Anti-inflammatory soothing efficacy of cyclic dipeptide and application of cyclic dipeptide

Abstract

The invention provides anti-inflammatory soothing application of cyclic dipeptide. The cyclic dipeptide is selected from cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination of the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide. The anti-inflammatory soothing of the present invention is achieved by inhibiting TNF [alpha], IL-1[ beta], IL-6, PGE2, COX2, TSLP and / or IL-33. The invention also relates to an application of the cyclic dipeptide in preparation of external skin agents, medicines and health-care foods with anti-inflammatory and soothing effects. The external skin agents are selected from face cream, emulsion, gel, toning lotion, essence, masks, eye cream, aerosol cleaning foam, spray, shower gel or facial cleanser.

Description

Technical Field

[0001] The present invention belongs to the application of cyclic dipeptides, and specifically relates to cyclic dipeptides with anti-inflammatory and soothing effects. The structure of the cyclic dipeptide is cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide, which has the effect of inhibiting the expression of inflammatory factors and can be used as a soothing ingredient in cosmetics, food or medicine. Background Art

[0002] Sensitive skin is a syndrome in which the skin experiences paroxysmal or periodic burning, flushing, stinging, itching and tightness, with or without persistent erythema, after being slightly stimulated by the outside world. Sensitive skin is most likely to occur on the face.

[0004] The pathogenesis behind sensitive skin is a complex process, in which immune inflammatory response is one of the main reasons. Inflammatory factors refer to various cytokines involved in inflammatory response. Among the many inflammatory factors, the main concerns at the skin level are TNF-α, IL-1β, IL-6, PGE2, COX2, TSLP, IL-33, etc. TNF-α is the earliest and most important inflammatory mediator in the inflammatory response process. It can activate neutrophils and lymphocytes, increase the permeability of vascular endothelial cells, regulate the metabolic activity of other tissues, and promote the synthesis and release of other cytokines. IL-6 can induce B cell differentiation and antibody production, and induce T cell activation, proliferation and differentiation, participate in the body's immune response, and is a promoter of inflammatory response. IL-1β is one of the key proinflammatory factors in immune inflammatory skin diseases. It can participate in pathological and physiological processes such as immune inflammatory response, cell proliferation and differentiation alone or by inducing the secretion of other cytokines. Prostaglandin E2 (PGE2) can induce the expression of MMPs, reduce matrix protein synthesis, and inhibit collagen synthesis by fibroblasts, while cyclooxygenase (COX) is the rate-limiting enzyme for PG synthesis. Cyclooxygenase (COX)-2 and its catalytic product, proinflammatory factor prostaglandin (PEG), participate in the inflammatory response of human skin keratinocytes (HaCaT) induced by external stimuli. TSLP (thymic stromal lymphopoietin) is an interleukin (IL-7-like) inflammatory cytokine with a four-a-helical bundle structure. Studies have shown that TSLP can recruit helper T cells (T helper, Th) by activating dendritic cells, type 2 innate lymphocytes, etc., enhance Th2-mediated hypersensitivity reactions, and help to produce memory Th2 cell subsets characterized by the expression of TSLP receptor (TSLP receptor, TSLPR). It plays an important role in allergic diseases such as asthma, atopic dermatitis, and allergic conjunctivitis. In recent years, studies have gradually found that TSLP can also act on various types of immune cells and tumor cells, and participate in a variety of inflammatory diseases. IL-33 is a member of the IL-1 family and plays an important role in the pathogenesis of atopic dermatitis. Studies have shown that the IL-33 level in the serum of patients with atopic dermatitis is significantly higher than that in the control group; the continuous expression of IL-33 in keratinocytes can cause AD-like inflammation in mice.

[0005] Therefore, inhibiting the release of inflammatory factors or limiting their effects can soothe the skin, which is important for people with sensitive skin or skin irritation symptoms.

[0006] Cyclic peptides are widely present in nature and have strong biological activity. Most of them can be used as drugs or drug precursors, playing an extremely important role in the life activities of organisms. Cyclic dipeptides are the smallest class of compounds among cyclic peptide members and are widely present in nature. Cyclic dipeptides have a diketopiperazine structure and therefore have high stability. The special spatial conformation of cyclic dipeptides gives them obvious physiological activity and has many potential application values ​​in the fields of chemistry, medicinal chemistry, biochemistry, life sciences, etc., so they have attracted the attention of many chemists, biologists and pharmacists at home and abroad.

[0007] Applicants are particularly interested in cyclo(L-leucine-L-proline) dipeptides and cyclo(D-leucine-L-proline) dipeptides.

[0008] Cyclic (L-leucine-L-proline) dipeptide is a microbial metabolite that can be metabolized and produced in many bacteria, fungi and actinomycetes. Qi Shuhua et al. isolated cyclic (L-leucine-L-proline) compounds from the metabolites of marine bacteria Pseudomonas sp. (Qi Shuhua, Qian Peiyuan, Zhang Si, Research on antimicrobial metabolites of marine bacteria Pseudomonas sp. [J], Natural Product Research and Development, 2009, 21(3): 420-423); Wu Xiuli et al. isolated this substance from the liquid culture of Pseudomonas pyrophyll (Wu Xiuli, Lin Sheng, Zhu Chenggen et al., Research on the chemical composition of the liquid culture of Pseudomonas pyrophyll [J], Chinese Journal of Traditional Chinese Medicine, 2011, 36(7): 874-880); Li Haifeng et al. also isolated this compound from Bacillus subtilis 7Ze3 (Li Haifeng, Ye Yong Hao, Guo Jianhua, Isolation and Identification of Cyclic Dipeptide from Bacillus subtilis 7Ze3 [J], Jiangsu Agricultural Science, 2010 (2): 107-109); Wang Jing et al. reported that cyclic (L-leucine-L-proline) dipeptide was isolated from endophytic fungi of Platycladus orientalis (Wang Jing, Wei Yan, Yi Xiaohua et al., Investigation of Herbicidal Selectivity of Endophytic Fungi J46 of Platycladus orientalis and Isolation of Active Substances [C], Proceedings of the 13th National Pesticide Teaching and Research Symposium, 2013: 260-266). Zhu Jiansheng et al. isolated cyclic (D-leucine-L-proline) compounds from marine bacteria (Zhu Jiansheng, Study on Cyclic Dipeptide and Its Biological Activity in Marine Bacteria Pseudomonas putida [D], Jiangsu: Yangzhou University, 2013).

[0009] However, none of these documents studies or mentions the activity research of cyclo(L-leucine-L-proline) dipeptide and / or cyclo(D-leucine-L-proline) dipeptide in beauty and skin care.

[0010] The present invention unexpectedly discovered that cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide have the effect of inhibiting the expression of inflammatory factors such as TNFα, IL-1β, IL-6, PGE2, COX2, TSLP, IL-33, etc., and can be used as a soothing ingredient in cosmetics, food or medicine. Summary of the invention

[0011] In one aspect, the present invention relates to the anti-inflammatory and soothing application of cyclic dipeptides, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination thereof.

[0012] In a preferred embodiment, the anti-inflammatory relief is achieved by inhibiting TNFα, IL-1β, IL-6, PGE2, COX2, TSLP and / or IL-33.

[0013] In a preferred embodiment, the cyclic dipeptide is used at a concentration of 5 ppm to 40 ppm, more preferably 20 ppm to 40 ppm.

[0014] In a preferred embodiment, the cyclic dipeptide is used at a concentration of 4 ppm.

[0015] On the other hand, the present invention also relates to the use of cyclic dipeptides in the preparation of external skin preparations, medicines and health foods with anti-inflammatory and soothing effects, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination thereof.

[0016] In a preferred embodiment, the content of the cyclic dipeptide in the external skin preparation, medicine and health food is 0.0001% to 20% by weight.

[0017] In a preferred embodiment, the skin external preparation is selected from the following forms: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser. DETAILED DESCRIPTION

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to implement or test the present invention, preferred methods and materials are described herein. For purposes of the present invention, the following terms are defined below.

[0019] As used herein, the term "about" refers to an amount, level, value, dimension, size, or amount that differs by up to 30%, 20%, or 10% compared to the amount, level, value, dimension, size, or amount of a reference product. Percentages used herein, unless otherwise indicated, are by weight.

[0020] Throughout the specification and claims, unless otherwise required, the following words "comprise" and variations "comprising" and "including" will be understood to imply the inclusion of a stated integer or step, or a group of integers or steps, but not the exclusion of any other integer or step, or other group of integers or steps.

[0021] The cyclic dipeptides described herein may optionally be in the form of finished product packaging. In one embodiment, the packaging is a container such as a plastic, metal or glass tube or jar 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 comprises the cyclic dipeptide and has instructions for guiding 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.

[0022] As used herein, "topical application" means applying or spreading directly onto the external skin, scalp, or hair, for example, using the hands or an application device such as a wipe, roller, or spray.

[0023] As used herein, "cosmetically acceptable" means that the ingredients described by the term are suitable for use in contact with tissue (eg, skin or hair) without undue toxicity, incompatibility, instability, irritation, allergic response, or the like.

[0024] The present invention is based on the following unexpected discovery: cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide have the effect of inhibiting the expression of inflammatory factors such as TNFα, IL-1β, IL-6, PGE2, COX2, TSLP, IL-33, etc., and can be used as a soothing ingredient in cosmetics, food or medicine to help improve skin redness, tingling, allergies, etc.

[0025] Cyclic dipeptide

[0026] The invention provides a cyclic dipeptide component, the structure of which is: cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide.

[0027] Table 1 shows the structural information of cyclo(L-leucine-L-proline) dipeptide and cyclo(D-leucine-L-proline) dipeptide.

[0028] Table 1

[0029]

[0030] In a specific embodiment, the composition of the present invention uses cyclo(L-leucine-L-proline) dipeptide and cyclo(D-leucine-L-proline) dipeptide provided by Donghua University.

[0031] Skin topical products

[0032] In some embodiments, the cyclic dipeptide can be used for the preparation of a skin topical agent. The skin topical agent is preferably a cosmetic composition, including but not limited to products in the form of cream, lotion, gel, toner, essence, mask, eye cream, aerosol (cleansing foam), spray, shower gel and facial cleanser.

[0033] The content of the cyclic dipeptide in the external preparation for skin is 0.0001% to 20% by weight, preferably 0.005% to 10% by weight, more preferably 0.01% to 5% by weight.

[0034] 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.

[0035] The skin topical preparation comprising the cyclic dipeptide 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 about 50% by weight to about 99.99% by weight of the skin topical preparation (e.g., 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.

[0036] 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.

[0037] Skin topical preparations containing cyclic dipeptides can be formulated as solutions. Solutions generally contain aqueous solvents or organic solvents (e.g., about 50% to about 99.99% or about 90% to about 99% of cosmetically acceptable aqueous solvents or organic solvents). 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Although it is preferred that the skin external preparation containing the cyclic dipeptide contains water, as an alternative, the skin external preparation may be anhydrous or an ointment containing no water but organic and / or silicone solvents, oils, lipids and waxes. The ointment may contain a simple base of animal or vegetable oils or semisolid 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] 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.

[0043] 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.

[0044] 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.

[0045] Skin topical preparations containing cyclic dipeptides can also be formulated into gels (e.g., aqueous gels, alcoholic gels, alcohol / water gels or oily gels using suitable gelling agents). Suitable gelling agents for aqueous gels and / or alcoholic gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gelling agents typically contain between about 0.1% and 5% by weight of such gelling agents.

[0046] The external preparation for skin application comprising the cyclic dipeptide may also be formulated as a solid preparation (eg, a wax-based stick, a bar soap, a powder, or a wipe containing a powder).

[0047] 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.

[0048] The skin external preparation of the present invention may contain additional components commonly found in skin care compositions, such as emollients, skin regulators, emulsifiers, preservatives, antioxidants, fragrances, chelating agents, etc., as long as they are physically and chemically compatible with other components in the skin external preparation and do not affect the effects of the cyclic dipeptide of the present invention.

[0049] 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 %.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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 %.

[0055] 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.

[0056] Additional cosmetic active agents

[0057] In some embodiments, the topical skin preparation may also include additional cosmetic active agents. As used herein, "cosmetic active agents" are compounds (such as synthetic compounds or compounds isolated from natural sources or natural extracts) that have cosmetic or therapeutic effects on skin or hair, including but not limited to anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, antifungal agents, antiparasitic agents, topical analgesics, sunscreens, photoprotectants, antioxidants, keratolytic agents, surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, curing agents, anti-hard cuticle agents, and agents for hair and / or skin conditioning.

[0058] In one embodiment, these cosmetic actives are selected from (but not limited to): hydroxy acids, benzoyl peroxide, D-panthenol, octyl methoxycinnamate, titanium dioxide, octyl salicylate, homosalate, avobenzone, carotenoids, free radical scavengers, spin traps, amines, retinoids such as retinol and retinyl palmitate, ceramides, polyunsaturated fatty acids, essential fatty acids, enzymes, enzyme inhibitors, minerals, hormones such as estrogen, steroids such as hydrocortisone, 2-dimethylaminoethanol, copper salts such as copper chloride, copper-containing peptides such as Cu:Gly-His-Lys, coenzyme Q10, peptides, amino acids such as proline, vitamins, lactobionic acid, acetyl-CoA, niacin, riboflavin, thiamine, ribose, electron transfer substances such as NADH and FADH2, and other plant extracts such as aloe, feverfew, oatmeal, and their derivatives and mixtures. The cosmetic active agent is generally present in an amount of about 0.001% to about 20%, for example about 0.005% to about 10%, such as about 0.01% to about 5%, by weight of the topical skin preparation of the present invention.

[0059] Examples of vitamins include, but are not limited to, vitamin A, vitamin B (e.g., vitamin B3, vitamin B5, and vitamin B12), vitamin C, vitamin K, and different forms of vitamin E (e.g., α, β, γ, or Δ tocopherol) or mixtures thereof and derivatives thereof.

[0060] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0061] Examples of antioxidants include, but are not limited to, water-soluble antioxidants such as sulfhydryl compounds and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine), lipoic acid and dihydrolipoic acid, resveratrol, lactoferrin and ascorbic acid and ascorbic acid derivatives (e.g., ascorbyl palmitate and ascorbic acid polypeptide). Oil-soluble antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, butylated hydroxytoluene, retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g., tocopherol ethyl ester), tocotrienols and ubiquinone. Natural extracts containing antioxidants suitable for use in the skin topical preparations of the present invention include, but are not limited to, extracts containing flavonoids and isoflavones and their derivatives (e.g., genistein and di-zein), extracts containing resveratrol, and the like. Examples of such natural extracts include grape seeds, green tea, pine bark, and propolis.

[0062] How to use

[0063] The skin topical of the present invention can be topically applied to mammalian skin in need of treating one or more signs of skin aging as described above. In one embodiment, the skin topical can be applied to skin in need of treating fine lines and wrinkles and / or loss of elasticity. The skin topical can be applied to skin in need of such treatment according to a suitable treatment regimen, such as monthly, weekly, every other day, daily, twice a day, etc.

[0064] In certain embodiments, the skin topical preparations of the present invention can also be used to treat other needs related to the skin. For example, the skin topical preparations of the present invention can be used to treat hyperpigmentation after inflammation, to reduce pore size, to reduce sebum production, and to alleviate scars. In certain other embodiments, the skin topical preparations of the present invention can be applied simultaneously with a mechanical or physical epidermal exfoliation treatment (e.g., microdermabrasion treatment) or within a few hours of the treatment, or simultaneously with a chemical peel or keratolytic agent such as salicylic acid. In certain other embodiments, the skin topical preparations of the present invention can be applied to mucous membranes or other tissues such as vaginal tissue, oral tissue, or eye tissue. In certain other embodiments, the skin topical preparations of the present invention can be applied to mild wounds or postoperative sites to promote healing, to insect bites, to poison ivy skin disorders or similar skin conditions, or generally used to relieve itching.

[0065] Example

[0066] The present invention is further described below in conjunction with specific examples. It is necessary to point out that the examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments based on the content of the present invention described above. The test methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, all percentages and parts are by weight.

[0067] Cyclic (L-leucine-L-proline) dipeptide (purity ≥ 98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0068] Cyclic (D-leucine-L-proline) dipeptide (purity ≥ 98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0069] Embodiment 1:

[0070] Cyclic (L-leucine-L-proline) dipeptide was accurately weighed and prepared with PBS phosphate buffer to a concentration of 400 ppm as the test stock solution.

[0071] Embodiment 2:

[0072] Cyclic (D-leucine-L-proline) dipeptide was accurately weighed and prepared with PBS phosphate buffer to a concentration of 400 ppm as the test stock solution.

[0073] Embodiment 3:

[0074] Example 1 was diluted with culture medium to prepare a concentration of 40 ppm as a test solution.

[0075] Embodiment 4:

[0076] Example 1 was diluted with culture medium to prepare a concentration of 20 ppm as a test solution.

[0077] Embodiment 5:

[0078] Example 1 was diluted with culture medium to prepare a concentration of 10 ppm as a test solution.

[0079] Embodiment 6:

[0080] Example 1 was diluted with culture medium to prepare a concentration of 5 ppm as a test solution.

[0081] Embodiment 7:

[0082] Example 1 was diluted with culture medium to prepare a concentration of 2.5 ppm as a test solution.

[0083] Embodiment 8:

[0084] Example 1 was diluted with culture medium to prepare a concentration of 4 ppm as a test solution.

[0085] Embodiment 9:

[0086] Example 2 was diluted with culture medium to prepare a concentration of 4 ppm as a test solution.

[0087] Test Example 1: Inhibitory effect on the expression of inflammatory factors in keratinocytes induced by UVB stimulation (2D cells)

[0088] Materials and Reagents:

[0089] Keratinocytes (Batch No.: Ep23082203) were provided by Guangdong Boxi Biotechnology Co., Ltd., KcGrowth culture medium (Guangdong Boxi Biotechnology), PBS (Solabo), RNAisoPlus (Acori Biotechnology), reverse transcription kit (Acori Biotechnology), and fluorescent dye (Acori Biotechnology).

[0090] Experimental equipment:

[0091] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), UVB irradiator (Philips), microplate reader (BioTek, Epoch), and fluorescence quantitative PCR instrument (BioRad, CFX-96).

[0092] The experiment was divided into a blank control group (BC), a model group (NC) and a sample group (Examples 3-7). After the cells were revived, when the plating rate reached about 60%, the cells were inoculated into a 6-well plate and incubated overnight in a CO2 incubator (37°C, 5% CO2). According to the test grouping, when the cell plating rate in the 6-well plate reached 30% to 50%, the drugs were administered in groups, with 3 replicates per group. 2 mL of culture medium was added to each well of the blank control group and the model group, and 2 mL of culture medium containing the corresponding concentration of the sample to be tested (Examples 3-7) was added to each well of the sample group. After the administration was completed, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours. According to the test grouping, except for the blank control group, the remaining groups were irradiated with UVB, and the irradiation dose was 300 mJ / cm 2 After irradiation, the cells were placed in a CO2 incubator (37°C, 5% CO2) and cultured for 24 hours. After incubation, the cells were washed twice with PBS, 1 mL of RNAisoPlus was added to each well, and the cells were lysed by blowing and then collected. RNA was extracted, reverse transcribed to cDNA, and then fluorescent quantitative PCR was performed. -△△CT Methods The results were calculated and expressed as Mean±SD. t-test was used for statistical analysis among the groups, p<0.05 indicated significant difference, p<0.01 indicated extremely significant difference.

[0093] Table 2: TNF-α gene expression results

[0094]

[0095] According to the results in the above table, compared with the BC group, the TNF-α gene expression in the NC group was significantly upregulated, indicating that this test was effective. Compared with the NC group, the TNF-α gene expression in Examples 3-6 was significantly downregulated, with the downregulation rates being 16.15% in Example 6, 24.62% in Example 5, 30.77% in Example 4, and 33.85% in Example 3; there was no significant change in the TNF-α gene expression in Example 7. This indicates that the cyclic dipeptide has a significant inhibitory effect on TNF-α expression in the range of 5-40ppm and has a skin soothing effect.

[0096] Table 3: IL-1β gene expression results

[0097]

[0098] According to the results in the above table, compared with the BC group, the IL-1β gene expression in the NC group was significantly upregulated, indicating that this test was effective. Compared with the NC group, the IL-1β gene expression in Examples 3-6 was significantly downregulated, with the downregulation rates being 45.16% in Example 3, 43.23% in Example 4, 41.29% in Example 5, and 14.84% in Example 6; there was no significant change in the IL-1β gene expression in Example 7. This indicates that the cyclic dipeptide has a significant inhibitory effect on IL-1β expression in the range of 5-40ppm and has a skin soothing effect.

[0099] Table 4: IL-6 gene expression results

[0100]

[0101] According to the results in the above table, compared with the BC group, the IL-6 gene expression in the NC group was significantly upregulated, indicating that this test was effective. Compared with the NC group, the IL-6 gene expression in Examples 3-4 was significantly downregulated, with the downregulation rates being 33.97% in Example 3 and 27.56% in Example 4; there was no significant change in the IL-6 gene expression in Examples 5, 6, and 7. This indicates that the cyclic dipeptide has a significant inhibitory effect on IL-6 expression in the range of 20-40ppm and has a skin soothing effect.

[0102] Table 5: PGE2 gene expression results

[0103]

[0104] According to the results in the above table, compared with the BC group, the PGE2 gene expression of the NC group was significantly upregulated, indicating that this test was effective. Compared with the NC group, the PGE2 gene expression of Examples 3-4 was significantly downregulated, with the downregulation rates of 73.71% for Example 3 and 65.73% for Example 4; there was no significant change in the PGE2 gene expression of Examples 5, 6, and 7. This shows that the cyclic dipeptide has a significant inhibitory effect on PGE2 expression within the range of 20-40ppm and has a skin soothing effect.

[0105] Table 6: COX2 gene expression results

[0106]

[0107] According to the results in the above table, compared with the BC group, the COX2 gene expression in the NC group was significantly upregulated, indicating that this test was effective. Compared with the NC group, the COX2 gene expression in Examples 3-4 was significantly downregulated, with the downregulation rates of 21.21% in Example 3 and 19.70% in Example 4; there was no significant change in the COX2 gene expression in Examples 5, 6, and 7. This indicates that the cyclic dipeptide has a significant inhibitory effect on COX2 expression in the range of 20-40ppm and has a skin soothing effect.

[0108] Test Example 2: Inhibition of the expression of inflammatory factors TSLP and IL-33 based on 3D epidermal skin model

[0109] This test is based on a 3D epidermal model to detect changes in TSLP and IL-33 protein expression after the action of Examples 8-9. A blank control group (BC), a model group (NC), and Examples 8-9 were set as sample groups.

[0110] 1. Test materials, reagents and main equipment

[0111] EpiKutis 3D epidermal skin model, PBS, DMSO (Sigma), culture medium (Gibco), TSLP ELISA kit (Abcam), IL-33 ELISA kit (Boster), isopropanol (Sinopharm), PolyI:C (Sigma), lipopolysaccharide (E.Coli.Sigma), anhydrous ethanol (Sinopharm), DEPC water (Beyotime).

[0112] CO2 incubator (Thermo), clean bench (Sujing Antai), inverted microscope (Olympus), microplate reader (BioTek), high-speed refrigerated centrifuge (Changsha Xiangyi), digital display constant temperature water bath (Changzhou Guohua).

[0113] 2. Experimental methods:

[0114] PolyI:C+LPS induction working solution: Take 1.44mL 5mg / mL PolyI:C stock solution and 3mL 2mg / mL LPS stock solution, dissolve in the culture medium, the total volume is 300mL, so that the final concentration of PolyI:C is 24μg / mL, and the final concentration of LPS is 20μg / mL.

[0115] The skin model was transferred to a 6-well plate according to the grouping (a small mold was added in advance, and 3.7 mL of culture solution was added), and the test group number was marked on the 6-well plate. The BC group and the NC group added culture solution, and the sample group evenly distributed Examples 8-9 on the surface of the model and placed in a CO2 incubator (37°C, 5% CO2) for incubation for 24 hours. After the administration and incubation were completed, the 6-well plate was removed from the incubator, the surface of the skin model was cleaned with sterile PBS, and the residual PBS liquid was wiped with a sterile cotton swab. Polyl:C and LPS induction working solution were added to the NC group and the sample group, and culture solution was added to the BC group. After the end, the 6-well plate was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours. The model culture solution was collected in a centrifuge tube. After collection, the samples for ELISA detection were placed in a -80°C refrigerator for freezing and storage, and the detection and analysis were performed according to the operating instructions of the ELISA kit. The t-test statistical analysis was used between the groups, p<0.05 indicated a significant difference, and p<0.01 indicated an extremely significant difference.

[0116] Table 7: TSLP protein detection results of 3D skin model

[0117]

[0118] According to the results in Table 7, after model induction, the TSLP content of the NC group increased significantly compared with the BC group, indicating that the stimulation conditions of this experiment were effective. Compared with the NC group, the cyclic (L-leucine-L-proline) dipeptide of Example 8 and the cyclic (D-leucine-L-proline) dipeptide of Example 9 at a concentration of 4ppm can significantly reduce the expression of the inflammatory factor TSLP, and the inhibition rates are 65.56% and 82.71%, respectively, indicating that the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide can achieve the effect of skin soothing by inhibiting the expression of the inflammatory factor TSLP.

[0119] Table 8: IL-33 protein detection results of 3D skin model

[0120]

[0121] According to the results in Table 8, after model induction, the IL-33 content of the NC group increased significantly compared with the BC group, indicating that the stimulation conditions of this experiment were effective. Compared with the NC group, the cyclic (L-leucine-L-proline) dipeptide of Example 8 and the cyclic (D-leucine-L-proline) dipeptide of Example 9 at a concentration of 4ppm can significantly reduce the expression of the inflammatory factor IL-33, and the inhibition rates are 37.22% and 46.40%, respectively, indicating that the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide can achieve the effect of skin soothing by inhibiting the expression of the inflammatory factor IL-33.

[0122] Application Examples

[0123] Cyclic (L-leucine-L-proline) dipeptide and cyclic (D-leucine-L-proline) dipeptide can be used for the preparation of external skin preparations.

[0124] The skin external preparation is preferably a cosmetic composition, such as a face cream, lotion, gel, toner, essence, mask, eye cream, aerosol (cleansing foam), spray, shower gel, facial cleanser, etc. The weight percentage of cyclo(L-leucine-L-proline) dipeptide and cyclo(D-leucine-L-proline) dipeptide in the skin external preparation is 0.001-20% (w / w). The preferred weight percentage is 0.1%-10% (w / w). The more preferred weight percentage is 0.01%-5% (w / w). The most preferred weight percentage is 0.1%-5% (w / w).

[0125] The following are specific application examples of cyclo(L-leucine-L-proline) dipeptide and cyclo(D-leucine-L-proline) dipeptide in skin external preparations, and the formulations and preparation methods of these preparations. In the following tables, "-" means no addition.

[0126] Application Example 1: Preparation of facial cream

[0127]

[0128]

[0129] Application Example 2: Preparation of Emulsion

[0130]

[0131] Application Example 3: Preparation of Gel

[0132]

[0133] Application Example 4: Preparation of lotion

[0134]

[0135]

[0136] Application Example 5: Preparation of Essence

[0137]

[0138] Application Example 6: Preparation of facial mask

[0139]

[0140]

[0141] Application Example 7: Preparation of Eye Cream

[0142]

[0143] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0144]

[0145] Application Example 9: Preparation of Spray

[0146]

[0147] Application Example 10: Preparation of Shower Gel

[0148]

[0149]

[0150] Application Example 11: Preparation of facial cleanser

[0151]

[0152]

Claims

1. Anti-inflammatory and soothing application of cyclic dipeptides, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination thereof.

2. The use according to claim 1, characterized in that The anti-inflammatory relief is achieved by inhibiting TNFα, IL-1β, IL-6, PGE2, COX2, TSLP and / or IL-33.

3. The use according to claim 1, characterized in that The cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide.

4. The use according to claim 1, characterized in that The cyclic dipeptide is a cyclo(D-leucine-L-proline) dipeptide.

5. The use according to claim 1, characterized in that The cyclic dipeptide is used at a concentration of 5 ppm to 40 ppm.

6. The use according to claim 5, characterized in that The cyclic dipeptide is used at a concentration of 20 ppm to 40 ppm.

7. The use according to claim 1, characterized in that The cyclic dipeptide was used at a concentration of 4 ppm.

8. Use of a cyclic dipeptide in the preparation of a skin topical agent, medicine and health food with anti-inflammatory and soothing effects, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination thereof.

9. The use according to claim 8, characterized in that The content of the cyclic dipeptide in external skin preparations, medicines and health foods is 0.0001%-20% by weight.

10. The use according to claim 8, characterized in that The skin external preparation is selected from the following forms: facial cream, lotion, gel, toner, essence, facial mask, eye cream, aerosol cleansing foam, spray, shower gel, or facial cleanser.

Images