Anti-wrinkle combined cyclic peptide and application thereof in cosmetics

Abstract

The invention provides an anti-wrinkle combined cyclic peptide and application thereof in cosmetics, the anti-wrinkle combined cyclic peptide comprises cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9, the combined cyclic peptide has a remarkable synergistic effect in the aspects of promoting expression of collagen genes Collagen I, Collagen III and Collagen IV and inhibiting acetylcholine secretion, the skin elasticity and glossiness are remarkably improved, and the anti-wrinkle combined cyclic peptide has a good anti-wrinkle effect. The generation of wrinkles is inhibited, and good anti-aging and wrinkle-removing effects are achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of cosmetics, and in particular relates to an anti-wrinkle combination cyclic peptide and application thereof in cosmetics. Background Art

[0002] With the advancement of modern science and technology and the improvement of people's living standards, the demand for anti-aging skincare products is increasing. Peptides, a class of small molecule proteins with significant biological activity, have demonstrated remarkable performance in delaying skin aging, improving wrinkles, and enhancing skin texture, and have gradually become a key ingredient in the cosmetics industry. Anti-wrinkle active peptides enhance skin elasticity and firmness by stimulating collagen and elastin synthesis; mitigate skin damage caused by free radicals and inflammation through antioxidant and anti-inflammatory effects; and promote cell regeneration and repair by regulating cytokine secretion.

[0003] However, linear peptides often have poor transdermal permeability and low stability, necessitating physical or chemical modification to improve metabolic stability. Cyclization of linear peptides via amide and ester bonds to produce cyclic active peptides significantly improves target affinity, metabolic stability, specificity, cell penetration, and molecular diversity. These advantages give cyclic peptides broad application prospects in drug development and skincare technology.

[0004] Classic anti-wrinkle peptides known on the market, such as pentapeptide-4 / palmitoyl pentapeptide-4, are signaling peptides that stimulate fibroblasts by sending signals, promoting the production of collagen fibers, elastic fibers, and glycosaminoglycans in the skin, thereby reducing wrinkles and improving skin firmness and smoothness. Tripeptide-5 / palmitoyl tripeptide-5 promotes collagen synthesis by activating growth factor (TGF-β). It can also inhibit the activity of matrix metalloproteinases and the production of inflammatory factors, thereby preventing collagen breakdown. This dual mechanism of action makes tripeptide-5 and palmitoyl tripeptide-5 exhibit significant anti-wrinkle effects in anti-aging skincare products. Palmitoyl is a lipophilic group, and peptides modified with palmitoyl can improve skin permeability, bioactivity, and economic feasibility. Hexapeptide-9 is a collagen peptide that improves epidermal aging by promoting the expression of tight junction proteins and regulating cell growth and differentiation; it improves inflammatory aging by downregulating the expression of inflammatory factors and regulating cellular energy metabolism; it activates collagen stem cells to promote the expression of various collagens, effectively improving the problem of aging caused by the loss of skin collagen. The above-mentioned linear peptides are cyclized head to tail through amide bonds, which greatly enhances the molecular stability, increases skin permeability, and better matches the target, thereby obtaining cyclic peptides with anti-wrinkle functions, such as cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9. This will further enhance their efficacy and utilization rate, and have broad application prospects in the field of beauty and skin care. However, there are currently few reports on the combination of the above-mentioned cyclic peptides for anti-wrinkle and firming.

[0005] Therefore, providing a cyclic peptide composition that can significantly improve skin elasticity and inhibit the formation of skin wrinkles is of great significance to the current fields of skin care products, cosmetics, etc. Summary of the Invention

[0006] The purpose of the present invention is to provide an anti-wrinkle combination cyclic peptide with high permeability, high stability and excellent collagen-promoting effect, and can significantly inhibit the release of the neurotransmitter acetylcholine, reduce the generation of dynamic lines, and thus have a better anti-aging and wrinkle-removing effect on the skin.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In one aspect, the present invention provides an anti-wrinkle combination cyclic peptide, comprising any one or more cyclic peptides of cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0009] Pentapeptide-4, tripeptide-5 and hexapeptide-9 are classic anti-wrinkle peptides known on the market, which have the effects of increasing collagen content and inhibiting the production of inflammatory factors. Cyclic peptides with anti-wrinkle function are prepared by cyclizing linear peptides, and their functionality and utilization rate will be further improved. Therefore, the present invention combines these three polypeptides after cyclization to obtain a combined cyclic peptide, and conducts a comparative study on the effects of different cyclic peptide compositions. It is verified that the combined cyclic peptide composed of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 has a significant synergistic effect in anti-wrinkle and firming.

[0010] Furthermore, the structural formula of the cyclic pentapeptide-4 is shown in formula (1), the structural formula of the cyclic tripeptide-5 is shown in formula (2), and the structural formula of the cyclic hexapeptide-9 is shown in formula (3):

[0011]

[0012]

[0013] In some embodiments, the present invention has been demonstrated through in vitro tests that a combination of cyclic peptides consisting of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 has a significant synergistic effect in promoting the expression of collagen genes I, III, and IV, inhibiting acetylcholine secretion, improving skin elasticity and reducing skin wrinkles. Compared with a combination of one or two cyclic peptides or other cyclic peptide compositions, the effect of the composition consisting of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 is more significant.

[0014] Furthermore, the anti-wrinkle combination cyclic peptide comprises, by weight percentage, 0.05-1% of cyclic pentapeptide-4, 0.05-1% of cyclic tripeptide-5 and 0.05-2% of cyclic hexapeptide-9.

[0015] In some embodiments, the present invention conducts experiments on a composition of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 within a certain proportion range. Studies have shown that a composition consisting of 0.05-1% cyclic pentapeptide-4, 0.05-1% cyclic tripeptide-5 and 0.05-2% cyclic hexapeptide-9 can achieve satisfactory results in promoting the expression of Collagen I, Collagen III, and Collagen IV, inhibiting acetylcholine secretion, improving skin elasticity and inhibiting the formation of wrinkles, reflecting a good synergistic effect.

[0016] In another aspect, the present invention provides an anti-aging and anti-wrinkle product comprising the anti-wrinkle combination cyclic peptide described above.

[0017] Within the scope that does not impair the effects of the present invention, various ingredients commonly used in the field of cosmetics and quasi-drugs can be appropriately compounded as needed, such as powder components, moisturizers, emulsifiers, thickeners, metal ion masking agents, pigments, pH regulators, skin nutrients, vitamins, preservatives, antioxidants, antioxidant adjuvants, fragrances, etc.

[0018] In addition, the polypeptide composition provided by the present invention can also be prepared in the form of a lyophilized powder, which can greatly improve the stability of the polypeptide during storage and use, and still maintain a high degree of activity during use, thereby exerting its maximum efficacy.

[0019] In another aspect, the present invention provides a method for preparing the anti-wrinkle combination cyclic peptide as described above.

[0020] In some embodiments, the preparation method of the cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9 includes first synthesizing a linear polypeptide by amide condensation or solid phase synthesis, and then cyclizing the linear peptide head-to-tail by an amide bond cyclization reaction to obtain a cyclic peptide.

[0021] In some embodiments, the linear polypeptides can be synthesized using solid-phase synthesis, liquid-phase synthesis, solid-phase and liquid-phase combined methods, carboxylactone methods, combinatorial chemistry, or natural coupling methods. They can also be prepared using biosynthetic methods aimed at producing the desired sequence, including but not limited to enzymatic hydrolysis, genetic engineering, fermentation, and enzyme-catalyzed methods. Methods for cyclizing linear polypeptides can also include but are not limited to disulfide bond formation or chemoselective ligation-mediated peptide cyclization reactions. In addition to synthesizing linear polypeptides and then cyclizing them, cyclic peptides can also be prepared by synthesizing two or more short linear peptides and then linking these linear peptides via amide bonds to form a cyclic peptide.

[0022] In some embodiments, in addition to synthesizing the cyclic peptide shown in the structural formula, its stereoisomers, or a mixture of its stereoisomers, or a salt thereof can also be synthesized.

[0023] In another aspect, the present invention provides a composition for preparing an agent for increasing skin collagen content, wherein the composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0024] Furthermore, the composition includes cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0025] Furthermore, the composition increases skin collagen content by promoting the expression of collagen I, collagen III and collagen IV genes.

[0026] In another aspect, the present invention provides a composition for preparing an agent for inhibiting acetylcholine secretion, wherein the composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0027] Furthermore, the composition includes cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0028] In another aspect, the present invention provides a composition for preparing an agent for improving skin elasticity and glossiness, wherein the composition comprises any one or more cyclic peptides selected from cyclopentapeptide-4, cyclotripeptide-5, and cyclohexapeptide-9.

[0029] Furthermore, the composition includes cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0030] In another aspect, the present invention provides a composition for preparing an agent for inhibiting the generation of dynamic skin lines, wherein the composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

[0031] The present invention has the following beneficial effects:

[0032] 1. The present invention provides a combination cyclic peptide composed of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9. The combination cyclic peptide has a significant synergistic effect in promoting the expression of collagen genes Collagen I, Collagen III and Collagen IV, providing a theoretical basis for the production of collagen in the skin. Human experiments have shown that the combination cyclic peptide significantly improves skin elasticity and glossiness.

[0033] 2. The combined cyclic peptides provided by the present invention, consisting of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9, have a significant synergistic effect in inhibiting the secretion of acetylcholine. Human experiments have shown that the combined cyclic peptides significantly reduce the volume and area of ​​skin wrinkles and inhibit the formation of dynamic lines, thereby being able to have a better anti-aging and wrinkle-removing effect on the skin.

[0034] 3. The combined cyclic peptide components provided by the present invention are green and safe, have low cytotoxicity, and have excellent effects, thus meeting the needs of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a cell viability curve of cyclic pentapeptide-4 on fibroblasts and neuronal cells at different concentrations.

[0036] Figure 2 This is a cell viability curve diagram of cyclic tripeptide-5 on fibroblasts and neuronal cells at different concentrations.

[0037] Figure 3This is a cell viability curve of fibroblasts and neuronal cells at different concentrations of cyclohexapeptide-9.

[0038] Figure 4 It is a bar graph of Collagen I gene expression.

[0039] Figure 5 It is a bar graph of Collagen III gene expression.

[0040] Figure 6 This is a bar graph of Collagen IV gene expression.

[0041] Figure 7 A histogram showing the acetylcholine content in neuronal cells. DETAILED DESCRIPTION

[0042] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary embodiments of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0043] Unless otherwise specified, the raw materials used in the examples were purchased commercially.

[0044] Example 1: Preparation of cyclic peptide

[0045] The reagent information used in the preparation process of this example is shown in Table 1.

[0046] Table 1 Reagent information used

[0047]

[0048]

[0049] (1) Preparation of cyclopentapeptide-4

[0050] Step 1: Resin pretreatment

[0051] 1.71 g of Wang resin was placed in a solid-phase synthesis reactor, washed once with 30 mL of DMF, and dried; then swelled with 30 mL of DCM at room temperature, stirred with N2 for 30 min until the resin was fully swollen, and dried; then washed three times with 30 mL of DMF in sequence to remove impurities and residual solvents in the resin, and dried.

[0052] Step 2: Coupling of amino acids to resin

[0053] Place Fmoc-Ser-OH (4.91 g, 15 mmol) and HOBt (2.03 g, 15 mmol) in a 100 mL beaker, cool to 4°C, add 25 mL of DMF solution and 2.3 mL of DIC solution, and let stand to mix for 20 minutes. The mixed solution was added to the pre-treated resin in the solid-phase synthesizer and stirred for 1.5 hours to allow the amino acid and resin to couple. After the reaction, wash the resin three times with 50 mL of DMF solution each time to remove unreacted amino acid and other impurities.

[0054] Step 3: Removal of Fmoc protecting group

[0055] After washing, add 50 mL of a 20% Pip / DMF solution to the resin and stir for 30 minutes to remove the Fmoc protecting group. Filter to remove the deprotection solution, then wash five times with 50 mL of DMF to remove piperidine and other byproducts. Dried solvent was evaporated under reduced pressure and added back to the solid-phase synthesizer reaction tube.

[0056] Step 4: Subsequent amino acid coupling

[0057] Repeat steps 2 and 3, and add Fmoc-Lys(Boc)-OH (7.03 g, 15 mmol), Fmoc-Thr-OH (5.12 g, 15 mmol), Fmoc-Thr-OH (5.12 g, 15 mmol), and Boc-Lys(Fmoc)-OH (7.03 g, 15 mmol) in sequence according to the amino acid sequence of pentapeptide-4. After each coupling and deprotection reaction, the resin was thoroughly washed with DMF.

[0058] Step 5: Peptide chain cleavage

[0059] Add 200 mL of a cleavage solution (TFA:TIS:H2O) at a ratio of 90:5:5 to the reactor and shake at 30°C for 2 h to cleave the peptide from the resin. Filter the resulting filtrate, decompress the filtrate and evaporate the solvent to yield the fully protected linear peptide H-Lys(Boc)-Thr-Thr-Lys(Boc)-Ser-OH.

[0060] Step 6: Cyclization of linear peptide

[0061] The fully protected peptide was dissolved in 200 mL of dichloromethane, and 1.54 mL of DIC, 1.35 g of HOBt, and 1.74 mL of DIEA were added. The mixture was stirred at 30°C for 14 h to form the cyclic peptide Cyclo (Lys(Boc)-Thr-Thr-Lys(Boc)-Ser). DCM was removed by rotary evaporation under reduced pressure.

[0062] Step 7: Removal of protecting groups

[0063] The product from step 6 was dissolved in 70 mL of TFA:DCM (1:3) and allowed to react for 2.5 h. The reaction solution was added dropwise to 300 mL of anhydrous ether under an ice bath to precipitate the peptide. The peptide was centrifuged to obtain a crude white solid peptide. The precipitate was washed three times with 20 mL of cold anhydrous ether to remove residual TFA and other impurities. The crude white solid peptide was dried in a vacuum oven to obtain a crude peptide powder.

[0064] Step 8: Purification and identification

[0065] After purification by reverse phase HPLC preparative chromatography and freeze drying, the pure cyclopentapeptide-4 was obtained. The structural formula is shown in formula (1). The structure of the sample was confirmed by nuclear magnetic resonance spectroscopy. 1H NMR: δ1.17-1.85(17H, 1.23(d, J=6.2Hz), 1.23(d, J=6.2Hz), 1.27(ddddd, J=13.4, 4.5, 4.3, 3.3, 1.8Hz), 1.39(tt, J=7.4, 7.0Hz), 1.39(tt, J=7.4, 7.0Hz), 1.49(ddddd, J=12.7, 10.2, 9.8, 3.1, 1.8Hz), 1.51( tt,J=7.4,7.3Hz),1.51(tt,J=7.4,7.3Hz),1.60(ddddd,J=13.4,9.8,8.0,4.5,1.7Hz),1.63(ddddd,J =12.7,10.2,4.3,2.5,1.7Hz),1.75(dddd,J=13.0,9.7,8.0,4.5Hz),1.74(td,J=7.0,4.7Hz),1.74(td, J=7.0,4.7Hz)),1.95(1H,dddd,J=13.0,4.5,3.3,1.6Hz),2.57-2.69(2H,2.63(t,J=7.3Hz),2.63(t,J =7.3Hz)),3.14(1H,ddd,J=14.4,10.2,3.1Hz),3.33(1H,ddd,J=14.4,10.2,2.5Hz),3.59(1H,dd,J=9.7 ,1.6Hz),3.95-4.15(4H,4.00(d,J=4.6Hz),4.00(d,J=4.6Hz),4.08(qd,J=6.2,4.4Hz),4.08(qd,J=6.2 ,4.6Hz)),4.21-4.52(4H,4.27(d,J=4.4Hz),4.31(t,J=4.7Hz),4.39(d,J=4.6Hz),4.47(t,J=4.6Hz)).

[0066]

[0067] (2) Preparation of cyclotripeptide-5

[0068] The preparation process of cyclic tripeptide-5 is basically the same as that of the above-mentioned cyclic pentapeptide-4, except that all amino acids are replaced with the amino acid sequence of tripeptide-5, namely Fmoc-Lys(Boc)-OH (7.03 g, 15 mmol), Fmoc-Val-OH (5.09 g, 15 mmol), and Boc-Lys(Fmoc)-OH (7.03 g, 15 mmol). The structural formula is shown in formula (2). The structure of the sample was confirmed by nuclear magnetic resonance spectroscopy. 1H NMR: δ0.81-0.93(6H,0.87(d,J=6.9Hz),0.87(d,J=6.9Hz)), 1.32-2.00(12H,1.39(tt,J=7 .4,7.0Hz),1.39(tt,J=7.4,7.0Hz),1.51(tt,J=7.4,7.3Hz),1.51(tt,J=7.4,7.3Hz),1.62 (ddddd,J=9.3,5.9,4.8,3.1,2.3Hz),1.63(ddddd,J=9.3,8.0,7.7,4.8,2.3Hz),1.71(dddd d,J=13.3,7.7,5.7,2.3,1.4Hz),1.75(td,J=7.0,5.7Hz),1.75(td,J=7.0,5.7Hz),1.81(dd dddd,J=13.3,8.8,5.9,2.3,1.4Hz),1.81(dddd,J=13.3,4.8,3.1,2.5Hz),1.91(dddd,J=13. 3,10.2,8.0,4.8Hz)),2.08(1H,septd,J=6.9,5.3Hz),2.57-2.69(2H,2.63(t,J=7.3Hz),2. 63(t,J=7.3Hz)),3.13-3.42(2H,3.21(ddd,J=13.5,5.7,1.4Hz),3.34(ddd,J=13.5,8.8,1. 4Hz)), 3.58 (1H, dd, J = 10.2, 2.5Hz), 4.62-4.74 (2H, 4.67 (d, J = 5.3Hz), 4.68 (t, J = 5.7Hz)).

[0069]

[0070] (3) Preparation of cyclohexapeptide-9

[0071] The preparation process of cyclic hexapeptide-9 is basically the same as that of the above-mentioned cyclic pentapeptide-4, except that all amino acids are replaced by the amino acid sequence of hexapeptide-9, namely Fmoc-Gln(Trt)-OH (9.16 g, 15 mmol), Fmoc-Pro-OH (5.06 g, 15 mmol), Fmoc-Gly-OH (4.46 g, 15 mmol), Fmoc-Gln(Trt)-OH (9.16 g, 15 mmol), Fmoc-Pro-OH (5.06 g, 15 mmol), and Fmoc-Gly-OH (4.46 g, 15 mmol). The other reagents and preparation methods are basically the same. The structural formula is shown in formula (3). The structure of the sample was confirmed by nuclear magnetic resonance spectroscopy. 1H NMR: δ1.84-2.17(12H,1.93(dddd,J=13.1,4.3,4.0,1.6,1.5Hz), 1.93(dddd,J=13.7,6.6,4.2,1.5Hz), 2.02(dddd,J=13 .7,10.1,8.4,4.3Hz),2.01(dtdd,J=13.1,10.2,4.2,3.8Hz),2.10(dt,J=8.0,7.4Hz),2.10(dt,J=8.0,7.4Hz)),2.28-2.4 0(4H,2.34(t,J=7.4Hz),2.34(t,J=7.4Hz)),3.30-3.56(4H,3.37(ddd,J=15.8,3.8,1.6Hz),3.48(ddd,J=15.8,10.2,4.0H z)), 4.22-4.49(4H, 4.29(d,J=16.9Hz), 4.41(d,J=16.9Hz)), 4.54-4.68(4H, 4.60(t,J=8.0Hz), 4.61(dd,J=8.4,6.6Hz)).

[0072]

[0073] Example 2: Cytotoxicity test

[0074] In this example, the MTT method was used to investigate the safety of cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9 within a concentration range of 0.05% to 10%.

[0075] 1. Experimental methods:

[0076] (1) Cell inoculation: Fibroblasts or neuronal cells were plated at 8×10 3 The cells were seeded into a 96-well plate at a density of 1000 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0077] (2) Experimental Grouping: The experiment set up a zero adjustment group, a solvent control group, a positive control group, and a sample group. The sample group was set up with 8 concentration gradients, and 3 replicate wells were set up under each concentration gradient. The test concentration settings of the sample group are shown in Table 2.

[0078] Table 2 Sample group concentration gradient setting table

[0079]

[0080] (3) Dosing: Dosing was performed when the cell plating rate in the 96-well plate reached 40% to 60%. For the solvent control group, 200 μL of culture medium was added to each well; for the positive control group, 200 μL of culture medium containing 10% DMSO was added to each well; for the sample group, 200 μL of culture medium containing the corresponding concentration of sample was added to each well; for the zero adjustment group, no cells were inoculated, and only 200 μL of cell culture medium was added. After dosing, the 96-well plate was placed in an incubator (37°C, 5% CO2) and cultured for 24 hours.

[0081] (4) Detection: After the cells were incubated for 24 h, the supernatant was discarded and MTT working solution (0.5 mg / mL) was added. The cells were incubated at 37°C in the dark for 4 h. After the incubation, the supernatant was discarded and 150 μL DMSO was added to each well. The OD value was read at 490 nm.

[0082] (5) Calculation of relative cell viability: Calculated according to the formula, relative cell viability (%) = (OD of sample well - OD of zero adjustment well) / (OD of solvent control well - OD of zero adjustment well) × 100%. The MTT test results are as follows Figures 1 to 3 shown.

[0083] 2. Experimental Results

[0084] according to Figure 1 The results showed that within the concentration range of 1%, the survival rates of fibroblasts and neuronal cells were greater than 90%, and no obvious toxicity was shown. Figure 2 The results showed that the sample cyclic tripeptide-5 showed no obvious toxicity to fibroblasts and neuronal cells within the concentration range of 1%. Figure 3 As a result, when the concentration of the sample cyclohexapeptide-9 reached 2%, the fibroblasts and neurons still maintained a high viability. After the sample concentration continued to increase, the viability of the two cells dropped below 90%. Therefore, it is believed that the sample cyclohexapeptide-9 did not show obvious cytotoxicity based on fibroblasts and neurons within the concentration range of 2%.

[0085] Taking into account cytotoxicity, solubility and economic factors, 0.5% cyclopentapeptide-4, 0.5% cyclotripeptide-5 and 1% cyclohexapeptide-9 were subsequently selected to explore the efficacy of the cyclic peptide composition.

[0086] Example 3: Comparison of anti-wrinkle efficacy of cyclic peptides and linear peptides in vitro

[0087] Collagen, primarily produced by fibroblasts in the dermis, is a crucial component of the skin's support. Type I collagen (Collagen I) accounts for approximately 80% of the dermis, contributing to the skin's plumpness and volume. Increasing Collagen I content can help reduce wrinkles. Therefore, by treating fibroblasts with the aforementioned linear peptides and cyclized cyclic peptides and measuring changes in Collagen I, we can assess the effectiveness of the test substance in promoting collagen expression, thereby achieving anti-wrinkle efficacy.

[0088] 1. Experimental Methods

[0089] (1) Cell seeding: 4×10 4 Fibroblasts were seeded into 24-well plates at a seeding density of 100 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0090] (2) Liquid preparation: Prepare the working solution of the test substance according to the test group (Table 3).

[0091] Table 3 Comparative test scheme of anti-wrinkle efficacy of cyclic peptides and linear peptides in vitro

[0092]

[0093] (3) Administration: According to the test protocol in Table 3, when the cell plating rate in the 24-well plate reaches 40% to 60%, group administration is carried out, with three replicate wells per group, and incubated in an incubator (37°C, 5% CO2) for 24 hours. Note: The linear peptides in this example were purchased from Nanjing Source Peptide Biotechnology Co., Ltd.

[0094] (4) UVA irradiation: According to the test group, the group that needs to be irradiated is irradiated with UVA, and the irradiation dose is 30J / cm 2 After irradiation, the cells were placed in an incubator and cultured for 24 hours.

[0095] (5) Sample collection: After the incubation, the cells were fixed with 4% paraformaldehyde for subsequent immunofluorescence testing.

[0096] (6) Immunofluorescence test: After 24 hours of fixation, perform Collagen I immunofluorescence detection, take pictures with a fluorescence microscope and use The data were analyzed using Sigma Plus image processing software. Statistical analysis was performed using the t-test method. Compared with the BC group, significance is indicated by #, and P-value < 0.01 is indicated by ##. Compared with the NC group, significance is indicated by *, and P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0097] 2. Experimental Results

[0098] The test results of each group are shown in Table 4.

[0099] Table 4 Summary of Collagen I Immunofluorescence Analysis Results

[0100]

[0101] Compared with the BC group, the Collagen I protein content in the NC group decreased significantly, indicating that the stimulation conditions of this test were effective. Compared with the NC group, the Collagen I protein content in the PC group increased significantly, indicating that the positive control of this test was effective.

[0102] This example evaluates the anti-wrinkle efficacy of three cyclic peptides (cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9) and their linear forms (pentapeptide-4, tripeptide-5 and hexapeptide-9). As can be seen from Table 4, compared with the linear peptides (the improvement rate is between 59% and 73%), the cyclized cyclic peptides (the improvement rate is between 90% and 98%) have a significant improvement in increasing the expression of collagen I. This shows that the cyclization modification of pentapeptide-4, tripeptide-5 and hexapeptide-9 is meaningful and has significant advantages. It may be that the structure of the cyclic polypeptide increases the rigidity and stability of the active molecule; it may also be due to reasons such as a better match with the target site and stronger targeting. Overall, cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 can exert anti-wrinkle effects by significantly increasing the expression of type I collagen.

[0103] Example 4: Comparison of in vitro permeability between cyclic peptides and linear peptides

[0104] In the development of anti-aging cosmetics, the core significance of substance permeability research lies in overcoming the limitations of the skin barrier to achieve effective anti-aging effects. The dense lipid structure and molecular weight selection properties of the skin's stratum corneum make it difficult for most anti-aging ingredients (such as large peptides and hydrophilic antioxidants) to penetrate their target sites in the dermis. Dermal fibroblast aging and collagen degradation are key factors in wrinkle formation and skin sagging. Therefore, improving the permeability of active ingredients is a major research strategy for developing anti-aging and wrinkle-reducing cosmetics.

[0105] 1. Experimental Methods

[0106] This example analyzes the in vitro transdermal absorption of free amino-containing polypeptides using a Franz diffusion cell. Using an artificial membrane as the research model, HPLC quantitative detection was used to measure the target content in the receiving fluid to calculate the cumulative permeation volume, permeation rate, and diffusion percentage. The transdermal absorption of the test sample was quantified to analyze the in vitro transdermal absorption of the polypeptides.

[0107] Test system: artificial membrane; Materials and equipment: TK-12D Franz diffusion cell for transdermal drug absorption, constant temperature magnetic stirrer. Experimental test groups are shown in Table 5.

[0108] Table 5 Comparative test scheme of in vitro permeability of cyclic peptides and linear peptides

[0109]

[0110] (1) The artificial membrane was fixed between the donor chamber and the receiving chamber of the Franz diffusion cell, with the stratum corneum facing the donor chamber and the dermis facing the receiving chamber.

[0111] (2) Add 7.0 mL of receiving solution to the receiving chamber. After tightening the artificial membrane, add 1.0 mL of receiving solution (PBS) to the receiving chamber through the sampler. Expel all air to ensure close contact between the dermis and the receiving solution.

[0112] (3) Add the sample to the surface of the artificial membrane in the supply chamber. The effective permeation area S is about 1.77 cm 2 The sample was added to the surface of the artificial membrane and evenly spread from the center of the artificial membrane to the edge. Each sample was repeated three times in parallel (three replicates were performed using skin from specific parts of independent donors).

[0113] (4) Infiltration: Turn on the electromagnetic stirrer and stir at a speed of 300 rpm, maintain a constant temperature water bath of (32±1)℃, and ensure that there are no bubbles in the water bath interlayer.

[0114] (5) Take the sample solution at 2 h, 8 h, and 24 h respectively, use the sampler to extract 2.0 mL of receiving solution through the sampling tube, and then place it in a 2 mL EP tube. In addition, add an equal amount of receiving solution after each sampling.

[0115] (6)① The calculation formula for the cumulative permeation volume Q is as follows: Q = [Cn × V + ΣCi × V0] / S (i = 1…n-1); where: Q represents the cumulative permeation volume; Cn represents the sample concentration measured at the nth sampling point; V represents the volume of the receiving liquid in the receiving chamber; Ci represents the sample concentration in the receiving liquid from the 1st to the n-1th sampling point; V0 represents the volume of each sampling; and S represents the effective diffusion area.

[0116] ②The diffusion percentage P is calculated as follows: P = Q / P0 × 100%;

[0117] Where: P represents the diffusion percentage; Q represents the cumulative permeation volume in the receiving chamber at each time point; P0 represents the initial sample load in the donor chamber.

[0118] 2. Experimental Results

[0119] The transdermal permeability test results of the samples at different times are shown in Table 6.

[0120] Table 6 Comparison of in vitro permeability of cyclic peptides and linear peptides

[0121]

[0122] As can be seen from Table 6, the diffusion degree of cyclic peptides is higher than that of linear peptides at each time point. The penetration effect of cyclic pentapeptide-4 after 24 hours is 2.47 times that of the original linear pentapeptide-4, the penetration effect of cyclic tripeptide-5 after 24 hours is 2.37 times that of the original linear tripeptide-5, and the penetration effect of cyclic hexapeptide-9 after 24 hours is 3.12 times that of the original linear hexapeptide-9, which proves that cyclization is beneficial and can achieve the effect of enhancing transdermal absorption, thereby improving its efficacy.

[0123] Example 5: In vitro anti-wrinkle efficacy test of combined cyclic peptides

[0124] In order to further verify that the composition composed of the above three cyclic peptides can significantly promote collagen expression, the present invention further explores the difference in the effects of the combined cyclic peptides. Type I collagen (Collagen I) accounts for about 80% of the weight of the dermis of the skin, making the skin plump and full, and promoting the increase in the content of Collagen I can achieve a certain effect of resisting wrinkles. Type III collagen (Collagen III) is also a major component of the dermal extracellular matrix, accounting for 10% of dermal collagen. Collagen III and collagen I are assembled into collagen fibers, which play an important role in the toughness of the skin. Type IV collagen (Collagen IV) is one of the main components of the hemidesmosome basement membrane complex and is a key protein at the junction of the true epidermis. Therefore, by treating fibroblasts with the above cyclic peptides and detecting the changes in Collagen I, III, and IV, the effect of the test substance in promoting collagen expression can be evaluated, thereby achieving anti-wrinkle efficacy.

[0125] 1. Experimental Methods

[0126] (1) Cell seeding: Fibroblasts were plated at 2×10 5 The cells were seeded into 6-well plates at a density of 1000 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0127] (2) Experimental Grouping: A blank control group and a sample group were set up. Three replicate wells were set up for each concentration gradient in the sample group. The experimental design is shown in Table 7.

[0128] Table 7 In vitro anti-wrinkle efficacy test scheme of combined cyclic peptides

[0129]

[0130] (3) Drug administration: According to the test scheme in Table 7, when the cell plating rate in the 6-well plate reaches 40% to 60%, group drug administration is carried out, with a dosage of 2 mL per well, 3 replicate wells per group, and incubation in an incubator (37°C, 5% CO2) for 24 h.

[0131] (4) Gene detection: After 24 hours of culture, discard the supernatant, wash twice with 1 mL / well PBS, add 1 mL RNAisoPlus to each well, blow and lyse the cells, and collect the samples. Extract RNA, reverse transcribe to cDNA, and perform fluorescence quantitative PCR detection. Method to calculate the results.

[0132] (5) Data Analysis: GraphPad Prism was used for graphing, and the results are expressed as mean ± SD. Statistical analysis was performed using the t-test for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 is indicated by * and considered to be significantly different; P < 0.01 is indicated by **, and P < 0.001 is indicated by *** and considered to be extremely significantly different.

[0133] 2. Experimental Results

[0134] The test results of each group are shown in Table 8. Figures 4-6 shown.

[0135] Table 8 Results of promoting collagen gene expression in different sample groups

[0136]

[0137]

[0138] This example evaluated the effects of three cyclic peptides and any combination thereof in promoting collagen expression and thus improving anti-wrinkle effects. The results in Table 4 show that when cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9 were used alone to treat fibroblasts, the amplification factor in promoting the expression of Collagen I, Collagen III, and Collagen IV genes was approximately 1.1 to 1.4, which is difficult to achieve a satisfactory collagen-promoting effect.

[0139] Comparing sample groups D, E, F, and G, after compounding the three cyclic peptide combinations of cyclopentapeptide-4, cyclotripeptide-5, and cyclohexapeptide-9 to treat fibroblasts, it can be found that when using any two cyclic peptide combinations, the degree of promoting the expression of Collagen I, Collagen III, and Collagen IV genes is better than that of single cyclic peptides, indicating that the cyclic peptide compositions designed by the present invention have played a good compounding synergistic effect. In addition, when 0.5% cyclopentapeptide-4+0.5% cyclotripeptide-5+1% cyclohexapeptide-9 (sample group G) was used to treat fibroblasts, the amplification factor for promoting the expression of the three collagen genes reached about 3.6 to 4.0 times, which was significantly different from other combinations. It can be seen that the cyclic peptide composition of sample group G has played a very significant synergistic effect in promoting collagen expression, significantly improving the expression of type I, type III, and type IV collagen, thereby helping the skin to maintain elasticity and firmness, reducing wrinkles and sagging. However, when 1% acetyl hexapeptide-8 was used in combination with 0.5% cyclopentapeptide-4, 0.5% cyclotripeptide-5, and 1% cyclohexapeptide-9 (sample group H), the amplification factor of promoting collagen gene expression was not as good as that of sample group G, indicating that adding 1% acetyl hexapeptide-8 to the three-cyclic peptide combination did not produce a more significant synergistic effect.

[0140] In addition, this example experiments were conducted on a composition of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 within a certain proportion range. The study showed that a composition of 0.05-1% cyclic pentapeptide-4, 0.05-1% cyclic tripeptide-5 and 0.05-2% cyclic hexapeptide-9 can achieve satisfactory effects in promoting the expression of Collagen I, Collagen III and Collagen IV, and can exhibit a good synergistic effect. However, the best effect was achieved with a composition of 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9.

[0141] Example 6: In vitro anti-skin dynamic wrinkle test of combined cyclic peptides

[0142] This example is based on neuronal cells to detect the inhibitory effect of compounds on acetylcholine release. Using neuronal cells as a research model, after treatment, the inhibitory effect of the test substance on expression lines is evaluated by detecting the changes in acetylcholine content. Acetylcholine is a neurotransmitter. In nerve cells, acetylcholine is synthesized by choline and acetyl-CoA under the catalysis of choline acetyltransferase (choline acetylase); the release of acetylcholine is caused by the depolarization of nerve endings. 2+ The influx of acetylcholine into the skin induces the secretion of acetylcholine, which then acts on muscle cells, causing them to contract. Therefore, inhibiting acetylcholine secretion inhibits muscle contraction, thereby smoothing wrinkles.

[0143] 1. Experimental Methods

[0144] (1) Cell inoculation: Neuronal cells were plated at a rate of 2×10 5 The cells were seeded into 6-well plates at a density of 1000 cells / well and incubated overnight in an incubator (37° C., 5% CO 2 ).

[0145] (2) Experimental Grouping: The blank control group and sample group were the same as those in Table 2 of Example 3. Three replicate wells were set for each concentration gradient in the sample group. The acetylcholine content of the neurons in the corresponding groups was detected by ELSA.

[0146] (3) Dosing: According to the test protocol, when the cell plating rate in the 6-well plate reaches 40% to 60%, group dosing is carried out, with a dosing volume of 2 mL per well. Each group has 3 replicate wells and is incubated in an incubator (37°C, 5% CO2) for 24 h.

[0147] (4) Kit detection: Collect the cell culture supernatant and perform ELISA to detect the acetylcholine content according to the instructions of the detection kit.

[0148] (5) Data Analysis: GraphPad Prism was used for graphing, and the results are expressed as mean ± SD. Statistical analysis was performed using the t-test for comparisons between groups. All statistical analyses were two-tailed. P < 0.05 is indicated by *, indicating a significant difference; P < 0.01 is indicated by **, indicating an extremely significant difference.

[0149] 2. Experimental Results

[0150] The data of each group are shown in Table 9. Figure 7 shown.

[0151] Table 9 Results of different sample groups inhibiting acetylcholine release

[0152]

[0153]

[0154] As shown in Table 9, when cyclic pentapeptide-4 and cyclic tripeptide-5 were used alone to treat neuronal cells, they were unable to inhibit acetylcholine secretion. When 1% cyclic hexapeptide-9 was used alone, acetylcholine content decreased significantly to 459 μg / mL, with an inhibition rate of 20.9%. According to previous research results, the linear form of hexapeptide-9 did not inhibit acetylcholine secretion, indicating that cyclization is beneficial and can inhibit the release of the neurotransmitter acetylcholine, thereby inhibiting dynamic wrinkles. When three cyclic peptides were compounded, such as sample group G, 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9 were combined to treat neuronal cells. The acetylcholine content was 336 μg / mL, and the inhibition rate was 42.1%, which was significantly different from the blank control group and the single cyclic peptide. The inhibition rates of the combination of any two cyclic peptides (sample groups D, E, and F) on acetylcholine release were 35.9%, 34.5%, and 9.1%, respectively, which were significantly lower than those of sample group G, indicating that the combination composed of two cyclic peptides was not as effective as sample group G in inhibiting acetylcholine secretion. This further indicates that the combined cyclic peptide composed of 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9 has a significant synergistic effect on inhibiting acetylcholine secretion.

[0155] When 1% acetyl hexapeptide-8 was added to the combination of 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9 (sample group H), the inhibition rate was only 43.3%, which was the same as the effect of the three-cyclic peptide combination in sample group G, indicating that the addition of the classic anti-wrinkle peptide on the market (acetyl hexapeptide-8) did not increase the efficacy of inhibiting dynamic lines. The above results show that the combination of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 has a significant synergistic effect in inhibiting acetylcholine secretion, thereby inhibiting the formation of dynamic lines and achieving a good anti-wrinkle effect.

[0156] In addition, this example experiments on a combination of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 within a certain proportion range. The study shows that a combination of 0.05-1% cyclic pentapeptide-4, 0.05-1% cyclic tripeptide-5 and 0.05-2% cyclic hexapeptide-9 can exert a significant synergistic effect on inhibiting acetylcholine, but the best effect is the combination of 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9.

[0157] Example 7: Combination Cyclic Peptide Human Testing

[0158] In order to investigate the effects of the three cyclic peptides and their compositions provided by the present invention on the human body when used as skin care products, this example further recruited volunteers to conduct tests on the efficacy of the cyclic peptides on the human body.

[0159] 1. Experimental Methods

[0160] (1) Preparation of an anti-wrinkle and anti-aging essence: 4% 1,3-butylene glycol, 0.5% betaine, 0.02% sodium hyaluronate, 0.08% xanthan gum, 0.3% phenoxyethanol, and deionized water (added to 100%) were stirred and mixed to prepare a base essence. The base essence only serves as a solvent for the combined cyclic peptides and has no effect on improving or inhibiting the moisture, elasticity, gloss, etc. of human skin.

[0161] (2) Experimental grouping: The experimental sample groups were consistent with sample groups A to H in Table 3 of Example 3. The compounds of sample A to H were added to the basic essence respectively.

[0162] (3) Volunteer selection: The selection of subjects followed the medical and ethical standards for human testing. All subjects must be willing to participate in the test and sign an informed consent form before the test. 240 healthy female subjects aged 18 to 40 were recruited and randomly divided into 8 groups, with 30 subjects in each group.

[0163] (4) Method of using experimental samples: After cleansing their faces in the morning and evening, the two groups of subjects used the essences from Samples A to H for skin care, using 0.2g±0.1g (one to two pumps) of essence each time and gently massaging until absorbed. Other anti-wrinkle skin care products were discontinued during the experiment.

[0164] (5) Before using the sample and 14 and 28 days after using the sample, facial images of the subjects were collected using VISIA-CR. VC20 Plus was used to collect images of the subjects and analyze the cheek skin roughness SEr and skin smoothness SEsm. Glossymeter CL200 was used to measure skin gloss. Cutometer MPA580 was used to measure the skin elasticity parameter R2 of the test site. The average value of the three measurements was taken. SPSS analysis software was used to compare the measured values ​​at different time points with the baseline values ​​before using the sample. The Shapiro-Wilk Test was used to perform a significance test for the normal distribution of the data improvement value.

[0165] The control group selected the subjects themselves for before and after comparison.

[0166] Before the test, the subjects washed their faces with the same cleansing product and kept them in a stabilization room with constant temperature and humidity (temperature: 21°C ± 1°C; humidity: 50% ± 10%) for 20 minutes to keep their skin in a stable condition, and then the above-mentioned test was performed.

[0167] 2. Experimental Results

[0168] The change rate after using the product = (data after use - data before use) / data before use × 100%. The test results are shown in Table 10.

[0169] Table 10 Effects of different essences on skin elasticity

[0170]

[0171] According to the data in Table 10, it can be seen that after using an essence containing a cyclic peptide component or a combination of cyclic peptides, the skin elasticity and glossiness are improved to varying degrees, while the average volume and area of ​​cheek wrinkles are significantly reduced. Using a triple combination of cyclic peptides (sample group G), after 14 days, the skin elasticity value increased by 6.9%, the skin glossiness increased by 40.2%, the average volume of wrinkles decreased by 20.8%, and the average area of ​​wrinkles decreased by 11.3%. The instant brightening and wrinkle removal effects are very significant; after 28 days of use, the anti-wrinkle effect is further improved, the skin elasticity value increased by 8.5%, the skin glossiness increased by 49.9%, the average volume of wrinkles decreased by 30.3%, and the average area of ​​wrinkles decreased by 15.6%. The anti-aging effect of the triple cyclic peptide is more significant than that of a single cyclic peptide or a combination of two cyclic peptides, and the three ingredients have a significant synergistic effect. However, when 1% of the classic anti-wrinkle peptide acetyl hexapeptide-8 was added to sample group G, the changes in various parameters were basically the same as those in sample group G, indicating that further adding acetyl hexapeptide-8 to the combined cyclic peptide composed of cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9 did not achieve a better improvement effect. The above results show that the combined cyclic peptide composed of 0.5% cyclic pentapeptide-4, 0.5% cyclic tripeptide-5 and 1% cyclic hexapeptide-9 provided by the present invention has a compound synergistic effect in reducing skin wrinkles, improving skin gloss, improving skin elasticity, etc., thereby making the skin look more vibrant and younger in appearance, with significant anti-aging and repair effects, and the combined cyclic peptide is preferably used to prepare skin care products, etc.

[0172] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An anti-wrinkle combination cyclic peptide, characterized in that: It includes any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

2. The anti-wrinkle combination cyclic peptide according to claim 1, wherein The structural formula of the cyclic pentapeptide-4 is shown in formula (1), the structural formula of the cyclic tripeptide-5 is shown in formula (2), and the structural formula of the cyclic hexapeptide-9 is shown in formula (3):

3. The anti-wrinkle combination cyclic peptide according to claim 2, wherein Calculated by weight, the invention comprises 0.05-1% of cyclopentapeptide-4, 0.05-1% of cyclotripeptide-5 and 0.05-2% of cyclohexapeptide-9.

4. An anti-aging and anti-wrinkle product, characterized in that: The invention comprises the anti-wrinkle combination cyclic peptide according to any one of claims 1 to 3.

5. A composition for preparing an agent for increasing skin collagen content, characterized in that: The composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

6. The use according to claim 5, characterized in that The composition increases skin collagen content by promoting the expression of collagen I, collagen III and collagen IV genes.

7. Use of a composition for preparing an agent for inhibiting acetylcholine secretion, characterized in that: The composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

8. A composition for preparing an agent for improving skin elasticity and glossiness, characterized in that: The composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

9. The use according to claim 8, characterized in that The composition includes cyclic pentapeptide-4, cyclic tripeptide-5 and cyclic hexapeptide-9.

10. A composition for preparing an agent for inhibiting the generation of dynamic skin lines, characterized in that: The composition comprises any one or more cyclic peptides selected from cyclic pentapeptide-4, cyclic tripeptide-5, and cyclic hexapeptide-9.

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