Cyclic peptide composition with anti-aging effect

Abstract

The invention provides a cyclic peptide composition with an anti-aging effect, and belongs to the field of medicines and cosmetics. The polypeptide components of the cyclic peptide composition are cyclic peptide, snake venom-like peptide and collagen peptide, the concentration of the polypeptide components is 0.00001 ppm to 20% (w / w), and the cyclic peptide composition comprises a polypeptide combination and a tripeptide combination in pairs. Compared with a single polypeptide group, the polypeptide composition can more effectively promote the generation of collagen in the in-vitro skin and inhibit the generation of neuronal acetylcholine, the effect of the combination of the cyclopeptide, the snake venom-like peptide and the collagen peptide is optimal, and the effect of the combination of the cyclopeptide, the snake venom-like peptide and the collagen peptide is the second order, so that the combination of the cyclopeptide, the snake venom-like peptide and the collagen peptide has a synergistic interaction effect. Therefore, the cyclopeptide composition can be added into various products such as essence, face cream, sunscreen cream, toning lotion, emulsion, eye cream, facial mask, facial cleanser, face cleaning honey, gel, spray and the like, so that the anti-wrinkle and tightening effects of the products are improved.

Description

Technical Field

[0001] The invention belongs to the field of medicine and cosmetics, and in particular relates to a cyclic peptide composition with anti-aging effect. Technical Background

[0002] Skin aging is an ongoing process associated with the depletion of the physiological functions of the skin. Over time, both natural and unnatural factors cause physiological changes in different organs of humans and animals. Skin aging is a multifactorial process that depends on intrinsic factors (genetics, hormones, and metabolism) and extrinsic factors (long-term exposure to ultraviolet rays, smoking, air pollution, chemicals, and malnutrition). Although multiple intrinsic processes affect the aging process, factors such as sun exposure, fluid intake, lifestyle, and pollution can exacerbate the aging process. In addition, due to the reduction of collagen, the thickness of the dermis decreases over time, and skin wrinkles occur [1]. Aging has an adverse effect on the connective tissue in the skin, resulting in a decrease in elastin and collagen fibers, resulting in fine lines and wrinkles; in addition, aging reduces the production of proteoglycans and glycosaminoglycans (such as hyaluronic acid) in the skin and cartilage, which can weaken the skin tissue and lose its integrity. The skin becomes dry and cannot retain enough water.

[0003] Collagen is an important scaffolding protein that makes the skin smooth and elastic, but its production decreases with age[2]. Collagen is considered essential for skin health because both photoaging and intrinsic aging reduce its presence in the body[1], which in turn leads to a decrease in skin thickness and loss of elasticity and flexibility. Therefore, boosting collagen is an important approach to skin anti-aging. Most collagen supplements are rich in peptides containing the amino acids proline, glycine, and hydroxyproline, which are considered essential components of collagen. Therefore, their presence in the body helps maintain the amount of collagen in the skin; in addition, it can also induce fibroblast migration and enhance collagen by increasing the production of hyaluronic acid peptides in skin fibroblasts, thereby improving the moisture of the stratum corneum.

[0004] Peptides and proteins are polymers of amino acids, and peptides are short chains of amino acids. Naturally occurring human peptides act as signaling molecules, such as regulating processes such as cell proliferation, cell migration, inflammation, angiogenesis, and melanogenesis; they also mediate various physiological processes, including defense, immunity, stress, growth, homeostasis, and reproduction. With the continuous understanding of natural peptides and the continuous maturity of synthetic peptide technology, different synthetic peptides have been developed: Glycine-histidine-lysine copper (Cu-GHK) was invented by Loren Pickard in 1973; in the late 1980s, the first copper peptide was added to skin care products; in the early 2000s, palmitoyl pentapeptide-4 was used; since then, research and industry have developed many short, stable, and synthetic peptides that can not only play a role in extracellular matrix synthesis, pigmentation, innate immunity, and inflammation, but can also be used to stimulate collagen production, wound healing, and "botulinum-like" wrinkle smoothing to achieve antioxidant, antibacterial, and whitening effects [3].

[0005] External cosmetic peptides can be divided into signal peptides, carrier peptides, neurotransmitter inhibitor peptides and enzyme inhibitor peptides. In recent years, the development and utilization of cyclic peptides in the cosmetic field has gradually become popular. According to the principle of action, they can be classified into signal peptides, enzyme inhibitor peptides, etc. [3]. Cyclic peptides (or cyclic peptides) belong to a type of polypeptide category. For example, the simplest cyclic peptide is a peptide main chain that is closed by the reaction of two terminal groups together to form a peptide bond; some cyclic peptides produce covalent bonds between their terminal amine and side chain or between the terminal carboxyl group and the side chain or between two side chains, thereby producing a cyclic structure inside the peptide molecule. Compared with conventional peptides, cyclic peptides have many advantages, such as they are insensitive to exopeptidases and are more stable than their linear counterparts; due to the bending of their backbone and the smaller degree of freedom, the rotation of many bonds is blocked, and they have side chains that are more available for interaction with external targets, the strength of interaction with their targets is increased, and adjacent amino acids do not interfere with molecular interactions. Compared with linear peptides, cyclic peptides have strong bioavailability and are widely used in the fields of medicine and biology. In recent years, scientists have confirmed that cyclic peptides have the characteristics of long half-life in the body, relatively stable structure and strong permeability, and external use products containing cyclic peptides can play a certain role in care and defense, and slow down aging. Therefore, the development of cyclic peptides for use in the field of beauty and skin care has certain innovation and practicality. In addition, as a unique skin care ingredient, snake venom peptides belonging to neurotransmitter inhibitor peptides have also attracted widespread attention in the beauty field in recent years. They are not directly extracted from snake venom, but simulate certain active ingredients in snake venom through scientific methods, so as to achieve anti-inflammatory, promote wound healing, improve blood circulation, and anti-wrinkle and anti-aging effects. Collagen peptides belong to signal peptides, which can usually play a variety of functions and effects such as anti-aging, wrinkle removal, and skin whitening, which is beneficial to improve facial skin problems. In addition to continuously developing new peptides with anti-aging effects, combining different types of cosmetics with available peptides, such as cyclic peptides, neurotransmitter inhibitor peptides, signal peptides, etc., is a strategy to solve skin anti-aging from different dimensions.

[0006] There are relevant patents and literatures that demonstrate the anti-aging ability of cyclic peptides, but there are few studies to test whether the combination of cyclic peptides, neurotransmitter inhibitor peptides and collagen peptides has better anti-aging ability. In other words, it is still unknown whether the combination of different types of peptides can achieve a synergistic effect. Summary of the invention

[0007] In view of the above problems, the present invention provides a cyclic peptide composition with anti-aging effect, which belongs to the field of medicine and cosmetics. The polypeptide components of the cyclic peptide composition include cyclic peptides, snake venom peptides and collagen peptides, and the concentration of the polypeptide components is 0.00001ppm~20% (w / w), and the cyclic peptide composition comprises a combination of two polypeptides and a combination of tripeptides. Compared with a single polypeptide group, the polypeptide composition can more effectively promote the production of collagen in the skin in vitro and inhibit the production of neuronal acetylcholine, among which the effect of the combination of cyclic peptide + snake venom peptide + collagen peptide is the best, and the combination of cyclic peptide + snake venom peptide is second, indicating that the combination of cyclic peptide, snake venom peptide and collagen peptide has a synergistic effect. Therefore, the cyclic peptide composition can be added to essences, creams, sunscreens, lotions, lotions, eye creams, facial masks, facial cleansers, cleansing honey, gels, sprays and other products to improve the anti-wrinkle and firming effects of the products.

[0008] In order to achieve the above object, the present invention adopts the following scheme:

[0009] On the one hand, the present invention provides a cyclic peptide composition with anti-aging effect, characterized in that the cyclic peptide composition comprises any one or more of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides.

[0010] Skin aging is mainly caused by age, unhealthy lifestyle, ultraviolet radiation, preference for sweets, endocrine disorders and other reasons. With age, the collagen and moisture of the skin will be lost, the elasticity of the skin will decrease, wrinkles and loose skin will appear under the influence of long-term facial expressions, and pigmentation will also appear to form spots. In view of the above-mentioned characteristics of skin aging, skin care products with anti-aging effects can be used to delay skin aging by adding cyclic peptides or neurotransmitter inhibitor peptides or collagen peptides, but it has not been reported that the composition between cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides has a synergistic effect. Therefore, the present invention has explored this and confirmed that the composition comprising any two or three of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides has a synergistic effect. Further, the synergistic effect of cyclic peptides is the most significant; and it is found that the composition mainly prevents skin aging by increasing the content of different collagens in the skin and inhibiting the production of acetylcholine in neuronal cells.

[0011] Further, the cyclic peptide comprises any one or more of cyclic hexapeptide-9, cyclic palmitoyl pentapeptide-4, cyclic myristoyl pentapeptide-4, and cyclic palmitoyl tripeptide-5. The structural characteristics and preparation methods of the four polypeptides are detailed in Chinese patent CN116162135B, and the cyclic hexapeptide-9, cyclic palmitoyl pentapeptide-4, cyclic myristoyl pentapeptide-4, and cyclic palmitoyl tripeptide-5 in the present invention correspond to the hexapeptide-9 cyclic peptide, palmitoyl pentapeptide-4 cyclic peptide, myristoyl pentapeptide-4 cyclic peptide, and palmitoyl tripeptide-5 cyclic peptide in "Patent CN116162135B" respectively.

[0012] Furthermore, the neurotransmitter inhibitor peptide comprises any one or more of snake venom peptide, acetyl hexapeptide-8, and conopeptide.

[0013] Furthermore, the collagen peptides include any one or more of recombinant type I collagen peptides, recombinant type III collagen peptides, recombinant type IV collagen peptides, recombinant type V collagen peptides, recombinant type VII collagen peptides, and recombinant type XVII collagen peptides. These six protein peptides are prepared by hydrolyzing the corresponding collagen by specific collagenases.

[0014] It is understandable that there are many types of known cyclic peptides, neurotransmitter inhibitor peptides and collagen peptides, and the combinations of these three types of polypeptides are endless. Therefore, in order to explore whether the combination of these polypeptides has a synergistic effect, in some specific embodiments, the cyclic peptide is cyclic hexapeptide-9 (see the hexapeptide-9 cyclic peptide of Chinese patent CN116162135B for details); the neurotransmitter inhibitor peptide is a snake venom peptide, and its specific chemical name is dipeptide diaminobutyryl benzylamide diacetate; the collagen peptide is a product generated by collagenase digestion of recombinant type III humanized collagen having an amino acid sequence as shown in SEQ ID NO: 1 in the sequence table (see Chinese patents 2024108845669 and 2024108904525 for details), and the molecular weight of the collagen peptide is 2-10 kDa, preferably 2.8-3 kDa.

[0015] Specifically, the cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

[0016] Furthermore, the cyclic peptide composition is preferably a combination of cyclic peptide + neurotransmitter inhibitor peptide + collagen peptide, specifically a combination of cyclic peptide + snake venom peptide + recombinant type III collagen peptide; among the combinations of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide, the best anti-aging effect is the combination of cyclic peptide + neurotransmitter inhibitor peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide.

[0017] Furthermore, the concentration of each polypeptide component in the cyclic peptide composition is 0.00001 ppm to 20% (w / w).

[0018] ppm, fraction, is defined as one part per million, 1ppm means one part per million.

[0019] On the other hand, the present invention provides a composition for preparing an agent for synergistically improving anti-wrinkle efficacy and / or firming efficacy, wherein the cyclic peptide composition comprises any one or more of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides.

[0020] Specifically, the cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

[0021] Preferably, the cyclic peptide composition is preferably a combination of cyclic peptide + neurotransmitter inhibitor peptide + collagen peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide + recombinant type III collagen peptide; among the combinations of cyclic peptides, neurotransmitter inhibitor peptides and collagen peptides, the best anti-aging effect is the combination of cyclic peptide + neurotransmitter inhibitor peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide.

[0022] On the other hand, the present invention provides a composition for preparing an agent for synergistically promoting collagen production in the skin, wherein the cyclic peptide composition comprises any one or more of a cyclic peptide, a neurotransmitter inhibitor peptide, and a collagen peptide.

[0023] Specifically, the cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

[0024] Furthermore, the cyclic peptide composition is preferably a combination of cyclic peptide + neurotransmitter inhibitor peptide + collagen peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide + recombinant type III collagen peptide; among the combinations of cyclic peptides, neurotransmitter inhibitor peptides and collagen peptides, the best anti-aging effect is the combination of cyclic peptide + neurotransmitter inhibitor peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide.

[0025] On the other hand, the present invention provides a composition for use as an agent for inhibiting acetylcholine production in neuronal cells, wherein the cyclic peptide composition comprises any one or more of cyclic peptides, snake venom peptides, and collagen peptides.

[0026] Specifically, the cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

[0027] Furthermore, the cyclic peptide composition is preferably a combination of cyclic peptide + neurotransmitter inhibitor peptide + collagen peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide + recombinant type III collagen peptide; among the combinations of cyclic peptides, neurotransmitter inhibitor peptides and collagen peptides, the best anti-aging effect is the combination of cyclic peptide + neurotransmitter inhibitor peptide, specifically a combination of cyclic hexapeptide-9 + snake venom peptide.

[0028] The beneficial effects of the present invention include:

[0029] 1. The present invention provides a cyclic peptide composition with anti-aging effect, which has high biocompatibility and is safe and non-irritating to the human body, and can be applied to the field of beauty and skin care;

[0030] 2. The present invention confirms that compared with a single polypeptide group, a cyclic peptide combination (a combination of two polypeptides and a combination of three peptides) can simultaneously and significantly increase the content of multiple collagens in an in vitro skin model after ultraviolet radiation, indicating that the above cyclic peptide combination has excellent anti-wrinkle and firming effects and synergistic effects; and the anti-aging effect of the tripeptide combination is also better than that of the combination of two polypeptides and a single component, further confirming that the combination of cyclic peptides, snake venom peptides, and collagen peptides has a synergistic effect;

[0031] 3. The present invention confirms that compared with a single polypeptide group, the above cyclic peptide combination (peptide combination of two or three peptides) can significantly reduce the content of acetylcholine in neurons, indicating that the above cyclic peptide combination has anti-wrinkle effect and the combination of each polypeptide component has a synergistic effect; and the anti-wrinkle effect of the tripeptide combination is the best, further confirming that the combination of cyclic peptides, snake venom peptides, and collagen peptides has a synergistic effect;

[0032] 4. By comparing the firming and anti-wrinkle properties of the above-mentioned cyclic peptide compositions (peptide combinations of two peptides and tripeptide combinations), it was found that the firming effects of the two types of peptide compositions were superior to their anti-wrinkle effects.

[0033] 5. The present invention compares the synergistic effects of polypeptide components and finds that cyclic peptides have the best synergistic effect, that is, the anti-aging effect of the composition containing cyclic peptides is better than that without cyclic peptides. DETAILED DESCRIPTION

[0034] The present invention is further elaborated in detail below in conjunction with the specification and specific examples. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. Based on the examples in the present invention, all other examples obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0035] The reagents and manufacturers used in the present invention are as follows:

[0036] (1) Experimental reagents required for the in vitro skin model: in vitro skin model culture medium (Guangdong Boxi Biotechnology), PBS buffer (Solaibao), vitamin C (VC, Sigma), vitamin E (VE, Sigma), Masson kit (Yike Biotechnology), Collagen I antibody (CST), Collagen III antibody (Wuhan Sanying), Collagen IV antibody (Abcam), Collagen V antibody (Abcam), Collagen VII antibody (Abcam), Collagen XVII antibody (Abcam), paraformaldehyde (Biosharp), xylene (Sinopharm Group), sodium citrate antigen retrieval solution (Zhonghui Hecai).

[0037] (2) Reagents required for neuronal cell experiments: DMEM culture medium (Gibco), fetal bovine serum (Lanzhou Rongye), PBS (Solebo), acetylcholine kit (Nanjing Jiancheng), MTT (Sigma), DMSO (Sigma).

[0038] The instruments and manufacturers used in the present invention are as follows:

[0039] (1) Based on in vitro skin model: CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), UVA irradiator (Philips), UVB irradiator (Philips), fluorescence microscope (Olympus, BX43), upright microscope (Olympus, BX53).

[0040] (2) Based on neuronal cells: CO 2 Incubator (Thermo, 150I), clean bench (Sujing Antai, SW-CJ-1F), and microplate reader (BioTek, Epoch).

[0041] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] Example 1: Determination of collagen fibers and different collagens

[0043] Because the content of collagen type I (CollagenⅠ) and collagen type III (CollagenⅢ) in the skin is the highest, and collagen type V (Collagen V) is the key collagen connecting type I and type III collagen, it is generally believed in the industry that the changes in the content of type I, type III and type V collagen reflect the "anti-wrinkle" effect of skin care products; in addition, the content of other collagens in the skin is relatively small, but their functions are also very important. Therefore, the present invention is based on an in vitro skin model, and evaluates the anti-wrinkle effect of the sample to be tested by detecting the changes in the content of collagen fibers, type I collagen, type III collagen, and type V collagen; and evaluates the firming effect of the sample to be tested by detecting the changes in the content of type IV collagen (CollagenⅣ), type VII collagen (CollagenVII), and type XVII collagen (Collagen XVII). It should be noted that "anti-wrinkle" and "firming" are just a term in the industry.

[0044] Table 1 Summary of the parameters, determination indicators and detection methods of the experiments carried out in this embodiment

[0045]

[0046] The sample combination, experimental parameters, etc. of this example are detailed in Table 1, wherein the solvent in the polypeptide solution is PBS buffer (pH 7.4), and the steps for detecting the changes in the content of collagen fibers and the above-mentioned different collagens in the in vitro skin model are as follows:

[0047] A. Tissue processing: Cut the ex vivo skin tissue from human abdomen into 24±2 mm 2 The tissue block was placed in a culture mold with the epidermis facing up and the dermis facing down. The culture mold was then transferred to a 6-well plate. 3.7 mL of in vitro skin model culture medium was added to each well and incubated at 37°C with 5% CO 2 Culture in an incubator and change the medium every day;

[0048] B. Irradiation and administration: After 2 days of tissue culture in step A, irradiation and administration were started with reference to the test groups and corresponding treatment conditions in Table 1. The cyclic peptide in Table 1 is cyclohexapeptide-9 (hexapeptide-9 cyclic peptide), and its chemical structure is The specific preparation method is detailed in patent CN116162135B; the snake venom peptide is dipeptide diaminobutyryl benzylamide diacetate (the peptide is composed of β-alanine, diaminobutyric acid, and proline arranged in a specific order and purchased from Shenzhen Vicky Technology Co., Ltd.); the collagen peptide is a hydrolyzate with a molecular weight of 2-10 kDa (preferably 2.8-3 kDa in this embodiment) prepared by digesting the recombinant type III humanized collagen with an amino acid sequence as shown in SEQ ID NO.1 by collagenase. The preparation method and function of the recombinant type III humanized collagen and its hydrolyzate are detailed in patents 2024108845669 and 2024108904525; the irradiation dose is UVA (30 J / cm 2 ) and UVB (50mJ / cm 2 ), irradiated continuously for 4 days, replaced with fresh ex vivo skin model culture medium after each irradiation, and administered, the positive control group was administered by submerged administration, and the sample group was administered by surface administration. After 4 days of continuous irradiation, the culture was continued for another 3 days, during which no irradiation was performed, and only sample administration was performed;

[0049] C. Washing: After the incubation, wash the remaining test substance with a wash bottle containing sterile PBS solution, and gently wipe away the residual liquid with a sterile cotton swab;

[0050] D. Collagen fiber detection: Take the in vitro skin model for detection, fix it with 4% paraformaldehyde, embed the tissue, slice it, and perform Masson staining according to the instructions in the kit to prepare tissue sections, and use an upright microscope to photograph and observe it. Plus image processing software for analysis;

[0051] E. Immunofluorescence (IF) was used to detect different types of collagen. The specific experimental steps are as follows:

[0052] 1) After administration, the skin tissue was fixed with 4% paraformaldehyde, and then embedded and sliced;

[0053] 2) Dewaxing: Place the paraffin sections in a 70°C baking machine and bake for 4 hours;

[0054] 3) Dewaxing and hydration: Soak the sections in xylene for 10 min, then replace xylene and soak for another 10 min, soak in anhydrous ethanol for 5 min, soak in 95% ethanol for 5 min, and soak in 75% ethanol for 5 min; then wash with PBS buffer (pH 7.4) 3 times, 5 min each time;

[0055] 4) Antigen repair: Place the paraffin sections in 0.01M sodium citrate antigen repair solution and use high pressure repair. After cooling, take out the sections and wash them 3 times with PBS buffer solution, 5 min / time;

[0056] 5) Block peroxidase: add 1 drop of 3% H 2 O 2 , incubate at room temperature for 30 min to block the activity of endogenous peroxidase, and then wash with PBS buffer solution three times, 5 min each time;

[0057] 6) Serum blocking: Add serum homologous to the secondary antibody and block at 37°C for 60 minutes. No need to rinse.

[0058] 7) Primary antibody incubation: add primary antibody working solution and incubate overnight at 4°C; wash 3 times with PBS buffer, 5 min / time;

[0059] 8) Secondary antibody incubation: add secondary antibody working solution and incubate at room temperature for 1 hour; wash with PBS buffer 3 times, 5 minutes each time;

[0060] 9) Nuclear staining: After the secondary antibody incubation, wash with PBS buffer for 3 times, 5 min each time. Shake off the PBS solution attached to the slide, add 100 μL of Hochest working solution to each section, and incubate at room temperature for 5 min.

[0061] 10) Wash with PBS buffer 3 times, 5 min / time. Wipe off the PBS solution with absorbent paper and seal the slide with a drop of antifade agent. Take photos with a fluorescence microscope within 24 hours, collect the images and use Plus image processing software for analysis.

[0062] F. Data processing: improvement rate (%) = (sample group - negative control group) / negative control group × 100%;

[0063] G. Statistical analysis of results: GraphPad Prism was used for drawing, and the results were expressed as Mean±SD. The t-test was used for comparison among the groups, and the statistical analysis was two-tailed; P<0.05 was considered to be significantly different, and P<0.01 was considered to be extremely significantly different.

[0064] Example 2: Effect of polypeptide composition on collagen fibers

[0065] In this example, the changes in collagen fiber content in the in vitro skin model were detected according to the experimental steps described in Example 1. The results are shown in Table 2. Compared with the BC group, the collagen fiber content of the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the collagen fiber content of the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the collagen fiber content in the skin model treated with each combination in the sample group could increase the collagen fiber content in the skin model, indicating that the above sample groups all have anti-wrinkle effects; further, the collagen enhancement rate was arranged from high to low as follows: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and two-peptide combinations containing cyclic peptides are superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or two-peptide combinations without cyclic peptides), indicating that cyclic peptides have the strongest anti-wrinkle synergistic effect; in summary, the anti-wrinkle effect of the combination of cyclic peptides + snake venom peptides + collagen peptides is the best, followed by the combination of cyclic peptides + snake venom peptides, and the collagen fiber enhancement rates of these two combinations are 104.35% and 80.43%, respectively.

[0066] Table 2 Summary of collagen fiber analysis results

[0067]

[0068]

[0069] Note: When the t-test method was used for statistical analysis, compared with the BC group, the significance was indicated by #, P-value < 0.05 was indicated by #, and P-value < 0.01 was indicated by ##; compared with the NC group, the significance was indicated by *, P-value < 0.05 was indicated by *, and P-value < 0.01 was indicated by **.

[0070] Example 3: Effect of the polypeptide composition on type Ⅰ collagen (Collagen Ⅰ)

[0071] In this example, the experimental steps described in Example 1 were used to detect the changes in the content of type I collagen in the in vitro skin model. The results are shown in Table 3. Compared with the BC group, the type I collagen content of the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type I collagen content of the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the type I collagen content in the skin model treated with each combination in the sample group could increase the type I collagen content in the skin model, indicating that the above sample groups all have anti-wrinkle effects; further, the collagen enhancement rate was arranged from high to low as follows: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and two-peptide combinations containing cyclic peptides are superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or two-peptide combinations without cyclic peptides), indicating that cyclic peptides have the strongest anti-wrinkle synergistic effect; in summary, the anti-wrinkle effect of the combination of cyclic peptides + snake venom peptides + collagen peptides is the best, followed by the combination of cyclic peptides + snake venom peptides, and the type I collagen enhancement rates of these two combinations are 186.36% and 136.36%, respectively.

[0072] Table 3 Summary of CollagenⅠ immunofluorescence analysis results

[0073]

[0074]

[0075] Note: The integrated optical density (IOD) reflects the content of Collagen I. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0076] Example 4: Effect of the polypeptide composition on type III collagen (Collagen III)

[0077] In this example, the experimental steps described in Example 1 were used to detect the changes in the content of type III collagen in the ex vivo skin model. The results are shown in Table 4. Compared with the BC group, the type III collagen content of the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type III collagen content of the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the type III collagen content in the skin model treated with each combination in the sample group could increase the type III collagen content in the skin model, indicating that the above sample groups all have anti-wrinkle effects; further, the collagen enhancement rate was arranged from high to low as follows: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and two-peptide combinations containing cyclic peptides are superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or two-peptide combinations without cyclic peptides), indicating that cyclic peptides have the strongest anti-wrinkle synergistic effect; in summary, the anti-wrinkle effect of the combination of cyclic peptides + snake venom peptides + collagen peptides is the best, followed by the combination of cyclic peptides + snake venom peptides, and the type III collagen enhancement rates of these two combinations are 88.00% and 76.00%, respectively.

[0078] Table 4 Summary of CollagenⅢ immunofluorescence analysis results

[0079]

[0080]

[0081] Note: The integrated optical density (IOD) reflects the content of Collagen III. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0082] Example 5: Effect of the polypeptide composition on type IV collagen

[0083] In this example, the experimental steps described in Example 1 were followed to detect changes in the content of type IV collagen in the ex vivo skin model. The results are shown in Table 5. Compared with the BC group, the type IV collagen content of the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type IV collagen content of the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the type IV collagen content in the skin model treated with each combination in the sample group could increase the type IV collagen content in the skin model, indicating that the above sample groups all had a firming effect; further, the collagen enhancement rate was arranged from high to low as follows: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of the peptide components, the single cyclic peptide and the two-peptide combination containing cyclic peptides were superior to the parallel combination in the same group (i.e., the single peptide group without cyclic peptide or the two-peptide combination without cyclic peptide), indicating that the cyclic peptide had the strongest firming effect; in summary, the combination of cyclic peptide + snake venom peptide + collagen peptide had the best firming effect, followed by the combination of cyclic peptide + snake venom peptide, and the type IV collagen enhancement rates of these two combinations were 162.86% and 120.00%, respectively.

[0084] Table 5 Summary of Collagen IV Immunofluorescence Analysis Results

[0085] Group Relative IOD / Area Average SD P-value Improvement rate (vs NC) BC 1.00 0.12 / / NC 0.35 0.04 0.001## / PC 1.05 0.07 0.000** / Cyclic peptides 0.55 0.02 0.003** 57.14％ Snake venom peptide 0.52 0.05 0.002** 48.57％ Collagen Peptides 0.53 0.04 0.000** 51.43％ Cyclic peptide + snake venom peptide 0.77 0.08 0.001** 120.00％ Cyclic peptide + collagen peptide 0.72 0.05 0.002** 105.71％ Snake venom peptide + collagen peptide 0.70 0.07 0.004** 100.00％ Cyclic peptide + snake venom peptide + collagen peptide 0.92 0.08 0.000** 162.86％

[0086] Note: The integrated optical density (IOD) / area reflects the content of Collagen IV. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0087] Example 6: Effect of the polypeptide composition on type V collagen (Collagen V)

[0088] In this example, the changes in the content of type V collagen in the in vitro skin model were detected according to the experimental steps described in Example 1. The results are shown in Table 6. Compared with the BC group, the type V collagen content of the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type V collagen content of the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the type V collagen content in the skin model treated with each combination in the sample group could increase the type V collagen content in the skin model, indicating that the above sample groups all have anti-wrinkle effects; further, the collagen enhancement rate was arranged from high to low as follows: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and two-peptide combinations containing cyclic peptides are respectively superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or two-peptide combinations without cyclic peptides), indicating that cyclic peptides have the strongest anti-wrinkle synergistic effect; in summary, the anti-wrinkle effect of the combination of cyclic peptides + snake venom peptides + collagen peptides is the best, followed by the combination of cyclic peptides + snake venom peptides, and the type V collagen enhancement rates of these two combinations are 123.91% and 71.74%, respectively.

[0089] Table 6 Summary of Collagen V Immunofluorescence Analysis Results

[0090]

[0091] Note: Integrated optical density (IOD) / area, its value reflects the content of Collagen V. When the t-test method is used for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, and P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, and P-value < 0.01 is indicated by **.

[0092] Example 7: Effect of the polypeptide composition on type VII collagen

[0093] In this example, the changes in the type VII collagen content in the ex vivo skin model were detected according to the experimental steps described in Example 1. The results are shown in Table 7. Compared with the BC group, the type VII collagen content in the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type VII collagen content in the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the in vitro skin model treated with each combination in the sample group could increase the type VII collagen content in the skin model, indicating that the above sample groups all have a firming effect; further, the collagen enhancement rate is arranged from high to low in the following order: tripeptide combination > two-peptide combination > single peptide group , indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and the combination of two peptides containing cyclic peptides are respectively superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or the combination of two peptides without cyclic peptides), indicating that cyclic peptides have the strongest firming and synergistic effect; in summary, the combination of cyclic peptides + snake venom peptides + collagen peptides has the best firming effect, followed by the combination of cyclic peptides + snake venom peptides. The type VII collagen enhancement rates of these two combinations are 239.29% and 182.14%, respectively.

[0094] Table 7 Summary of Collagen VII Immunofluorescence Analysis Results

[0095] Group Relative IOD / Area Average SD P-value Improvement rate (vs NC) BC 1.00 0.11 / / NC 0.28 0.03 0.000## / PC 0.77 0.06 0.000** / Cyclic peptides 0.51 0.04 0.001** 82.14％ Snake venom peptide 0.49 0.02 0.003** 75.00％ Collagen Peptides 0.50 0.03 0.001** 78.57％ Cyclic peptide + snake venom peptide 0.79 0.03 0.000** 182.14％ Cyclic peptide + collagen peptide 0.73 0.02 0.000** 160.71％ Snake venom peptide + collagen peptide 0.74 0.04 0.002** 164.29％ Cyclic peptide + snake venom peptide + collagen peptide 0.95 0.04 0.000** 239.29％

[0096] Note: Integrated optical density (IOD) / area, its value reflects the content of Collagen VII. When using t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **.

[0097] Example 8: Effect of the polypeptide combination on type XVII collagen

[0098] In this example, the changes in the type VII collagen content in the ex vivo skin model were detected according to the experimental steps described in Example 1. The results are shown in Table 8. Compared with the BC group, the type XVII collagen content in the NC group decreased significantly, indicating that the stimulation conditions of this test were effective; compared with the NC group, the type XVII collagen content in the PC group increased significantly, indicating that the positive control of this test was effective; compared with the NC group, the in vitro skin model treated with each combination in the sample group could increase the type XVII collagen content in the skin model, indicating that the above sample groups all have a firming effect; further, the collagen enhancement rate is arranged from high to low in the following order: tripeptide combination > two-peptide combination > single peptide group, indicating that the combination of these three peptides has a synergistic effect; furthermore, in terms of the synergistic effect of peptide components, single cyclic peptides and the combination of two peptides containing cyclic peptides are respectively superior to the parallel combinations in the same group (i.e., single peptide groups without cyclic peptides or the combination of two peptides without cyclic peptides), indicating that cyclic peptides have the strongest firming and synergistic effect; in summary, the combination of cyclic peptides + snake venom peptides + collagen peptides has the best firming effect, followed by the combination of cyclic peptides + snake venom peptides. The XVII collagen enhancement rates of these two combinations are 258.82% and 197.06%, respectively.

[0099] Table 8 Summary of Collagen XVII Immunofluorescence Analysis Results

[0100]

[0101] Note: Integrated optical density (IOD) / area, its value reflects the content of Collagen XVII. When using t-test method for statistical analysis, compared with the BC group, the significance is indicated by #, P-value < 0.05 is indicated by #, P-value < 0.01 is indicated by ##; compared with the NC group, the significance is indicated by *, P-value < 0.05 is indicated by *, P-value < 0.01 is indicated by **.

[0102] Combining the test results of the two combinations with the best anti-aging effects in the in vitro skin model in Examples 1 to 8, it can be seen (compared with the control group):

[0103] A. The combination of cyclic peptide + snake venom peptide significantly increased the content of type IV collagen, type VII collagen, and type XVII collagen in the in vitro skin model, with the increase rates being 120.00%, 182.14%, and 197.06%, respectively, indicating that the sample can increase the content of type IV collagen, type VII collagen, and type XVII collagen at this concentration, and has a firming effect;

[0104] B. The sample cyclic peptide + snake venom peptide + collagen peptide significantly increased the content of type IV collagen, type VII collagen, and type XVII collagen in the in vitro skin model, with the increase rates being 162.86%, 239.29%, and 258.82%, respectively, indicating that the sample can increase the content of type IV collagen, type VII collagen, and type XVII collagen at this concentration, and has a firming effect;

[0105] C. Cyclic peptide + snake venom peptide significantly increased the contents of collagen fibers, type I collagen, type III collagen, and type V collagen in the in vitro skin model, with the increase rates being 80.43%, 136.36%, 76.00%, and 71.74%, respectively. This indicates that the sample can increase the contents of collagen fibers, type I collagen, type III collagen, and type V collagen at this concentration, and has anti-wrinkle effects.

[0106] D. Cyclic peptide + snake venom peptide + collagen peptide significantly increased the contents of collagen fibers, type I collagen, type III collagen, and type V collagen in the in vitro skin model, with the increase rates being 104.35%, 186.36%, 88.00%, and 123.91%, respectively. This indicates that the sample can increase the contents of collagen fibers, type I collagen, type III collagen, and type V collagen at this concentration, and has anti-wrinkle effects.

[0107] In addition, by comparing the firming and anti-wrinkle effects between polypeptide combinations, it was found that the firming and anti-wrinkle effects of the cyclic peptide + snake venom peptide + collagen peptide combination were significantly better than those of the cyclic peptide + snake venom peptide combination or other combinations of two polypeptides, that is, the improvement rate of the former was 10% to 60% higher than that of the latter, further illustrating that there is a coordinated and synergistic effect between cyclic peptides, snake venom peptides and collagen peptides.

[0108] In terms of efficacy, it is obvious that the firming efficacy of the two polypeptide compositions is superior to their anti-wrinkle efficacy, which may be because the cyclic peptides may act on the basement membrane site in the skin, thereby greatly promoting the production of collagen types associated with the basement membrane.

[0109] In addition, the present invention also carried out the experiments and result analysis described in Examples 1 to 8 on polypeptide components (cyclic peptides, snake venom peptides and collagen peptides) with a concentration of 0.00001ppm to 20% (w / w). The results showed that the polypeptide components with a concentration range of 0.00001ppm to 20% (w / w) all have the effects described in Examples 2 to 8.

[0110] Example 9: Method for determining neuronal acetylcholine content

[0111] Acetylcholine is a neurotransmitter that plays a role in transmitting signals in the nervous system. For example, in muscle tissue, acetylcholine transmits signals generated by nerve endings to muscles, causing muscle contraction. The formation of wrinkles is related to the repeated contraction of facial muscles. By reducing the content of acetylcholine, the transmission of nerve signals can be reduced, thereby relaxing the muscles and reducing the frequency and intensity of muscle contractions. Dynamic wrinkles are formed due to the activity of facial expression muscles. By inhibiting the release of acetylcholine and reducing muscle activity, the appearance of these wrinkles can be effectively reduced. Some anti-wrinkle products may contain ingredients that can affect the function of acetylcholine, which can achieve the effect of reducing wrinkles through topical application. Therefore, reducing the content of acetylcholine or inhibiting its function can achieve an anti-wrinkle effect by reducing the contraction of facial muscles.

[0112] The present invention evaluates the anti-wrinkle effect of the sample to be tested by detecting the change of acetylcholine content in neuronal cells.

[0113] Table 9 Summary of the parameters, determination indicators and detection methods of the experiments carried out in this embodiment

[0114]

[0115]

[0116] The sample combination and experimental parameters of this example are detailed in Table 9, wherein the solvent of the cyclic peptide polypeptide solution is PBS buffer (pH=7.4), and the steps for detecting the change of acetylcholine content in neurons are as follows:

[0117] A. Cell inoculation: After neuronal cells are revived, when the plating rate reaches about 60%, the cells are inoculated into 6-well plates and incubated in an incubator (37°C, 5% CO 2 ) and incubated overnight;

[0118] B. Solution preparation: Prepare the test substance working solution according to the test grouping (Table 9), wherein the cyclic peptide, snake venom peptide and collagen peptide described in Table 1 are the same as those described in Example 1.

[0119] C. Drug administration: Test groups according to Table 9. When the cell plating rate in the 6-well plate reaches 40% to 60%, group drug administration is carried out, and 3 replicate wells are set up for each group. 2 mL of culture medium is added to each well of the blank control group, 2 mL of culture medium containing acetyl hexapeptide-8 is added to each well of the positive control group, and 2 mL of culture medium containing the sample to be tested at the corresponding concentration is added to each well of the sample group. Place in CO 2 Incubator (37°C, 5% CO 2 ) and incubated for 24 h;

[0120] D. Detection: Collect cell culture supernatant and perform detection according to the instructions of the acetylcholine detection kit;

[0121] E. Calculation of inhibition rate:

[0122] F. Statistical analysis of results: GraphPad Prism was used for graphing, and the results were expressed as Mean±SD. The t-test was used for comparison among the groups, and all statistical analyses were two-tailed. P<0.05 was considered to be significantly different, and P<0.01 was considered to be extremely significantly different.

[0123] Example 10: Effect of the polypeptide composition on the acetylcholine content of neuronal cells

[0124] This example uses the method described in Example 9 to detect changes in acetylcholine content in neurons.

[0125] The results are shown in Table 10. Compared with the BC group, the acetylcholine content in the neuronal cells treated with each combination in the sample group decreased significantly, indicating that the above sample groups all have anti-wrinkle effects; further, the acetylcholine production inhibition rate is arranged from high to low as follows: tripeptide combination>peptide combination>single polypeptide group, indicating that the combination of these three polypeptides has a synergistic effect; furthermore, in terms of the synergistic effect of polypeptide components, single cyclic peptides and two-peptide combinations containing cyclic peptides are superior to the parallel combinations in the same group (i.e., single polypeptide groups without cyclic peptides or two-peptide combinations without cyclic peptides), indicating that cyclic peptides have the strongest anti-wrinkle synergistic effect; in summary, the anti-wrinkle effect of the combination of cyclic peptides + snake venom peptides + collagen peptides is the best, followed by the combination of cyclic peptides + snake venom peptides, and the inhibition rates of these two combinations are 42.18% and 30.87%, respectively.

[0126] Table 10 Summary of Acetylcholine Content Test Results

[0127]

[0128] Note: When the t-test method was used for statistical analysis, the significance was indicated by *, P-value < 0.05 was indicated by *, and P-value < 0.01 was indicated by ** compared with the BC group.

[0129] In summary, the test results of changes in the content of acetylcholine in collagen fibers and different collagens and neuronal cells in the skin ex vivo model confirmed that compared with a single polypeptide group, the combination of two polypeptides and the combination of three peptides significantly improved the firming and anti-wrinkle effects, and when the cyclic peptide, snake venom peptide, and collagen were used in combination, the composition had a synergistic effect. At the same time, the present invention also conducted the experiments and result analysis described in Examples 9 to 10 on polypeptide components (cyclic peptides, snake venom peptides, and collagen peptides) with a concentration of 0.00001ppm to 20% (w / w), and the results showed that the polypeptide components with a concentration in the range of 0.00001ppm to 20% (w / w) all had the effects described in Example 10.

[0130] It is understandable that other experimental indicators and / or models may be used to detect the content of collagen and acetylcholine.

[0131] It is understandable that the above-mentioned compositions (peptide combinations in pairs, tripeptide combinations) can be combined with different formula systems and used in essences, creams, sunscreens, lotions, emulsions, eye creams, facial masks, facial cleansers, facial cleansers, gels, sprays and other products.

[0132] The basic principles and main features of the present invention and the advantages of the present invention are shown and described above. For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and the present invention can be implemented in other specific forms without departing from the spirit or basic features of the present invention; therefore, no matter from which point of view, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is limited by the attached claims rather than the above description. Therefore, it is intended to include all changes within the meaning and scope of the equivalent elements of the claims in the present invention, and any marks in the claims should not be regarded as limiting the claims involved.

[0133] References

[0134] 1. Al-Atif, H., Collagen Supplements for Aging and Wrinkles: A ParadigmShift in the Field of Dermatology and Cosmetics. Dermatology Practical&Conceptual, 2022.

[0135] 2.Reilly,D.M.and J.Lozano,Skin collagen through the lifestages:importance for skin health and beauty.Plastic and Aesthetic Research,2021.8.

[0136] 3.Schagen,S.,Topical Peptide Treatments with Effective Anti-AgingResults.Cosmetics,2017.4(2)。

Claims

1. A cyclic peptide composition with anti-aging effect, characterized in that: The cyclic peptide composition comprises any one or more of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides.

2. The cyclic peptide composition according to claim 1, characterized in that The cyclic peptide comprises any one or more of cyclohexapeptide-9, cyclopalmitoyl pentapeptide-4, cyclomyristoyl pentapeptide-4, and cyclopalmitoyl tripeptide-5.

3. The cyclic peptide composition according to claim 2, characterized in that The neurotransmitter inhibitor peptide comprises any one or more of snake venom peptide, acetyl hexapeptide-8 and conopeptide.

4. The cyclic peptide composition according to claim 3, characterized in that The collagen peptides include any one or more of recombinant type I collagen peptides, recombinant type III collagen peptides, recombinant type IV collagen peptides, recombinant type V collagen peptides, recombinant type VII collagen peptides, and recombinant type XVII collagen peptides.

5. The cyclic peptide composition according to claim 1, characterized in that The cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

6. The cyclic peptide composition according to claim 1, characterized in that The concentration of each polypeptide component in the cyclic peptide composition is 0.00001 ppm to 20% (w / w).

7. Use of a composition for preparing an agent for synergistically improving anti-wrinkle efficacy and / or firming efficacy, characterized in that: The cyclic peptide composition comprises any one or more of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides.

8. The use according to claim 7, characterized in that The cyclic peptide composition is a combination of cyclic peptide+neurotransmitter inhibitor peptide+collagen peptide, or a combination of cyclic peptide, neurotransmitter inhibitor peptide and collagen peptide.

9. A composition for preparing an agent for synergistically promoting collagen production in the skin, characterized in that: The cyclic peptide composition comprises any one or more of cyclic peptides, neurotransmitter inhibitor peptides, and collagen peptides.

10. A composition for use as an agent for inhibiting acetylcholine production in neuronal cells, characterized in that: The cyclic peptide composition comprises any one or more of cyclic peptides, snake venom peptides, and collagen peptides.

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