Anti-aging effect and application of cyclic dipeptide

Abstract

The invention provides an anti-aging application of a cyclic dipeptide. The cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide. The anti-aging effect of the invention is achieved by promoting type I collagen, fibronectin, core proteoglycan Decrin and / or elastin, or the anti-aging effect is achieved by inhibiting a glycosylated end product. The invention also relates to application of the cyclic dipeptide in preparation of an external skin preparation, a medicine and a health-care food with an anti-aging effect. The external skin preparation is selected from face cream, emulsion, gel, toning lotion, essence, a mask, 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 a cyclic dipeptide with anti-aging effect. The structure of the cyclic dipeptide is cyclic (L-leucine-L-proline) dipeptide, which has the effect of promoting type I collagen, fibronectin, core protein polycan Decorin and elastin, and can be used as an anti-aging ingredient in cosmetics, food or medicine. Background Art

[0002] Beauty and anti-aging are the goals that people have always pursued. Collagen and elastin in the dermis are the main factors that affect skin aging. If the collagen content in the dermis decreases, the skin will show signs of aging, lose elasticity, become loose, and form wrinkles. Type I collagen is a fibrous collagen, which is contained in large quantities in bones and skin and participates in the elasticity of bones and skin. On the other hand, elastin has the function of supporting collagen fibers and is the main component of elastic fibers. It is particularly abundant in the connective tissue of the skin and exists at the same time as collagen fibers, giving the tissue elasticity and tension resistance, allowing the skin to stretch and fold, and is responsible for maintaining and supporting the elasticity of the skin. With age, or the influence of external factors such as UV, the content of collagen and elastin in the skin gradually decreases, causing the network support required to maintain the firmness of the skin to gradually disintegrate, the skin begins to sag, and wrinkles appear. Decorin, a core proteoglycan of the extracellular matrix, has a core protein structure that binds to type I collagen, and through the anti-parallel double helix structure formed between the GAG ​​side chains, it restricts the lateral assembly of collagen molecules, regulates the diameter of collagen fibers, and ensures their correct assembly; Fibronectin can promote the growth, differentiation and migration of skin cells, help the skin to regenerate in an orderly manner, enhance the expression of barrier proteins, repair the skin barrier, and improve the skin's self-protection ability. Therefore, they are all key targets for anti-aging and skin repair.

[0003] As we age, our metabolism slows down, life and work pressures increase, and with irregular lifestyles and eating habits, the sugar we consume will not be metabolized immediately and will accumulate in the body. In the absence of enzymes, free sugars react with collagen in the dermis to form some reversible primary "glycosylation products", which then form highly fluorescent, cross-linked and irreversible brown non-enzymatic glycation end products (AGEs). The whole process is called non-enzymatic glycosylation (NEG), and AGEs can further directly cross-link biological macromolecules in the body, causing functional damage.

[0004] When elastin and collagen in the skin participate in non-enzymatic glycosylation reactions to generate AGEs, collagen and elastin cross-linking products are formed. As collagen cross-linking gradually increases, the solubility of collagen decreases, insoluble collagen accumulates, and the permeability of skin tissue decreases, causing barriers to the exchange of nutrients and metabolites between cells, ultimately leading to a decrease in skin elasticity, sagging, and wrinkles. Not only that,

[0005] Glycated collagen turns yellow and becomes brittle. As we age, AGEs accumulate and our skin gradually turns yellow. In addition, when the receptors of AGEs (AGER) are activated, they induce the body to produce a large number of free radicals, which adds a lot of burden to the body's antioxidant system, further damaging the skin tissue and stimulating the production of melanin, putting the skin into a vicious cycle. Therefore, in recent years, the anti-glycation theory has become a hot mechanism and target for skin beauty. The anti-glycation effect is not only closely related to skin aging, but also closely related to the dark yellow color of the skin.

[0006] At present, the representative anti-glycation ingredients include aminoguanidine, carnosine, lipoic acid, niacinamide, quercetin and curcumin, etc. Their main mechanism is to block the development of AGEs and inhibit the production of AGEs.

[0007] In recent years, bioactive peptides have been increasingly used in the field of cosmetics and skin beauty. These bioactive peptides are often used to improve skin aging, such as stimulating the synthesis of collagen and elastin, improving wound healing, and increasing the proliferation of fibroblasts. However, the linear structure of the polypeptide determines that it is more hydrophilic and not easily absorbed through the skin. At the same time, polypeptide molecules are substrates for many protease hydrolysis. Most polypeptides will be degraded and inactivated by proteases in the blood and tissues, losing their efficacy. Therefore, the bioavailability of polypeptide raw materials is very low, so that they are greatly restricted in practical applications. At the same time, common beauty peptides on the market, such as the classic anti-wrinkle (dynamic lines) raw material acetyl hexapeptide-8, can inhibit dynamic lines. Its mechanism of action is mainly to inhibit the release of acetylcholine, but there is no relevant report on promoting collagen expression; for example, palmitoyl pentapeptide-4 mainly stimulates the production of collagen I, IIII and VI, and no data on the promotion of elastin is found. Furthermore, as small molecular linear peptides, hexapeptide-9, palmitoyl pentapeptide-4, myristoyl pentapeptide-4, and palmitoyl tripeptide-5 also have problems such as low stability, easy degradation, inactivation, and poor transdermal absorption. Therefore, it is necessary to discover other active peptides that are more stable, well absorbed, and have clear efficacy. For example, existing literature reports the efficacy of plant cyclic peptides, a class of cyclic compounds formed by peptide bonds or disulfide bonds, in improving skin (Yu Minyi, Huang Jiefang, Liu Ruijie, et al., Plant cyclic peptides and their application potential in the field of cosmetics [J], Flavors and Fragrances Cosmetics, 2023 (6): 76-81. DOI: 10.20099 / j.issn.1000-4475.2023.0170). For example, Chinese patent application 202211550526.8 provides a cyclic peptide with superior performance and its application, wherein the cyclic peptide includes hexapeptide-9 cyclic peptide, palmitoyl pentapeptide-4 cyclic peptide, myristoyl pentapeptide-4 cyclic peptide, and palmitoyl tripeptide-5 cyclic peptide, which have the functions of promoting the expression of collagen I, FLG and TGM1, and increasing skin moisture content (a group of cyclic peptides with superior performance and their applications, CN202211550526.8[P].2023-05-26).

[0008] Cyclo(L-Leucine-L-Proline)Dipeptide is a cyclic dipeptide. Cyclo(L-Leucine-L-Proline)Dipeptide is a microbial metabolite that can be metabolized and produced by many bacteria, fungi and actinomycetes.

[0009] Qi Shuhua et al. isolated the cyclic (L-leucine-L-proline) compound from the metabolites of marine bacteria Pseudomonas sp. (Qi Shuhua, Qian Peiyuan, Zhang Si, Research on antibacterial metabolites of marine bacteria Pseudomonas sp. [J], Natural Product Research and Development, 2009, 21(3): 420-423); Wu Xiuli et al. isolated the 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 the 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).

[0010] However, none of these documents studies or mentions the activity research of cyclo(L-leucine-L-proline) dipeptide in beauty and skin care.

[0011] The present invention unexpectedly found that cyclic (L-leucine-L-proline) dipeptide has excellent effects on promoting type I collagen, fibronectin, core protein polycan Decorin and elastin, and can be used as a functional additive in skin external preparations, medicines and health foods. Summary of the invention

[0012] In one aspect, the present invention relates to the anti-aging application of a cyclic dipeptide, wherein the cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide.

[0013] In a preferred embodiment, the anti-aging effect is achieved by promoting type I collagen, fibronectin, decorin and / or elastin. In a preferred embodiment, the anti-aging effect is achieved by inhibiting advanced glycation end products.

[0014] In a preferred embodiment, the cyclic dipeptide is used at a concentration of 0.4 ppm to 40 ppm. In a preferred embodiment, the cyclic dipeptide is used at a concentration of 1.6 ppm to 40 ppm, 0.4 ppm to 1.6 ppm, or 4 ppm.

[0015] In a preferred embodiment, the cyclic dipeptide is used at a concentration of 250 ppm.

[0016] On the other hand, the present invention also relates to the use of a cyclic dipeptide in the preparation of an external skin preparation, a medicine and a health food with anti-aging effects, wherein the cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide.

[0017] In a preferred embodiment, the content of the cyclic dipeptide in the external skin preparation, medicine and health food is 0.001-20% by weight, more preferably 0.01-5% by weight.

[0018] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The anti-glycation efficacy fluorescence staining images of the blank group (A), the model group (B) and the 250 ppm cyclo(L-leucine-L-proline) dipeptide (C) are shown. The immunofluorescence image was taken by a fluorescence microscope, and the image was quantitatively analyzed using Image J. The magnification is 20 times. DETAILED DESCRIPTION

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] The present invention has found that cyclic dipeptides are useful for treating signs of skin aging. As used herein, "signs of skin aging" include the presence of fine lines and wrinkles, loss of elasticity, uneven skin tone, and scarring. In a particularly preferred embodiment, the signs of aging are the presence of fine lines and wrinkles and / or loss of elasticity.

[0028] As used herein, "treating the signs of skin aging" means alleviating, reducing, preventing, ameliorating or eliminating the presence or signs of skin aging as described above.

[0029] As used herein, "wrinkles" include fine lines, micro wrinkles or macro wrinkles. Examples of wrinkles include, but are not limited to, fine lines around the eyes (e.g., "crow's feet"), forehead and cheek wrinkles, frown lines, and smile lines around the mouth.

[0030] As used herein, "loss of elasticity" includes loss of elasticity or structural integrity of skin or tissue, including but not limited to sagging, loose and loose tissue. Loss of elasticity or tissue structural integrity can be caused by a variety of factors, including but not limited to disease, aging, hormonal changes, mechanical trauma, environmental damage, or as a result of applying products such as cosmetics or drugs to the tissue.

[0031] As used herein, "uneven skin tone" refers to a skin condition associated with diffuse or mottled pigmentation that can be classified as hyperpigmentation, such as post-inflammatory hyperpigmentation.

[0032] As used herein, "scarring" means a skin condition associated with redness or erythema.

[0033] As used herein, "cosmetic" refers to cosmetic substances or preparations that maintain, restore, impart, stimulate, or enhance the physical appearance or seem to increase beauty or youthfulness, especially as it relates to the appearance of tissue or skin.

[0034] As used herein, "cosmetically effective amount" means an amount of a physiologically active compound or composition sufficient to treat one or more signs of skin aging, but low enough to avoid serious side effects. The cosmetically effective amount of a compound or composition will vary depending on the specific condition being treated, the age and physical condition of the end user, the severity of the condition being treated / prevented, the duration of the treatment, the nature of other treatments, the specific compound or product / composition employed, the specific cosmetically acceptable carrier utilized, and the like.

[0035] The present invention is based on the following unexpected discovery: cyclic (L-leucine-L-proline) dipeptide has the effect of promoting type I collagen, fibronectin, core protein polycan Decorin and elastin, and can be used as a functional additive for preparing skin topical preparations, medicines and health foods to help achieve the effects of maintaining skin firmness, improving skin sagging and / or wrinkles, and resisting skin aging.

[0036] Cyclic dipeptide

[0037] The present invention provides a cyclic dipeptide component, the structure of which is cyclic (L-leucine-L-proline) dipeptide, and its effect on promoting type I collagen, fibronectin, decorin and elastin is confirmed. Table 1 shows the structural information of cyclic (L-leucine-L-proline) dipeptide.

[0038] Table 1

[0039]

[0040]

[0041] In a specific embodiment, the composition of the present invention uses cyclo(L-leucine-L-proline) dipeptide provided by Donghua University.

[0042] Skin topical products

[0043] The cyclic dipeptide provided by the present invention has the effect of improving collagen and promoting the production of elastin to repair and resist aging. Therefore, it can be used as a skin external agent to help improve aging symptoms such as skin wrinkles and sagging, and can also be used as a medicine and health food.

[0044] 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.

[0045] The content of the cyclic dipeptide in the external preparation for skin may be 0.000001-1% by weight, preferably 0.00001-1% by weight, more preferably 0.001-1% by weight, and even more preferably 0.01-1% by weight.

[0046] In some embodiments, the content of the cyclic dipeptide in the external preparation for skin is 0.000008-20% by weight, preferably 0.00001-10% by weight.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] 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 %.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 %.

[0068] 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.

[0069] Additional cosmetic active agents

[0070] 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.

[0071] 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.

[0072] 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.

[0073] Examples of hydroxy acids include, but are not limited to, glycolic acid, lactic acid, malic acid, salicylic acid, citric acid, and tartaric acid.

[0074] 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.

[0075] How to use

[0076] 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.

[0077] 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.

[0078] Example

[0079] 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.

[0080] Cyclic (L-leucine-L-proline) dipeptide (purity ≥ 98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0081] Cyclic (D-leucine-L-proline) dipeptide (purity ≥ 98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0082] Embodiment 1:

[0083] 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.

[0084] Embodiment 2:

[0085] 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.

[0086] Embodiment 3:

[0087] Example 1 was diluted with culture medium to prepare a concentration of 40 ppm as a test solution.

[0088] Embodiment 4:

[0089] Example 1 was diluted with culture medium to prepare a concentration of 4 ppm as a test solution.

[0090] Embodiment 5:

[0091] Example 1 was diluted with culture medium to prepare a concentration of 1.6 ppm as a test solution.

[0092] Embodiment 6:

[0093] Example 1 was diluted with culture medium to prepare a concentration of 0.4 ppm as a test solution.

[0094] Embodiment 7:

[0095] Example 2 was diluted with culture medium to prepare a concentration of 4 ppm as a test solution.

[0096] Example 8

[0097] Example 1 was diluted with culture medium to prepare a concentration of 250 ppm as a test solution.

[0098] Test Example 1: Elastin content test based on fibroblasts

[0099] The experiment included a blank control group (BC) and a sample group (the sample of the embodiment).

[0100] Press 4×10 4 Inoculate cells into a 24-well plate at a seeding density of cells / well and incubate overnight in an incubator (37°C, 5% CO2). When the cell plating rate in the 24-well plate reaches 40% to 60%, group medication is performed. Add 2 mL of sample to each well, and set up 3 replicate wells in each group. After medication, place the 24-well plate in an incubator and culture for 24 hours. After the incubation, collect the cell culture supernatant in a centrifuge tube and store it in a -80°C refrigerator. Perform the test according to the operating instructions of the Elastin ELISA Kit (Human Elastin ELISA Kit, Abcam).

[0101] Elastin test results:

[0102] Table 2

[0103]

[0104] The experimental results show that the cyclic dipeptide of Example 4 has a significant effect on increasing the content of elastin at a concentration of 4 ppm, indicating that it has a promoting effect on elastin, while Comparative Example 7 has no effect.

[0105] Test Example 2: Gene expression detection of fibroblasts

[0106] Based on human dermal fibroblasts (FB), the anti-aging effect of promoting collagen / elasticity was evaluated by measuring the changes in the expression of collagen and other related genes by RT-PCR after the examples acted on FB cells.

[0107] 1) Inoculation: 2×10 5 The cells were seeded into 6-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0108] 2) Grouping: The experiment set up a blank BC group and a sample (example) group.

[0109] 3) Drug administration: When the cell plating rate in the 6-well plate reaches 40% to 60%, the drugs are administered in groups, with a dosage of 2 mL per well. Two replicate wells are set up for each group, and the cells are incubated in an incubator (37° C., 5% CO 2 ) for 24 h.

[0110] 4) Sample collection: After culturing for 24 hours, discard the supernatant, wash twice with PBS, add 1 mL of Trizol Reagent to each well, pipette and lyse the cells, and then collect the samples.

[0111] 5) Extraction: mRNA was separated and purified using chloroform and isopropanol respectively, and mRNA precipitate was obtained after washing three times with 75% ethanol, and enzyme-free water was added to dissolve it to obtain mRNA solution.

[0112] 6) Quality and concentration determination: Nanodrop was used to determine the concentration of mRNA and obtain preliminary RNA quality test results.

[0113] 7) Reverse transcription: The extracted mRNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (Acori Biotech).

[0114] 8) Fluorescence quantitative PCR detection: Prepare the test solution according to the instructions of the real-time fluorescence quantitative PCR kit and perform the detection on the machine.

[0115] The 2-△△CT method was used to calculate the results.

[0116] 9) Statistical analysis of results: GraphPad Prism was used for graphing, and the results were expressed as Mean ± SD. One-way ANOVA statistical analysis was used for comparison between groups. P < 0.05 was considered to be significantly different*, and P < 0.001 was considered to be extremely significantly different***.

[0117] Type I collagen test results:

[0118] Table 3

[0119]

[0120] FN1 fibronectin test results:

[0121] Table 4

[0122]

[0123] Decorin Experimental Results:

[0124] Table 5

[0125]

[0126] The experimental results showed that Examples 3, 5, and 6 significantly increased the gene expression of type I collagen and decorin protein, and significantly promoted the gene expression of fibronectin at two concentrations.

[0127] Test Example 3: Anti-glycated cell immunofluorescence experiment

[0128] 1. Test Principle

[0129] Carboxymethyl lysine (CML) is one of the representatives of advanced glycation end products. In the experiment, glyoxal was used to induce glycosylation in primary human skin fibroblasts, aminoguanidine was used as a positive control, and immunofluorescence staining was used to quantitatively analyze the expression of CML.

[0130] 2. Test Materials

[0131] 2.1 Main reagents: primary human dermal fibroblasts and culture medium ( Cell Technology), penicillin-streptomycin-amphotericin B solution (Bioind), DPBS (Gibco), CCK reagent (Tongren Biotechnology), CML antibody (Abcam), Alexa 568Donkey Anti-Mouse IgG (Invitrogen), bovine serum albumin BSA (Biyuntian), glyoxal (Adamas), aminoguanidine (Aladdin).

[0132] 2.2 Main equipment: cell culture incubator, inverted fluorescence microscope (Leica), biological safety cabinet, microplate reader.

[0133] 3. Test Methods

[0134] 3.1 Cell seeding:

[0135] After digestion, the cells were inoculated into a 24-well plate with cell slides added, with 1.5*10 4 cells.

[0136] 3.2 Glycation induction:

[0137] After 48 hours, the cells were treated with 0.5 mM glyoxal to induce glycation reaction as a negative control group. At the same time, a blank group (NT), a control group (glyoxal) and a sample group (glyoxal + sample) were set up and the cells were treated for 48 hours.

[0138] 3.3 Fixation and staining:

[0139] The induced cells were rinsed once with PBS, fixed with paraformaldehyde (PFA), and then CML fluorescent staining was performed. After BSA blocking, primary antibody incubation and secondary antibody incubation, the slides were finally sealed with an aqueous mounting medium containing DAPI. Fluorescence images were taken with a fluorescence microscope (Leica DMi8, Heidelberg, Germany) at a magnification of 20 times, and the images were quantitatively analyzed using Image J. IOD represents the cumulative optical density.

[0140] 4. Test Results

[0141] Table 6

[0142] Group IOD average SD Inhibition rate (%) Blank Group 797548 256278.9 / Model Group 1302625 115482.6 / Example 8 870356.3 90671.47 33.18

[0143] The results showed that the glycation inhibition rate of Example 8 was 33.18% (P<0.01), indicating that at this concentration, it has a strong inhibitory effect on the glycation reaction of fibroblasts induced by glyoxal and has anti-glycation efficacy.

[0144] In addition, it can be seen from the figure that compared with the blank group BC, the expression of the glycation end product CML in the control group NC after glyoxal induction in human primary dermal fibroblasts was significantly increased, that is, the orange CML staining fluorescence area and brightness were significantly increased. After the test of Example 8, the orange fluorescence area and brightness were reduced compared with the control group, indicating that the expression of the glycation end product CML in the cells was inhibited, which has an anti-glycation effect.

[0145] Application Examples

[0146] The cyclic dipeptide can be used in the preparation of medicines, health foods or external skin preparations.

[0147] The skin topical preparation is preferably a cosmetic composition, including but not limited to products in dosage forms such as cream, lotion, gel, toner, essence, mask, eye cream, aerosol (cleansing bubble), spray, shower gel and facial cleanser. The weight percentage of the cyclic dipeptide in the medicine, health food or skin topical preparation is 0.0001%-20% (w / w). The preferred weight percentage is 0.001%-10% (w / w). The more preferred weight percentage is 0.001%-5% (w / w). The most preferred weight percentage is 0.01%-5% (w / w).

[0148] The following are specific application examples of the cyclic dipeptide obtained in the examples in external skin preparations, as well as the formulations and preparation methods of these preparations. In the following tables, "-" means no addition.

[0149] Application Example 1: Preparation of facial cream

[0150]

[0151] Application Example 2: Preparation of Emulsion

[0152]

[0153]

[0154] Application Example 3: Preparation of Gel

[0155]

[0156]

[0157] Application Example 4: Preparation of lotion

[0158]

[0159] Application Example 5: Preparation of Essence

[0160]

[0161]

[0162] Application Example 6: Preparation of facial mask

[0163]

[0164]

[0165] Application Example 7: Preparation of Eye Cream

[0166]

[0167] Application Example 8: Preparation of Aerosol (Cleaning Bubble)

[0168]

[0169]

[0170] Application Example 9: Preparation of Spray

[0171]

[0172] Application Example 10: Preparation of shower gel

[0173]

[0174]

[0175] Application Example 11: Preparation of facial cleanser

[0176]

[0177]

Claims

1. Anti-aging application of cyclic dipeptide, wherein the cyclic dipeptide is cyclic (L-leucine-L-proline) dipeptide.

2. The use according to claim 1, characterized in that The anti-aging effect is achieved by promoting type I collagen, fibronectin, decorin and / or elastin.

3. The use according to claim 1, characterized in that The anti-aging effect is achieved by inhibiting advanced glycation end products.

4. The use according to claim 2, characterized in that The cyclic dipeptide is used at a concentration of 0.4 ppm to 40 ppm.

5. The use according to claim 4, characterized in that The cyclic dipeptide is used at a concentration of 1.6 ppm to 40 ppm.

6. The use according to claim 4, characterized in that The cyclic dipeptide is used at a concentration of 0.4 ppm to 1.6 ppm.

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

8. The use according to claim 3, characterized in that The cyclic dipeptide was used at a concentration of 250 ppm.

9. Use of a cyclic dipeptide in the preparation of an external skin preparation, a medicine and a health food with anti-aging effects, wherein the cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide.

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

11. The use according to claim 9, 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.

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