Use of cyclic dipeptides to improve barrier moisturization repair

Abstract

The present application provides the use of a cyclic dipeptide to improve barrier moisturization repair, the cyclic dipeptide is selected from the group consisting of: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or a combination thereof. The improved barrier moisturization repair of the present application is achieved by promoting the expression of skin barrier-related factors FLG, AQP3, LOR and / or IVL. The present application also relates to the use of a cyclic dipeptide in the preparation of a skin external preparation having the effect of improving barrier moisturization repair, the skin external preparation is selected from the group consisting of: a face cream, a lotion, a gel, a toner, an essence, a mask, an eye cream, an aerosol cleaning foam, a spray, a shower gel, or a facial cleanser.

Description

Technical Field

[0001] This invention pertains to the application of cyclic dipeptides, specifically involving cyclic dipeptides that improve barrier hydration and repair. These dipeptides have the structure of cyclic (L-leucine-L-proline) dipeptides and / or cyclic (D-leucine-L-proline) dipeptides, and promote the expression of skin barrier moisturizing factors such as FLG, AQP3, LOR, and IVL. They can be used as ingredients to improve barrier hydration and repair in cosmetics, food, or pharmaceuticals. Background Technology

[0002] The skin is the largest organ in the human body, accounting for approximately 16% of total body weight. It serves as the first line of defense against harmful external factors, protecting internal tissues and organs. The skin protects against environmental stimuli, such as physically blocking ultraviolet rays and harmful substances. It prevents excessive moisture loss, maintains the hydration level of the stratum corneum, and regulates the skin's moisture content. Normal skin moisture content is 20%–35%. When skin moisture content drops below 10%, the skin barrier function is impaired. Damaged skin barriers can lead to dryness, roughness, pigmentation, wrinkles, and aging. Therefore, protecting the skin, maintaining its barrier function, and improving skin hydration are crucial for skincare products.

[0003] The structural basis of the skin barrier is the stratum corneum formed by the outermost keratinocytes, the lipids between the keratinocytes, and natural protective factors, the so-called "brick wall theory," which consists of the stratum corneum and its keratin capsule (bricks and mortar) and the lipid bilayer between cells (mortar). The formation of the stratum corneum involves several important proteins, including filaggrin (FLG), involucrin (IVL), loricrin (LOR), keratinocyte transglutaminase, keratin 1 (K1), and keratin 10 (K10).

[0004] Fliquin (FLG), also known as microfilament aggregate protein, is mainly found in the granular layer and stratum lucidum of the epidermis. It is derived from inactive pFLG in the keratinocytes of the granular layer through the catalysis of specific phosphatases and proteases. It can interact with intermediate keratin filaments to aggregate into keratin fiber bundles, thus forming the flattened scaffold structure of the outermost keratinocytes. Studies have shown that FLG expression is decreased in patients with atopic dermatitis, indicating weakened or impaired skin barrier function. LOR (Leydigin of the Nail) is mainly expressed in the granular layer and stratum corneum and is a major component of the final differentiated keratinocytes (CEs). The formation of the keratin capsule marks the production of keratinocytes, the terminal product of keratinocyte differentiation, and is the basis of the epidermis as a defensive barrier. Inner keratin (IVL) is mainly expressed in the upper spinous layer and granular layer. It is a marker protein of keratinocyte differentiation, located in the outer layer of the keratin capsule, and covalently binds to ceramides containing -OH groups, thereby connecting the lipid matrix and keratinocytes to perform its function.

[0005] While the stratum corneum is considered the most important physiological barrier in skin structure, the basal layer of the epidermis is equally important for barrier function. Keratinocytes in the basal layer of the epidermis express aquaporin 3 (AQP3), a complete transmembrane protein channel. Water and glycerol circulating in the body can reach the epidermis via AQP3, promoting hydration of the stratum corneum and closely related to the skin's moisturizing barrier function.

[0006] Cyclic peptides are widely distributed in nature and possess strong biological activity. Many serve as drugs or drug prodrugs, playing crucial roles in the life processes of organisms. Cyclic dipeptides are the smallest class of cyclic peptides and are ubiquitous in nature. Cyclic dipeptides possess a diketopiperazine structure, thus exhibiting high stability. Their unique spatial conformation endows them with significant physiological activity, making them highly valuable for applications in chemistry, medicinal chemistry, biochemistry, and life sciences. Therefore, they have attracted considerable attention from chemists, biologists, and pharmacologists both domestically and internationally.

[0007] The applicant is particularly interested in cyclic (L-leucine-L-proline) dipeptide and cyclic dipeptide (D-leucine-L-proline) dipeptide.

[0008] Cyclic (L-leucine-L-proline) dipeptide is a microbial metabolite produced by many bacteria, fungi, and actinomycetes. Qi Shuhua et al. obtained this substance from marine bacteria. Pseudomonas Cyclic (L-leucine-L-proline) compounds were isolated from the metabolites of *Sp. sp.* (Qi Shuhua, Qian Peiyuan, Zhang Si, Marine Bacteria). PseudomonasResearch on antibacterial metabolites of *Sp.* [J], Research and Development of Natural Products, 2009, 21(3):420-423; Wu Xiuli et al. isolated this substance from the liquid culture of *Fotomyces tumefaciens* (Wu Xiuli, Lin Sheng, Zhu Chenggen et al., Study on chemical composition of liquid culture of *Fotomyces tumefaciens* [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 7Ze3 cyclic dipeptide from Bacillus subtilis [J], Jiangsu Agricultural Sciences, 2010(2):107-109; Wang Jing et al. reported the isolation of cyclic (L-leucine-L-proline) dipeptide from endophytic fungi of Platycladus orientalis (Wang Jing, Wei Yan, Yi Xiaohua et al., Study on the herbicidal selectivity of endophytic fungus J46 of Platycladus orientalis and isolation of active substances [C], Proceedings of the 13th National Symposium on Pesticide Science Teaching and Research, 2013:260-266). Zhu Jiansheng et al. isolated cyclic (D-leucine-L-proline) compound from marine bacteria (Zhu Jiansheng, Marine Bacteria Pseudomonas putida Study on the bioactivity of a medium-cyclic dipeptide [D], Jiangsu: Yangzhou University, 2013.

[0009] However, none of these studies investigated or mentioned the activity of cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide in cosmetic and skincare applications.

[0010] This invention unexpectedly discovered that cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide can promote the expression of skin barrier moisturizing factors such as FLG, AQP3, LOR, and IVL, and can be used as ingredients to improve barrier function and moisturize in cosmetics, food, or pharmaceuticals. Summary of the Invention

[0011] On one hand, the present invention relates to the application of cyclic dipeptides in improving barrier hydration and repair, wherein the cyclic dipeptides are selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or combinations thereof.

[0012] In a preferred embodiment, the improved barrier hydration and repair is achieved by promoting the expression of skin barrier-related factors FLG, AQP3, LOR and / or IVL.

[0013] 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 10 ppm to 40 ppm, 2.5 ppm to 20 ppm, or 4 ppm.

[0014] On the other hand, the present invention relates to the use of cyclic dipeptides in the preparation of topical skin agents with barrier-improving moisturizing and repairing effects, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or combinations thereof.

[0015] In a preferred embodiment, 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. Invention Details

[0017] 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.

[0018] 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.

[0019] 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.

[0020] This 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 promoting the expression of skin barrier moisturizing factors such as FLG, AQP3, LOR, and IVL, and can be used as ingredients to improve barrier function and moisturize in cosmetics, food or pharmaceuticals.

[0021] Cyclic dipeptide

[0022] This invention provides a cyclic dipeptide component with the structure of cyclic (L-leucine-L-proline) dipeptide and / or cyclic (D-leucine-L-proline) dipeptide.

[0023] Table 1 shows the structural information of the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide.

[0024] Table 1

[0025]

[0026] In a specific embodiment, the composition of the present invention uses cyclic (L-leucine-L-proline) dipeptide and cyclic (D-leucine-L-proline) dipeptide provided by Donghua University.

[0027] Topical skin agents

[0028] The present invention also provides a topical skin agent comprising a cyclic (L-leucine-L-proline) dipeptide and / or a cyclic (D-leucine-L-proline) dipeptide.

[0029] The term "topical skin agent" is a general term encompassing all ingredients typically used on the external surface of the skin, including cosmetic compositions and pharmaceutical compositions. Cosmetic compositions may include basic cosmetics, facial makeup, body cosmetics, hair care cosmetics, etc., with no specific restrictions on their dosage forms; appropriate formulations can be selected based on different purposes.

[0030] Cyclic (L-leucine-L-proline) dipeptides and / or cyclic (D-leucine-L-proline) dipeptides can be added as cosmetic additives to serums, creams, lotions, eye creams, facial oils, massage oils, and other cosmetics.

[0031] The cosmetic composition may also contain different cosmetically permissible media or matrix excipients, depending on the dosage form and purpose.

[0032] Cosmetic, dermatological, or pharmaceutically acceptable excipients that can be used in the topical skin formulations of this invention are in the form of an aqueous phase, oil phase, gel, water-in-wax emulsion, water-in-oil emulsion, or oil-in-water emulsion. The aqueous phase is a mixture of one or more water-soluble or dispersible components, which may be liquid, semi-solid, or solid at room temperature (25°C). Excipients include or may be in the form of suspensions, dispersions, or solutions in water or water-alcohol excipients, and may contain thickeners or gelling agents. Those skilled in the art can select appropriate product forms and the components contained therein based on their knowledge.

[0033] The composition may include an aqueous phase, which may contain water or a mixture of water and at least one hydrophilic organic solvent, such as alcohols, especially straight-chain or branched lower monohydric alcohols containing 2-5 carbon atoms, such as ethanol or propanol; polyhydric alcohols, such as propylene glycol, sorbitol, glycerol, panthenol, or polyethylene glycol and mixtures thereof.

[0034] When the composition of the invention is in the form of an emulsion, the composition may optionally contain a surfactant.

[0035] The composition may also comprise a film-forming polymer, such as a polyurethane, a polyacrylic acid homopolymer or copolymer, a polyester, a hydrocarbon-based resin, and / or a siloxane resin. The polymer may be dissolved or dispersed in a cosmetically acceptable excipient and optionally incorporated with a plasticizer.

[0036] The compositions of the present invention may further comprise any of the components commonly used in the cosmetics field. These components include preservatives, aqueous phase thickeners (extractive biopolymers, synthetic polymers) and fatty phase thickeners, fragrances, hydrophilic and lipophilic active agents, and mixtures thereof.

[0037] The compositions of the present invention may also contain an additional particulate phase, which may be pigments and / or pearlescent agents and / or fillers used in cosmetic compositions.

[0038] Pigments can be present in the composition. Suitable inorganic pigments include titanium oxide, zirconium oxide, and cerium oxide, as well as zinc oxide, iron oxide, and iron blue; suitable organic pigments include barium, strontium, calcium, and aluminum lakes and carbon black.

[0039] Pearlizing agents can be present in the composition, and suitable pearlizing agents include mica coated with titanium dioxide, iron oxide, or natural pigments.

[0040] Fillers may be present in the composition. Suitable fillers include talc, silica, zinc stearate, mica, kaolin, nylon powder, polyethylene powder, Teflon, starch, boron nitride, and copolymer microspheres, such as siloxane resin microspheres.

[0041] The compositions of the present invention can be formulated into any suitable product form. Such product forms include, but are not limited to, aerosol sprays, creams, lotions, solids, liquids, dispersions, foams, gels, lotions, mousses, ointments, powders, patches, hair oils, solutions, pump sprays, sticks, masks, and wet wipes. The compositions of the present invention can be conveniently used in the preparation or as topical cosmetic, dermatological, or pharmaceutical products by various methods well known in the art.

[0042] The topical skin composition of the present invention may include one or more of the following components: anti-allergic agents, anti-inflammatory agents, moisturizers, antimicrobial agents, antioxidants, chelating agents, colorants, depigmenting agents, emollients, emulsifiers, epidermal exfoliants, film-forming agents, fragrances, insect repellents, lubricants, pharmaceutical activators, humectants, lightfastness agents, preservatives, skin care agents, skin penetration enhancers, sunscreens, stabilizers, surfactants, thickeners, viscosity modifiers, vitamins, or any combination thereof. Detailed Implementation

[0043] The present invention is further illustrated below with reference to specific embodiments. However, the scope of the present invention is not limited to these embodiments, which are intended to illustrate the technical solutions of the present invention and do not constitute a limitation on the scope of the present invention. Experimental 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.

[0044] Cyclic (L-leucine-L-proline) dipeptide (purity ≥98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0045] Cyclic (D-leucine-L-proline) dipeptide (purity ≥98%), naturally extracted or chemically synthesized, provided by Donghua University.

[0046] Example 1:

[0047] 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.

[0048] Example 2:

[0049] 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.

[0050] Example 3:

[0051] Take Example 1, dilute it with culture medium to prepare a concentration of 40 ppm, and use it as the test solution.

[0052] Example 4:

[0053] Take Example 1, dilute it with culture medium to prepare a concentration of 20 ppm, and use it as the test solution.

[0054] Example 5:

[0055] Take Example 1, dilute it with culture medium to prepare a 10 ppm concentration, and use it as the test solution.

[0056] Example 6:

[0057] Take Example 1, dilute it with culture medium to prepare a 5 ppm concentration, and use it as the test solution.

[0058] Example 7:

[0059] Take Example 1, dilute it with culture medium to prepare a concentration of 2.5 ppm, and use it as the test solution.

[0060] Example 8:

[0061] Take Example 1, dilute it with culture medium to prepare a 4 ppm concentration, and use it as the test solution.

[0062] Example 9:

[0063] Take Example 2, dilute it with culture medium to prepare a 4 ppm concentration, and use it as the test solution.

[0064] Test Example 1: Improving Barrier and Moisturizing Gene Expression - 2D Cells

[0065] Materials and reagents:

[0066] Keratinocytes (batch number: Ep23082203) were provided by Guangdong Boxi Biotechnology Co., Ltd.; KcGrowth culture medium (Guangdong Boxi Biotechnology), PBS (Solepro), RNAisoPlus (Aikerui Biotechnology), and reverse transcription kit (Aikerui Biotechnology).

[0067] Experimental equipment:

[0068] CO2 incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), real-time PCR instrument (BioRad, CFX-96).

[0069] Promote FLG expression

[0070] This test, based on keratinocytes, examined changes in FLG gene expression levels after treatment with Examples 3-7. A blank control group (BC) was set up, and Examples 3-7 served as the sample groups.

[0071] Based on 2.0×10 5 Cells were seeded at a density of [number] cells / well into 6-well plates and incubated overnight in an incubator (37 ℃, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–60%, cells were divided into groups and treated with 2.0 mL of drug per well, with 3 replicates per group, and incubated for 24 h in an incubator (37 ℃, 5% CO2). The supernatant was then discarded, and the cells were washed twice with 1.0 mL / well of PBS. 1.0 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time PCR. -△△CT Results were calculated using the following methods. Results are expressed as mean ± SD. The sample group and the BC group were compared using t-test statistical analysis. All statistical analyses were two-tailed. A p-value < 0.05 was considered statistically significant. A p-value < 0.01 is considered highly significant and is expressed as follows: .

[0072] Upregulation rate (%) = (gene content sample group - gene content BC) / gene content BC × 100%.

[0073] The results are shown in Table 2.

[0074] Table 2: Results of FLG gene expression detection

[0075]

[0076] The experimental results show that, compared with the blank group, the FLG gene expression level in Examples 3-7 was significantly upregulated, with upregulation rates of 23.00%, 30.00%, 32.00%, 84.00%, and 86.00%, respectively. This indicates that the cyclic dipeptide has a promoting effect on FLG expression in keratinocytes within the range of 2.5ppm-40ppm, suggesting that the cyclic dipeptide has the function of improving skin barrier and repair.

[0077] Promote AQP3 expression

[0078] Changes in the AQP3 gene in keratinocytes were detected by qPCR.

[0079] This test, based on keratinocytes, examined changes in AQP3 gene expression levels after treatment with Examples 3-7. A blank control group (BC) was set up, and Examples 3-7 served as the sample groups.

[0080] Based on 2.0×10 5 Cells were seeded at a density of [number] cells / well into 6-well plates and incubated overnight in an incubator (37 ℃, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–60%, cells were divided into groups and treated with 2.0 mL of drug per well, with 3 replicates per group, and incubated for 24 h in an incubator (37 ℃, 5% CO2). The supernatant was then discarded, and the cells were washed twice with 1.0 mL / well of PBS. 1.0 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time PCR. -△△CT Results were calculated using the method described above. Results are expressed as mean ± SD. The sample group and the BC group were compared using t-test statistical analysis. All statistical analyses were two-tailed. A p-value < 0.05 was considered statistically significant. A p-value < 0.01 is considered highly significant and is expressed as follows: .

[0081] Upregulation rate (%) = (gene content sample group - gene content BC) / gene content BC × 100%.

[0082] The results are shown in Table 3.

[0083] Table 3: AQP3 gene expression detection results

[0084]

[0085] The experimental results show that, compared with the control group, the AQP3 gene expression level in Examples 3-5 was significantly upregulated, with upregulation rates of 38.00%, 61.00%, and 83.00%, respectively, indicating that the cyclic dipeptide promotes AQP3 expression in keratinocytes within the range of 10ppm-40ppm. The AQP3 gene expression level in Examples 6-7 was comparable to that in the control group, indicating that the cyclic dipeptide had no effect on AQP3 gene expression at concentrations of 5ppm and 2.5ppm. These results indicate that the cyclic dipeptide has skin moisturizing and repairing functions.

[0086] Promote LOR expression

[0087] Changes in the LOR gene in keratinocytes were detected by qPCR.

[0088] This test, based on keratinocytes, examined changes in LOR gene expression levels after treatment with Examples 3-7. A blank control group (BC) was set up, and Examples 3-7 served as the sample groups.

[0089] Based on 2.0×10 5 Cells were seeded at a density of [number] cells / well into 6-well plates and incubated overnight in an incubator (37 ℃, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–60%, cells were divided into groups and treated with 2.0 mL of drug per well, with 3 replicates per group, and incubated for 24 h in an incubator (37 ℃, 5% CO2). The supernatant was then discarded, and the cells were washed twice with 1.0 mL / well of PBS. 1.0 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time PCR. -△△CT Results were calculated using the method described above. Results are expressed as mean ± SD. The sample group and the BC group were compared using t-test statistical analysis. All statistical analyses were two-tailed. A p-value < 0.05 was considered statistically significant. A p-value < 0.01 is considered highly significant and is expressed as follows: .

[0090] Upregulation rate (%) = (gene content sample group - gene content BC) / gene content BC × 100%.

[0091] The results are shown in Table 4.

[0092] Table 4: Results of LOR gene expression detection

[0093]

[0094] The experimental results show that, compared with the blank group, the expression level of LOR gene in Examples 3-7 was significantly upregulated, with upregulation rates of 51.00%, 87.00%, 95.00%, 99.00%, and 135.00%, respectively. This indicates that the cyclic dipeptide has a promoting effect on LOR expression in keratinocytes within the range of 2.5ppm-40ppm, suggesting that the cyclic dipeptide has the function of improving skin barrier and repair.

[0095] Promote IVL expression

[0096] Changes in IVL gene in keratinocytes were detected by qPCR.

[0097] This test, based on keratinocytes, examined changes in IVL gene expression levels after treatment with Examples 3-7. A blank control group (BC) was set up, and Examples 3-7 served as the sample groups.

[0098] Press 2.0 × 10 5 Cells were seeded at a density of [number] cells / well into 6-well plates and incubated overnight in an incubator (37 ℃, 5% CO2). When the cell deposition rate in the 6-well plates reached 40%–60%, cells were divided into groups and treated with 2.0 mL of drug per well, with 3 replicates per group, and incubated for 24 h in an incubator (37 ℃, 5% CO2). The supernatant was then discarded, and the cells were washed twice with 1.0 mL / well of PBS. 1.0 mL of RNAiso Plus was added to each well, and the cells were lysed by pipetting. RNA was extracted, reverse transcribed into cDNA, and then detected by real-time PCR. -△△CT Results were calculated using the method described above. Results are expressed as mean ± SD. The sample group and the BC group were compared using t-test statistical analysis. All statistical analyses were two-tailed. A p-value < 0.05 was considered statistically significant. A p-value < 0.01 is considered highly significant and is expressed as follows: .

[0099] Upregulation rate (%) = (gene content sample group - gene content BC) / gene content BC × 100%.

[0100] The results are shown in Table 5.

[0101] Table 5: IVL gene expression detection results

[0102]

[0103] The experimental results show that, compared with the control group, the IVL gene expression level in Example 3 was significantly upregulated, with an upregulation rate of 50.00%, indicating that the cyclic dipeptide at a concentration of 40 ppm promotes IVL expression in keratinocytes. In contrast, the IVL gene expression levels in Examples 4-7 were comparable to those in the control group, indicating that the cyclic dipeptide had no effect on IVL gene expression at concentrations of 2.5 ppm to 20 ppm. These results demonstrate that the cyclic dipeptide has the function of improving the skin barrier and repairing the skin.

[0104] Test Example 2: Improving the skin barrier—Promoting FLG and LOR expression on a 3D skin model

[0105] This test, based on a 3D epidermal model, examined the changes in FLG and LOR gene expression levels after treatment with Examples 8-9. A blank control group (BC), a model group (NC), and Examples 8-9 were used as the sample group.

[0106] 1. Experimental materials, reagents and main equipment

[0107] EpiKutis 3D epidermal skin model, PBS, DMSO (Sigma), culture medium (Gibco), PolyI:C (Sigma), lipopolysaccharide (E. coli. Sigma), RNAiso Plus (TaKaRa), reverse transcription kit (TaKaRa).

[0108] CO2 incubator (Thermo), ultra-clean workbench (Sujing Antai), inverted microscope (Olympus), high-speed refrigerated centrifuge (Changsha Xiangyi), real-time fluorescence quantitative PCR instrument (BIO-RAD), digital display constant temperature water bath (Changzhou Guohua).

[0109] 2. Experimental Methods:

[0110] PolyI:C+LPS induction working solution: Take 1.44 mL of 5 mg / mL PolyI:C stock solution and 3 mL of 2 mg / mL LPS stock solution, dissolve them in the culture medium, with a total volume of 300 mL, so that the final concentration of PolyI:C is 24 μg / mL and the final concentration of LPS is 20 μg / mL.

[0111] According to the grouping, the skin models were transferred to 6-well plates (small molds were added in advance, along with 3.7 mL of culture medium), and the test group numbers were labeled on the 6-well plates. Culture medium was added to the BC and NC groups, while the sample group had the samples from Examples 8-9 evenly distributed on the model surface. The plates were then incubated in a CO2 incubator (37℃, 5% CO2) for 24 h. After drug administration and incubation, the 6-well plates were removed from the incubator, the skin model surface was cleaned with sterile PBS, and any residual PBS was wiped off with sterile cotton swabs. Polyl:C and LPS induction working solution were added to the NC and sample groups, and culture medium was added to the BC group. After completion, the 6-well plates were incubated in a CO2 incubator (37℃, 5% CO2) for 24 h. The model culture medium was collected in centrifuge tubes. After sample collection, RNA extraction, reverse transcription, and quantitative real-time PCR were performed according to the kit instructions. -△△CT Results were calculated using the following methods. t-tests were used for statistical analysis between groups; p < 0.05 indicated a significant difference, and p < 0.01 indicated an extremely significant difference.

[0112] Table 6: FLG gene detection results of 3D skin model

[0113]

[0114] According to the results in Table 6, after model induction, FLG expression decreased compared to the normal group, while FLG expression significantly increased after sample administration. The results showed that the cyclic (L-leucine-L-proline) dipeptide in Example 8 and the cyclic (D-leucine-L-proline) dipeptide in Example 9, at a concentration of 4 ppm, significantly enhanced the expression of the barrier factor FLG gene, with enhancement rates of 49.21% and 71.43%, respectively. This indicates that both the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide can improve skin barrier and repair functions by promoting FLG expression.

[0115] Table 7: LOR gene detection results of 3D skin models

[0116]

[0117] According to the results in Table 7, after model induction, the expression level of LOR decreased compared to the normal group, while the expression level of LOR significantly increased after sample administration. The results showed that the cyclic (L-leucine-L-proline) dipeptide in Example 8 and the cyclic (D-leucine-L-proline) dipeptide in Example 9, at a concentration of 4 ppm, significantly enhanced the expression of the barrier factor LOR gene, with enhancement rates of 98.46% and 87.69%, respectively. This indicates that both the cyclic (L-leucine-L-proline) dipeptide and the cyclic (D-leucine-L-proline) dipeptide can improve skin barrier and repair functions by promoting LOR expression.

[0118] Application examples

[0119] Cyclic (L-leucine-L-proline) dipeptide and cyclic (D-leucine-L-proline) dipeptide can be used in the preparation of topical skin agents.

[0120] The topical skin agent is preferably a cosmetic composition, such as a face cream, lotion, gel, toner, serum, mask, eye cream, aerosol (cleansing foam), spray, shower gel, facial cleanser, etc. The weight percentage of the cyclic (L-leucine-L-proline) dipeptide and cyclic (D-leucine-L-proline) dipeptide in the topical skin agent is 0.0001%-20% (w / w). A preferred weight percentage is 0.1%-10% (w / w). A more preferred weight percentage is 0.01%-5% (w / w). The most preferred weight percentage is 0.1%-5% (w / w).

[0121] The following are specific application examples of cyclic (L-leucine-L-proline) dipeptides and 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.

[0122] Application Example 1: Preparation of Face Cream

[0123]

[0124] Application Example 2: Emulsion Preparation

[0125]

[0126] Application Example 3: Preparation of Gel

[0127]

[0128] Application Example 4: Preparation of Toner

[0129]

[0130] Application Example 5: Preparation of Serum

[0131]

[0132] Application Example 6: Preparation of Facial Masks

[0133]

[0134] Application Example 7: Preparation of Eye Cream

[0135]

[0136] Application Example 8: Preparation of Spray

[0137]

[0138] Application Example 9: Preparation of Shower Gel

[0139]

[0140] Application Example 10: Preparation of Facial Cleanser

[0141]

Claims

1. The application of cyclic dipeptides for non-therapeutic purposes to improve barrier hydration and repair, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide or combinations thereof.

2. The application as described in claim 1, wherein, The improved barrier hydration and repair is achieved by promoting the expression of skin barrier-related factors FLG, AQP3, LOR and / or IVL.

3. The application as described in claim 1, wherein, The cyclic dipeptide is a cyclic (L-leucine-L-proline) dipeptide.

4. The application as described in claim 1, wherein, The cyclic dipeptide is a cyclic (D-leucine-L-proline) dipeptide.

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

6. The application as described in claim 5, characterized in that, The concentration of the cyclic dipeptide used is 10 ppm-40 ppm.

7. The application as described in claim 5, characterized in that, The concentration of the cyclic dipeptide used is 2.5 ppm-20 ppm.

8. The application as described in claim 1, characterized in that, The concentration of the cyclic dipeptide used is 4 ppm.

9. The use of cyclic dipeptides in the preparation of topical skin agents with barrier-improving moisturizing and repairing effects, wherein the cyclic dipeptide is selected from: cyclic (L-leucine-L-proline) dipeptide, cyclic (D-leucine-L-proline) dipeptide, or combinations thereof.

10. The application as described in claim 9, wherein 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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