Inhibitory efficacy of cyclic dipeptide against TRPV1 protein and its application

Abstract

This invention provides the application of a cyclic dipeptide in inhibiting the TRPV1 protein, wherein the cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide. This invention also relates to the application of the cyclic dipeptide in the preparation of topical skin agents or pharmaceuticals with TRPV1 protein inhibitory activity, wherein the topical skin agent is selected from: creams, lotions, gels, toners, serums, masks, eye creams, aerosol cleansing foams, sprays, shower gels, or facial cleansers.

Description

Technical Field

[0001] This invention pertains to the application of cyclic dipeptides, specifically relating to cyclic dipeptides with TRPV1 protein inhibitory effects. These dipeptides have a cyclic (D-leucine-L-proline) dipeptide structure and can inhibit Ca2+ protein production. 2+ The neurosensitive skin discomfort caused by endocrine disorders can be used as an active ingredient in the preparation of pharmaceuticals or topical skin agents to help improve skin sensitivity issues such as dryness, itching, and stinging, and to soothe the skin. Background Technology

[0002] Sensitive skin refers specifically to a hyperreactive state of the skin under physiological or pathological conditions, primarily occurring on the face. Subjective symptoms may include burning, stinging, itching, and tightness, with or without objective signs such as erythema, telangiectasia, and desquamation. With factors such as environmental pollution, excessive cleansing, and life stress, the proportion of people with sensitive skin is increasing.

[0003] The skin barrier is the skin's first line of defense. Damage or dysfunction of the skin barrier can lead to increased skin sensitivity and inflammatory reactions. In sensitive skin types, besides abnormal skin barrier function, another common issue is neuroreactivity. Keratinocytes express receptors such as transient receptor potentials (TRPs) that sense temperature and pain. TRPs can be activated by various physical, chemical, or thermal stimuli, which are also triggers for sensitive skin. On one hand, electrical signals are transmitted to the central nervous system, producing symptoms such as stinging and itching. On the other hand, the calcium ion influx caused by TRPV1 activation can also trigger neuroinflammation, ultimately leading to redness and erythema. The causes of sensitive skin are complex, but its formation is clearly correlated with the pain and itching receptors TRPV1. When stimulated by external factors, the skin experiences pain, burning, and itching. Therefore, repairing the skin barrier requires addressing the root cause of discomfort such as stinging and itching in sensitive skin, which can reduce erythema and inflammation and accelerate barrier repair. Therefore, the inhibition of TRPV1 is also a key target for evaluating cosmetic ingredients with soothing effects, and the ability to develop active ingredients with clear inhibition of TRPV1 protein has become the core development direction for skin care products for sensitive skin.

[0004] Currently, some literature and patents have reported the effects of certain components in this regard, mostly plant-based or plant-based combinations. Examples include combinations of extracts from Citrus reticulata peel, Gentiana scabra, Artemisia annua, and Anelica pubescens fruit, hydrolyzed algae extract; combinations of Camellia japonica and Portulaca oleracea; combinations of Patchouli leaf extract and Jasminum sambac flower extract; and Stephania tetrandra, etc. In addition, there are some reports on related peptides in this regard. For example, Chinese patent application 202310756911.6 provides an anti-aging composition that significantly soothes TRPV1 stimulation caused by retinol and its derivatives. By adding palmitoyl tripeptide-8 and / or acetyl dipeptide-1 cetyl ester to skin care products containing retinol / derivatives, it can significantly inhibit the expression of TRPV1 protein in epidermal keratinocytes. Chinese patent application 202310040156.1 provides a soothing and anti-allergic combination peptide and its application, wherein the soothing and anti-allergic combination peptide includes palmitoyl tripeptide-8 and acetyl dipeptide-1 cetyl ester. Chinese patent application 202310661203.4 discloses that a polypeptide TAT-TRPV1-C has the potential for application in the preparation of drugs for treating symptoms such as itching mediated by TRPV1. Chinese patent application 202211261864.X discloses a synthetic peptide that has the effect of inhibiting TRPV1.

[0005] Cyclic (D-leucine-L-proline) dipeptide is a type of cyclic dipeptide. Zhu Jiansheng et al. isolated cyclic (D-leucine-L-proline) compounds from marine bacteria (Zhu Jiansheng, Marine Bacteria). Pseudomonas putida Study on the bioactivity of a cyclic dipeptide [D], Jiangsu: Yangzhou University, 2013. A patent application filed by South China Agricultural University relates to the application of a cyclic (D-leucine-L-proline) dipeptide and its use as a plant antibacterial agent.

[0006] However, none of these studies have investigated or mentioned the activity of cyclic (D-leucine-L-proline) dipeptide in cosmetic and skincare applications.

[0007] This invention unexpectedly revealed that the cyclic (D-leucine-L-proline) dipeptide has an inhibitory effect on TRPV1 (transient receptor potential vanilloid 1, TRPV1) protein. The study found that the cyclic (D-leucine-L-proline) dipeptide has an excellent inhibitory effect on TRPV1 protein levels, thereby preventing Ca2+ oxidative stress. 2+ Influenza can cause neurosensitive skin discomfort. Furthermore, cyclic dipeptides can also be used as functional additives in the preparation of pharmaceuticals or topical skin agents to help improve skin sensitivity issues such as dryness, itching, and stinging, and to soothe the skin. Summary of the Invention

[0008] On one hand, the present invention relates to the application of cyclic dipeptides in the inhibition of TRPV1 protein, wherein the cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide.

[0009] In a preferred embodiment, the concentration of the cyclic dipeptide used is 2.5 ppm to 40 ppm. In another preferred embodiment, the concentration of the cyclic dipeptide used is 5 ppm to 40 ppm, or 20 ppm to 40 ppm.

[0010] On the other hand, the present invention also relates to the use of cyclic dipeptides in the preparation of topical skin agents or pharmaceuticals with TRPV1 protein inhibitory effects, wherein the cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide.

[0011] In a preferred embodiment, the cyclic dipeptide is present in a topical skin preparation or pharmaceutical product at a content of 0.0001-20% by weight, more preferably 0.01-5% by weight.

[0012] In a preferred embodiment, the topical skin agent is selected from the following forms: face cream, lotion, gel, toner, serum, face mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. Brief description of the attached figures

[0013] Figure 1 The results of TRPV1 immunofluorescence for blank control, negative control, and Examples 4, 3, 7, 6, and 5 are shown at a magnification of 20x.

[0014] Figure 2 The results of TRPV1 immunofluorescence in Example 8 are shown at a magnification of 20x. Detailed Implementation

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar or equivalent to those described herein may be used to practice or test the invention, preferred methods and materials are described herein. For the purposes of this invention, the following terms are defined.

[0016] As used herein, the term "about" means a quantity, level, value, dimension, size, or amount that differs from that of a reference by as much as 30%, 20%, or 10%. Percentages used herein, unless otherwise stated, are by weight.

[0017] Throughout this specification and claims, unless otherwise required, the words “comprising” and its variations “containing” and “including” shall be understood to mean including the said whole or step, or a group of whole or steps, but not excluding any other whole or step, or other group of whole or steps.

[0018] The cyclic dipeptide described herein may optionally be packaged as a finished product. In one embodiment, the packaging is a container such as a plastic, metal, or glass tube or wide-mouth bottle containing the cyclic dipeptide. The product may additionally have packaging such as a plastic or cardboard box for storing the container. In one embodiment, the product contains the cyclic dipeptide and includes instructions for the user to apply the cyclic dipeptide to the skin to treat signs of skin aging as discussed below. Such instructions may be printed on the container, a label insert, or any other packaging.

[0019] As used in this article, “topical application” means, for example, applying or distributing directly to the external skin, scalp, or hair using the hand or an application device such as a wiping cloth, roller, or sprayer.

[0020] As used herein, “cosmetically acceptable” means that the ingredient described by the term is suitable for contact with tissues (such as skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic reaction or similar symptoms.

[0021] This invention is based on the unexpected discovery that the cyclic (D-leucine-L-proline) dipeptide significantly inhibits TRPV1 protein, indicating its role in improving skin sensitivity conditions such as stinging, itching, and redness. Meanwhile, using the cyclic (L-leucine-L-proline) dipeptide as a control, it was found that it did not have this effect.

[0022] Cyclic dipeptide

[0023] This invention provides a cyclic dipeptide component with the structure of a cyclic (D-leucine-L-proline) dipeptide.

[0024] Table 1 shows the structural information of the cyclic (D-leucine-L-proline) dipeptide.

[0025] Table 1

[0026]

[0027] In a specific embodiment, the composition of the present invention uses a cyclic (D-leucine-L-proline) dipeptide provided by Donghua University.

[0028] Medicines or topical skin preparations containing cyclic dipeptides

[0029] The cyclic dipeptide of the present invention can be used as an efficacy additive in pharmaceuticals or topical skin preparations.

[0030] In some embodiments, the pharmaceutical product is selected from tablets, capsules, emulsions, suspensions, powders, granules, solutions, and various pharmaceutical dosage forms known in the art. Different dosages are added depending on the type of dosage form.

[0031] In some embodiments, the cyclic dipeptide can be used in the preparation of topical skin agents. These topical skin agents are preferably cosmetic compositions, including but not limited to products in the form of creams, lotions, gels, toners, serums, masks, eye creams, aerosols (cleansing foams), sprays, shower gels, and facial cleansers.

[0032] The content of the cyclic dipeptide in the topical skin preparation is 0.001-20% by weight, preferably 0.005-10% by weight, and more preferably 0.01-5% by weight.

[0033] The term "topical skin agent" is a general term encompassing all ingredients typically used on the external surface of the skin, such as cosmetic compositions. These cosmetic compositions can include basic cosmetics, facial makeup cosmetics, body cosmetics, hair care cosmetics, etc., with no specific restrictions on their dosage forms; they can be rationally selected according to different purposes. Depending on the dosage form and purpose, these cosmetic compositions may also contain different cosmetically permissible media or matrix excipients.

[0034] The term "topical skin agent" is a general term encompassing all ingredients typically used on the external surface of the skin, such as cosmetic compositions. These cosmetic compositions can include basic cosmetics, facial makeup cosmetics, body cosmetics, hair care cosmetics, etc., with no specific restrictions on their dosage forms; they can be rationally selected according to different purposes. Depending on the dosage form and purpose, these cosmetic compositions may also contain different cosmetically permissible media or matrix excipients.

[0035] A topical skin agent containing a cyclic dipeptide can be applied topically to human skin and / or hair. The topical skin agent may also contain a cosmetically acceptable topical carrier, which may be from about 50% to about 99.99% by weight of the topical skin agent (e.g., from about 80% to about 99% by weight of the topical skin agent). In a preferred embodiment of the invention, the cosmetically acceptable topical carrier comprises water. The cosmetically acceptable topical carrier may comprise one or more substances selected from wetting agents, emollients, oils, humectants, and similar substances. In one embodiment, the cosmetically acceptable topical carrier comprises a substrate such as a nonwoven fabric or membrane material.

[0036] Topical skin agents can be formulated into a variety of product types, including but not limited to lotions, creams, gels, sticks, sprays, ointments, cleansing liquids and solid soaps, shampoos and hair conditioners, hair fixatives, pastes, foams, powders, mousses, shaving creams, wipes, patches, hydrogels, film-forming products, masks and skin films, and cosmetics such as foundations and mascaras. These product types may 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.

[0037] Topical skin preparations containing cyclic dipeptides can be formulated as solutions. Solutions typically contain aqueous or organic solvents (e.g., about 50% to about 99.99% or about 90% to about 99% of cosmetically acceptable aqueous or organic solvents). Examples of suitable organic solvents include propylene glycol, polyethylene glycol, polypropylene glycol, glycerin, 1,2,4-butanetriol, sorbitan esters, 1,2,6-hexanetriol, ethanol, and mixtures thereof.

[0038] Topical skin preparations can be formulated as solutions containing emollients. Such topical skin preparations preferably contain about 2% to about 50% of one or more emollients. As used in this invention, "emollient" refers to a substance used to prevent or alleviate dryness, for example, by preventing the loss of moisture from the skin. Examples of emollients include, but are not limited to, plant oils, mineral oils, aliphatic esters, etc.

[0039] Lotions can be prepared from such solutions. Lotions typically contain about 1% to about 20% (e.g., about 5% to about 10%) of one or more emollients and about 50% to about 90% (e.g., about 60% to about 80%) of water.

[0040] Another type of product that can be formulated from a solution is a cream. Creams typically contain about 5% to about 50% (e.g., about 10% to about 20%) of one or more emollients and about 45% to about 85% (e.g., about 50% to about 75%) of water.

[0041] While water is preferred in topical skin preparations containing cyclic dipeptides, alternatively, the topical skin preparation may be anhydrous or an ointment that does not contain water but is composed of organic and / or organosilicon solvents, oils, lipids, and waxes. The ointment may contain a simple base of animal or vegetable oils or semi-solid hydrocarbons. The ointment may contain about 2% to about 10% of one or more emollients and about 0.1% to about 2% of one or more thickeners.

[0042] This topical skin agent 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 fields of personal care and cosmetic formulation.

[0043] Lotions and creams can be formulated into 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.

[0044] Water-in-oil and oil-in-water monoemulsion skincare formulations, such as lotions and creams, are well known in the cosmetics industry and can be used in this invention. Multiphase emulsion topical skincare formulations (e.g., water-in-oil-in-water and oil-in-water-in-oil) can also be used in this invention. Typically, such monophase or multiphase emulsions contain water, emollients, and emulsifiers as their basic components.

[0045] Topical skin preparations containing cyclic dipeptides can also be formulated as gels (e.g., aqueous gels, alcohol gels, alcohol / aqueous gels, or oil gels using suitable gelling agents). Suitable gelling agents for aqueous and / or alcohol gels include, but are not limited to, natural gums, acrylic 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 butene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gels typically contain between about 0.1% and 5% by weight of these gelling agents.

[0046] Topical skin preparations containing cyclic dipeptides can also be formulated into solid dosage forms (e.g., wax-based sticks, soap strips, powders, or wipes containing powder).

[0047] In addition to the components described above, the skin topical agents used in this invention may also contain a variety of other oil-soluble and / or water-soluble substances that are conventionally used in skin topical agents for use on skin and hair at levels determined in the technical field.

[0048] The topical skin formulation of the present invention may include additional components commonly found in skin care compositions, such as emollients, skin conditioners, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., provided that they are physically and chemically compatible with other components in the topical skin formulation and do not affect the efficacy of the cyclic dipeptide of the present invention.

[0049] In some embodiments of the topical skin formulation of the present invention, one or more preservatives may be used. Suitable preservatives include p-hydroxyacetophenone, C1-C4 alkyl p-hydroxybenzoate, and phenoxyethanol. Based on the total weight of the composition, the amount of preservative used is from about 0.5 to about 2% by weight, preferably from about 0.5 to 1% by weight.

[0050] In one example of the topical skin preparation of the present invention, one or more antioxidants may be used. Suitable antioxidants include butylated hydroxytoluene (BHT), ascorbyl palmitate (BHA), butylated hydroxyanisole, phenyl-α-naphthylamine, propyl gallate, nordihydroguaiacol, vitamin E or a derivative of vitamin E, vitamin C and its derivatives, calcium pantothenate, green tea extract, and mixed polyphenols, as well as mixtures of the substances described above. The antioxidant used is approximately 0.02 to 0.5% by weight of the total weight of the composition, more preferably in the range of approximately 0.002 to 0.1% by weight.

[0051] In one example of the topical skin preparation of the present invention, one or more emollients may be used, which, by virtue of their ability to remain on the skin surface or in the stratum corneum, act as lubricants to reduce exfoliation 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, not limited to, include polypropylene glycol (“PPG”)-15 octadecyl ether, PPG-10 hexadecyl ether, Steareth-10, Oleth-8, PPG-4 dodecyl ether, vitamin E acetate, lanolin, cetyl alcohol, cetearyl alcohol ethylhexanoate, cetearyl alcohol, glyceryl stearate, octyl hydroxystearate, dimethyl polysiloxane, and combinations thereof. Cetyl alcohol, cetearyl alcohol ethylhexanoate, cetearyl alcohol, glyceryl stearate, and combinations thereof are preferred. When used, the emollient is applied in an amount ranging from about 0.1% to about 30% by weight, preferably from about 1% to about 30% by weight, based on the total weight of the composition.

[0052] In one example of the topical skin preparation of the present invention, one or more moisturizers may be used. Moisturizers, also known as humectants, help enhance the effectiveness of emollients, reduce exfoliation, stimulate the removal of scaly skin, and improve skin feel. Polyols may be used as moisturizers, including, but not limited to, glycerin, polyalkylene glycols, alkylene polyols and their derivatives, including butylene glycol, propylene glycol, dipropylene glycol, glycerol, polyethylene glycol and their derivatives, sorbitol, hydroxypropyl sorbitol, hexanediol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerin, propoxylated glycerin, and combinations thereof. When used, the amount of moisturizer is from about 0.1% to about 20% by weight, preferably from about 1% to about 15% by weight, based on the total weight of the composition.

[0053] In one example of the topical skin formulation of the present invention, one or more emulsifiers may be used. The emulsifier may be used within a range of effective stable amounts. Preferably, the emulsifier is used at an amount of about 1.0 to about 10.0% by weight, more preferably about 3.0 to about 6.0% by weight, based on the total weight of the composition. Any emulsifier compatible with the components in the composition may be used. Suitable emulsifiers include stearic acid, cetyl alcohol, glyceryl stearate, lecithin, octadecyl alcohol, Steareth-2, Steareth-20, acrylate / C10-30 alkanol acrylate crosspolymers, and combinations thereof.

[0054] In one example of the topical skin formulation of the present invention, one or more pH adjusters may be used. Beneficial pH adjusters in the topical skin formulation of the present invention include tromethamine. When used, the amount of pH adjuster is approximately 0.1 to approximately 2% by weight, preferably approximately 0.1 to approximately 1% by weight, based on the total weight of the composition.

[0055] In one specific embodiment of the invention, the topical skin agent comprises an acrylate / C10-30 alkanol acrylate crosspolymer, glycerin, p-hydroxyacetophenone, glyceryl stearate and lecithin, cetearyl alcohol, cetearyl ethylhexanoate, tromethamine or a combination thereof.

[0056] Example

[0057] The present invention will be further illustrated below with reference to specific embodiments. It is important to note that these embodiments are for illustrative purposes only and should not be construed as limiting the scope of protection of the invention. Those skilled in the art can make non-essential improvements and adjustments based on the above description of the invention. Test methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0058] Cyclic (L-leucine-L-proline) dipeptide (purity ≥98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0059] Cyclic (D-leucine-L-proline) dipeptide (purity ≥98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0060] Example 1:

[0061] Accurately weigh the cyclic (D-leucine-L-proline) dipeptide and prepare it to a concentration of 400 ppm with PBS phosphate buffer as the test stock solution.

[0062] Example 2:

[0063] Accurately weigh the cyclic (L-leucine-L-proline) dipeptide and prepare it to a concentration of 400 ppm with PBS phosphate buffer as the test stock solution.

[0064] Example 3:

[0065] Take Example 1, dilute it with culture medium to prepare a concentration of 40 ppm, and use it as the test solution.

[0066] Example 4:

[0067] Take Example 1, dilute it with culture medium to prepare a concentration of 20 ppm, and use it as the test solution.

[0068] Example 5:

[0069] Take Example 1, dilute it with culture medium to prepare a 10 ppm concentration, and use it as the test solution.

[0070] Example 6:

[0071] Take Example 1, dilute it with culture medium to prepare a 5 ppm concentration, and use it as the test solution.

[0072] Example 7:

[0073] Take Example 1, dilute it with culture medium to prepare a concentration of 2.5 ppm, and use it as the test solution.

[0074] Example 8:

[0075] Take Example 2, dilute it with culture medium to prepare a 4 ppm concentration, and use it as the test solution.

[0076] Test Example 1: Protein content detection method based on capsaicin receptor (TRPV1) in keratinocytes

[0077] 1. Test principle:

[0078] Based on keratinocytes, the soothing efficacy of test samples was evaluated by detecting the protein content of capsaicin receptor 1 (TRPV1). The inhibition of TRPV1 after treatment with the test sample indicates that the test sample achieves its soothing effect by inhibiting TRPV1.

[0079] 2. Test materials:

[0080] Keratinocytes Ep21072702 (Guangdong Boxi), KC2500 (Guangdong Boxi), PBS (Solepro), MTT (Sigma), DMSO (Sigma), paraformaldehyde (Sinopharm), TRPV1 antibody (Abcam).

[0081] CO2 incubator (Thermo, 150I), clean bench (Suzhou Antai, SW-CJ-1F), inverted microscope (Olympus, CKX41), fluorescence microscope (Leica, DM2500).

[0082] 3. Testing System:

[0083] Table 2

[0084]

[0085] 4. Testing Method:

[0086] 1) Cell seeding: at a rate of 1.8 × 10⁻⁶ 5 Seed keratinocytes at a seeding density of cells / well into 6-well plates and incubate overnight in an incubator (37°C, 5% CO2).

[0087] 2) Drug administration: According to the test system in the table above, when the cell deposition rate in the 6-well plate reaches 40%~50%, the drugs are administered in groups, with 2 mL administered to each well, and 3 replicates per group. After drug administration, the 6-well plates are placed in an incubator (37℃, 5% CO2) for 24 h.

[0088] 3) TRPV1 detection: The sample was fixed with 4% paraformaldehyde for 30 min, and then the capsaicin receptor (TRPV1) was detected by immunofluorescence. The sample was photographed under a microscope and the images were collected and analyzed.

[0089] 4) Statistical analysis of results: GraphPad Prism was used for plotting, and the results are expressed as Mean ± SD. t-tests were used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.01 was considered highly statistically significant.

[0090] 5. Test Results

[0091] Based on the testing protocol and following the immunofluorescence procedure, TRPV1 detection was performed.

[0092] 1) TRPV1 immunofluorescence results

[0093] Figure 1-2 The results of TRPV1 immunofluorescence are shown, where green fluorescence represents the content of the target protein "TRPV1", and the higher the fluorescence intensity, the higher the protein content.

[0094] The experimental results showed that the green fluorescence intensity of the NC group was greater than that of the BC group, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the green fluorescence intensity of Examples 3, 4, 5, 6 and 7 was weakened, indicating that the examples had an inhibitory effect on TRPV1 protein. However, Example 8, as a comparative example, did not show a decrease in fluorescence, suggesting that it was ineffective.

[0095] 2) TRPV1 protein content detection results

[0096] The table below shows the results of TRPV1 protein content detection:

[0097] Table 3

[0098]

[0099] Note: When performing statistical analysis using the t-test method, significance compared to the BC group is indicated by #, with P-value < 0.05 indicated by # and P-value < 0.01 indicated by ##; significance compared to the NC group is indicated by... This indicates that a p-value < 0.05 is considered... A p-value < 0.01 indicates that... .

[0100] Experimental results showed that the TRPV1 protein content in the NC group was significantly increased compared to the BC group, indicating that the stimulation conditions in this test were effective. Compared to the NC group, Examples 3-7 all significantly reduced the TRPV1 protein content (p<0.01), and showed a dose-dependent effect with increasing concentration, indicating that the examples themselves had a highly significant inhibitory effect on TRPV1 protein. However, Example 8, as a comparative example, did not show a reduction in fluorescence, suggesting it was ineffective. This also indicates that not all leucine and proline linkages have the same efficacy; this invention is limited to D-leucine-L-proline.

[0101] Application examples

[0102] The cyclic dipeptide described in this invention can be used as an intermediate raw material or functional additive in the preparation of pharmaceuticals or topical skin agents. The topical skin agents are preferably cosmetic compositions, including but not limited to products in dosage forms such as creams, lotions, gels, toners, serums, masks, eye creams, aerosols (cleansing foams), sprays, shower gels, and facial cleansers. The weight percentage of the cyclic dipeptide in the topical skin agent can be 0.0001%-20% (w / w), preferably 0.001%-10% (w / w), more preferably 0.001%-5% (w / w), and most preferably 0.01%-5% (w / w).

[0103] The following are specific application examples of cyclic (D-leucine-L-proline) dipeptides in topical skin preparations, along with the formulations and preparation methods of these dosage forms. In the tables below, "-" indicates no additives.

[0104] Application Example 1: Preparation of Face Cream

[0105]

[0106] Application Example 2: Emulsion Preparation

[0107]

[0108] Application Example 3: Preparation of Gel

[0109]

[0110] Application Example 4: Preparation of Toner

[0111]

[0112] Application Example 5: Preparation of Serum

[0113]

[0114] Application Example 6: Preparation of Facial Masks

[0115]

[0116] Application Example 7: Preparation of Eye Cream

[0117]

[0118] Application Example 8: Preparation of Spray

[0119]

[0120] Application Example 9: Preparation of Shower Gel

[0121]

[0122] Application Example 10: Preparation of Facial Cleanser

[0123]

Claims

1. Non-therapeutic application of cyclic dipeptides to inhibit TRPV1 protein, among which, The cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide, and the inhibition of the TRPV1 protein can improve skin sensitivity.

2. The application as described in claim 1, characterized in that, The concentration of the cyclic dipeptide used is 2.5 ppm to 40 ppm.

3. The application as described in claim 2, characterized in that, The concentration of the cyclic dipeptide used is 5 ppm-40 ppm.

4. The application as described in claim 3, characterized in that, The concentration of the cyclic dipeptide used is 20 ppm-40 ppm.

5. The application of cyclic dipeptide in the preparation of a topical skin agent with TRPV1 protein inhibitory activity, wherein the cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide, and the TRPV1 protein inhibition can improve skin sensitivity.

6. The application as described in claim 5, characterized in that, The content of the cyclic dipeptide in the topical skin preparation is 0.0001-20% by weight.

7. The application as described in claim 6, characterized in that, The content of the cyclic dipeptide in the topical skin preparation is 0.01-5% by weight.

8. The application as described in claim 5, characterized in that, The topical skin agent is selected from the following forms: face cream, lotion, gel, toner, serum, mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

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