments, suppressing pain refers to suppressing skin stinging. For example, suppressing pain refers to relieving burning and stinging pain of sensitive skin. In some embodiments, suppressing pain is achieved by suppressing activation of human TRPV1 receptors.

[0056] 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 a substance with the effect of inhibiting the activation of TRPV1 receptors, which can alleviate the discomfort of burning and tingling of sensitive skin, and has important application value.

[0057] In some embodiments, the content of the long-chain fatty acid with a degree of unsaturation ≥ 3 in the external preparation for skin is 0.001-10 wt %, preferably 0.01-10 wt %.

[0058] 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.

[0059] 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.

[0060] The skin topical preparation comprising a long-chain fatty acid having a degree of unsaturation of ≥3 can be topically applied to human skin and / or hair. The skin topical preparation may also comprise a cosmetically acceptable topical carrier, which may be from about 50% by weight to about 99.99% by weight of the skin topical preparation (e.g., from 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 may comprise one or more selected from wetting agents, emollients, greases, moisturizers and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or a membrane material.

[0061] 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.

[0062] The skin topical preparation containing a long-chain fatty acid with a degree of unsaturation of ≥3 can be formulated as a solution. The solution generally contains 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] Although it is preferred that the skin external preparation containing long-chain fatty acids with a degree of unsaturation of ≥3 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 oils or semisolid hydrocarbons. 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] Skin topical preparations containing long-chain fatty acids with a degree of unsaturation of ≥3 can also be formulated into gels (e.g., aqueous gels, alcoholic gels, alcohol / water-based 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.

[0071] The topical preparation for skin application comprising a long-chain fatty acid having a degree of unsaturation of 3 or more may also be formulated into a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing a powder).

[0072] 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.

[0073] The skin topical preparation of the present invention may contain additional components commonly found in skin care compositions, such as moisturizers, skin regulators, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin topical preparation and do not affect the effects of the long-chain fatty acids with a degree of unsaturation ≥ 3 of the present invention.

[0074] 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 %.

[0075] 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.

[0076] 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 flaking and improve the appearance of the skin. Typical emollients include fatty esters, fatty alcohols, mineral oils, polyether silicone copolymers, and the like. Examples of suitable emollients include, but are not limited to, polypropylene glycol ("PPG")-15 stearyl ether, PPG-10 cetyl ether, Steareth-10, Oleth-8, PPG-4 lauryl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, hydroxystearate octyl, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl ethylhexanoate, cetearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is used in an amount ranging from about 0.1 to about 30% by weight, preferably about 1 to about 30% by weight, based on the total weight of the composition.

[0077] In one example of the skin external preparation of the present invention, one or more moisturizers can be used. Moisturizers are also called humectants, which help to improve the effect of emollients, reduce flaking, remove the scales of the composition and improve the touch of the skin. Polyols can be used as moisturizers, 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 moisturizer is about 0.1 to about 20% by weight, preferably about 1 to about 15% by weight, based on the total weight of the composition.

[0078] 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.

[0079] 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 %.

[0080] 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.

[0081] 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.

[0082] The reagents, consumables, instruments and purchasing manufacturers used in the following examples are as follows:

[0083] 1. Cells, consumables and reagents

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Stearic acid, petroselinic acid, linoleic acid, α-linolenic acid, arachidonic acid, and eicosapentaenoic acid were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0088] 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.

[0089] 2. Instruments

[0090] 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.

[0091] Example 1: Test on the inhibition of TRPV1 receptor activation by long-chain fatty acids with unsaturation ≥ 3

[0092] 1. Experimental Principle

[0093] 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 TRPV1 receptor, and then determine whether the detected substance has the effect of inhibiting stinging.

[0094] 2. Experimental Methods

[0095] 1. Cytotoxicity Test

[0096] 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.

[0097] 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%.

[0098] 2.2 Sample preparation: First prepare 200 mM fatty 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.

[0099] 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.

[0100] 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.

[0101] 2.5 Statistical analysis: After the test, the data was imported into Excel to calculate and analyze the cell viability.

[0102] 2. Suppress stinging evaluation

[0103] 2.1 Cell inoculation

[0104] 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%.

[0105] 2.2 Prepare samples to be tested

[0106] First, prepare a 200mM fatty acid sample stock solution with DMSO, and then dilute it with KRH buffer in an isocratic 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.

[0107] 2.3 Loading cells with green fluorescent calcium indicator

[0108] 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.

[0109] 2.4 Add the test sample and KRH buffer containing the inhibitor capsaicin

[0110] 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.

[0111] 2.5 Adding the activator dihydrocapsaicin

[0112] 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.

[0113] 2.6 Fluorescence microplate reader data collection

[0114] Immediately measure the fluorescence value of each well using a fluorescence microplate reader.

[0115] 2.7 Data Analysis

[0116] 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 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.

[0117] 3. Experimental Results

[0118] 1. The information of long-chain fatty acids with unsaturation ≥ 3 and the cytotoxicity results are shown in the following table:

[0119] The results of CCK-8 cytotoxicity test showed that α-linolenic acid, arachidonic acid, and eicosapentaenoic acid had no cytotoxicity within the test range of 1000 μM.

[0120] Table 1

[0121] Serial number name Carbon chain type Cytotoxicity results 1 α-linolenic acid 18：3(n-3) No cytotoxicity within the tested range of 1000 μM 2 Arachidonic acid 20：4(n-6) No cytotoxicity within the tested range of 1000 μM 3 Eicosapentaenoic acid 20：5(n-3) No cytotoxicity within the tested range of 1000 μM

[0122] 2. Test results of long-chain fatty acids with unsaturation ≥3 inhibiting stinging pain

[0123] The pain inhibition evaluation method based on the human TRPV1 receptor overexpression cell line was used to evaluate the long-chain fatty acids α-linolenic acid, arachidonic acid, and eicosapentaenoic acid with an unsaturation degree ≥ 3. The results are shown below.

[0124] exist Figure 1 In the figure, KRH represents KRH buffer control, 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 fatty acid samples to be tested, α-linolenic acid, arachidonic acid, and eicosapentaenoic acid. Compared with the KRH group (blank control group), the p value of DHC group (negative control group) <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "&&". Compared with the DHC group (negative control group), the p value of CZ+DHC group (positive control group) <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "##". Compared with the DHC group, the p value of the sample group <0.05 indicates that the difference between the values ​​of the two groups is statistically significant, indicated by "*", and the p value of <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "**".

[0125] from Figure 1The results show that TRPV1 receptors can be activated by dihydrocapsaicin and inhibited by capsaicin, and the difference is significant, indicating that the experimental method is reliable. The specific results of sample detection are as follows: the relative fluorescence intensity of the detected sample α-linolenic acid is -61.61% at a concentration of 1000μM, -51.74% at a concentration of 500μM, 58.38% at a concentration of 250μM, and 62.49% at a concentration of 125μM, which means that 0-linolenic acid has a certain inhibitory effect on the activation of TRPV1 receptors by dihydrocapsaicin at medium and high concentrations; the relative fluorescence intensity of the detected sample arachidonic acid is -27 at a concentration of 500μM. .35%, when the concentration was 250μM, the relative fluorescence intensity was 37.54%, and when the concentration was 125μM, the relative fluorescence intensity was 71.29%, that is, arachidonic acid has a certain inhibitory effect on the activation of TRPV1 receptor by dihydrocapsaicin at medium concentrations; the relative fluorescence intensity of the tested sample eicosapentaenoic acid was -36.31% at a concentration of 125μM, and the relative fluorescence intensity was -2.07% at a concentration of 62.5μM, that is, arachidonic acid has a certain inhibitory effect on the activation of TRPV1 receptor by dihydrocapsaicin at low concentrations.

[0126] In general, the results showed that long-chain fatty acids with unsaturation ≥ 3, such as α-linolenic acid, arachidonic acid, and eicosapentaenoic acid, showed strong inhibitory effects on the activation of TRPV1 receptors by dihydrocapsaicin and had strong TRPV1 receptor antagonist activity.

[0127] Comparative Example 1: Test on the inhibition of TRPV1 receptor activation by long-chain fatty acids with unsaturation of 1 and 2 I. Experimental Principle

[0128] 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 TRPV1 receptor, and then determine whether the detected substance has the effect of inhibiting stinging.

[0129] 2. Experimental Methods

[0130] 1. Cytotoxicity Test

[0131] 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.

[0132] 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%.

[0133] 2.2 Sample preparation: First prepare 200 mM fatty 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.

[0134] 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.

[0135] 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.

[0136] 2.5 Statistical analysis: After the test, the data was imported into Excel to calculate and analyze the cell viability.

[0137] 2. Suppress stinging evaluation

[0138] 2.1 Cell inoculation

[0139] 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%.

[0140] 2.2 Prepare samples to be tested

[0141] First, prepare a 200mM fatty acid sample stock solution with DMSO, and then dilute it with KRH buffer in an isocratic 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.

[0142] 2.3 Loading cells with green fluorescent calcium indicator

[0143] 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.

[0144] 2.4 Add the test sample and KRH buffer containing the inhibitor capsaicin

[0145] 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.

[0146] 2.5 Adding the activator dihydrocapsaicin

[0147] 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.

[0148] 2.6 Fluorescence microplate reader data collection

[0149] Immediately measure the fluorescence value of each well using a fluorescence microplate reader.

[0150] 2.7 Data Analysis

[0151] 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 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.

[0152] 3. Experimental Results

[0153] 1. The information and cytotoxicity results of long-chain fatty acids with unsaturation of 1 and 2 are shown in the following table:

[0154] The results of CCK-8 cytotoxicity test showed that petroselinic acid and linoleic acid had no cytotoxicity within the test range of 1000μM.

[0155] Table 2

[0156] Serial number name Carbon chain type Cytotoxicity results 1 Petrosellic acid 18：1(n-12) No cytotoxicity within the tested range of 1000 μM 2 Linoleic acid 18：2(n-6) No cytotoxicity within the tested range of 1000 μM

[0157] 2. Results of the stinging inhibition test of long-chain fatty acids with 1 and 2 unsaturations

[0158] The pain inhibition evaluation method based on the human TRPV1 receptor overexpression cell line was used to evaluate the long-chain fatty acids petroselinic acid and linoleic acid with unsaturation levels of 1 and 2. The results are shown below.

[0159] exist Figure 2 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 the fatty acid samples to be tested, petroselinic acid and linoleic acid. Compared with the KRH group (blank control group), the p value of DHC group (negative control group) <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "&&". Compared with the DHC group (negative control group), the p value of CZ+DHC group (positive control group) <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "##". Compared with the DHC group (negative control group), the p value of the sample group <0.05 indicates that the difference between the values ​​of the two groups is statistically significant, indicated by "*", and the p value of <0.01 indicates that the difference between the values ​​of the two groups is highly statistically significant, indicated by "**".

[0160] from Figure 2 The results show that TRPV1 receptors can be activated by dihydrocapsaicin and inhibited by capsaicin, and the difference is significant, indicating that the experimental method is reliable. The specific results of sample detection are as follows: the relative fluorescence intensity of the detected sample petroselinic acid is 39.90% at a concentration of 1000μM, and the relative fluorescence intensity is 61.28% at a concentration of 500μM, that is, petroselinic acid has a certain inhibitory effect on the activation of TRPV1 receptors by dihydrocapsaicin at high concentrations; the relative fluorescence intensity of the detected sample linoleic acid is 41.64% at a concentration of 1000μM, 55.63% at a concentration of 500μM, 79.71% at a concentration of 250μM, and 66.88% at a concentration of 125μM, that is, linoleic acid has a certain inhibitory effect on the activation of TRPV1 receptors by dihydrocapsaicin at high concentrations.

[0161] In general, the results showed that the long-chain fatty acids petroselinic acid and linoleic acid with unsaturation of 1 and 2 showed a certain weak inhibitory effect on the activation of TRPV1 receptor by dihydrocapsaicin and had weak TRPV1 receptor antagonist activity.

Claims

1. Use of a long-chain fatty acid with a degree of unsaturation of ≥3 for suppressing pain for non-therapeutic purposes, wherein the long-chain fatty acid has a chain length of 14 or more carbon atoms.

2. The use according to claim 1, wherein: The long chain fatty acids have a chain length of 18-20 carbon atoms.

3. The use according to claim 1, wherein: The long chain fatty acid with a degree of unsaturation of ≥3 is LCFA n3 or LCFA n6 with a length of C18-C20 and 3-5 double bonds.

4. The use according to claim 1, wherein: The long-chain fatty acid is selected from: α-linolenic acid, arachidonic acid, eicosapentaenoic acid or a combination thereof.

5. The use according to claim 1, wherein: The long chain fatty acid is used at a concentration of at least 30 μM.

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

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

8. Use of a long-chain fatty acid with a degree of unsaturation of ≥3 in the preparation of an external skin preparation for inhibiting pain, wherein the long-chain fatty acid has a chain length of 14 or more carbon atoms.

9. The use according to claim 8, wherein: The long chain fatty acids have a chain length of 18-20 carbon atoms.

10. The use according to claim 8, wherein: The long chain fatty acid with a degree of unsaturation of ≥3 is LCFA n3 or LCFA n6 with a length of C18-C20 and 3-5 double bonds.

11. The use according to claim 8, wherein: The long-chain fatty acid is selected from: α-linolenic acid, arachidonic acid, eicosapentaenoic acid or a combination thereof.

12. The use according to claim 8, wherein: The content of the long-chain fatty acid in the external preparation for skin is 0.001-10% by weight.

13. The use according to claim 8, 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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