A bicyclic peptide composition and its use in skin care

Abstract

Disclosed are a bicyclic peptide composition and application thereof in skin care, and relate to the technical field of polypeptides, the composition comprising s-mu-conotoxin CnIIIC and a cyclic tetrapeptide-24 aminocyclohexane formate ester.The composition can be used for oil control, acne removal, repair, tightening, wrinkle reduction or anti-aging.

Description

Technical Field

[0001] This disclosure relates to the field of peptide technology, and in particular to a bicyclic peptide composition and its application in skin care. Background Technology

[0002] Since the 1980s, functional skincare products have evolved from basic moisturizing to more specialized products. Early products primarily used occlusive ingredients such as glycerin and petrolatum, only meeting the skin's surface moisturizing needs. In the early 21st century, the rise of ingredient-conscious consumers drove the widespread use of single-function ingredients such as retinol and niacinamide. In recent years, with in-depth research into skin biology, skincare product development has shifted towards precise intervention through multi-pathway synergy, especially with a surge in demand for solutions to complex skin problems such as oily and aging skin.

[0003] Excessive sebum secretion is primarily triggered by androgens activating 5α-reductase and the SREBP signaling pathway within sebaceous gland cells. This excessive sebum secretion leads to clogged hair follicles, acne, and enlarged pores. Skin aging is the result of both endogenous (e.g., collagen loss, elastic fiber breakage) and exogenous (e.g., UV radiation, oxidative stress) factors. Dermal collagen is lost at a rate of 1% per year, leading to wrinkles and sagging; the decline in the epidermal barrier function accelerates moisture loss and the penetration of external irritants. With consumers' increasing demand for diversified skincare product efficacy, developing products with multi-dimensional effects has become a core challenge for the industry. Traditional small-molecule ingredients typically act on a single target; for example, kojic acid has a significant effect on inhibiting melanin synthesis but has not shown effects on other efficacy targets. Peptide active ingredients, as endogenous bioactive substances, have amino acid sequences highly homologous to natural human peptides and can achieve precise biological functions by directly regulating cell signaling pathways. Further research into compounding peptide active ingredients is needed to develop a composition that can synergistically target multiple targets to better address skin problems. This is a key technological direction for overcoming the limitations of traditional ingredients. Summary of the Invention

[0004] This disclosure relates to a cyclic peptide composition having effects such as skin or mucous membrane care.

[0005] On one hand, this disclosure provides a cyclic peptide composition comprising s-μ-conospiropeptide CnIIIC and cyclic tetrapeptide-24-aminocyclohexanecarboxylate.

[0006] s-μ-cono-peptide CnIIIC is a polypeptide consisting of 22 amino acid residues with the sequence pGlu-Gly-Cys-Cys-Asn-Gly-Pro-Lys-Gly-Cys-Ser-Ser-Lys-Trp-Cys-Arg-Asp-His-Ala-Arg-Cys-Cys-NH2. It is cyclically linked by three pairs of disulfide bonds, which are located at Cys3-Cys15, Cys4-Cys21, and Cys10-Cys22, respectively.

[0007] Cyclic tetrapeptide-24-aminocyclohexanecarbamate is an active peptide raw material registered with the National Medical Products Administration by Shenzhen Vikitech Co., Ltd., with registration number 20240043.

[0008] The inventors of this disclosure were surprised to discover that when s-μ-conospiropeptide CnIIIC and cyclic tetrapeptide-24-aminocyclohexanecarboxylate are combined, they produce a synergistic effect, which can significantly reduce the synthesis or secretion of sebum in sebaceous gland cells and significantly increase the content of collagen, resulting in a composite product that simultaneously has the effects of oil control, acne removal, repair, firming, wrinkle reduction or anti-aging.

[0009] In some embodiments, the concentration ratio of s-μ-conospiropeptide CnIIIC to cyclic tetrapeptide-24-aminocyclohexanecarboxylate is 3:(1.5~9).

[0010] In some embodiments, the concentration ratio of s-μ-conospiropeptide CnIIIC to cyclic tetrapeptide-24-aminocyclohexanecarboxylate is 3:(6~9).

[0011] The concentration ratio of s-μ-conospiropeptide CnIIIC to cyclic tetrapeptide-24-aminocyclohexanecarboxylate has a significant impact on the efficacy of this combination. The inventors of this disclosure have discovered that when the concentration ratio of s-μ-conospiropeptide CnIIIC to cyclic tetrapeptide-24-aminocyclohexanecarboxylate is 3:(1.5~9), the combination produces a synergistic effect.

[0012] In some embodiments, the composition exhibits a better synergistic effect when the concentration ratio of s-μ-conospiropeptide CnIIIC to cyclic tetrapeptide-24-aminocyclohexanecarbamate is 3:(6~9).

[0013] In another aspect of this disclosure, a cosmetic product is provided, comprising an effective amount of the above-described cyclic peptide composition, and at least one additional ingredient.

[0014] "Effective amount" means an amount of the cyclic peptide composition of this disclosure that is non-toxic but sufficient to provide the desired effect. The cyclic peptide composition of this disclosure is used in the cosmetics of this disclosure at an effective concentration to obtain the desired effect. In some embodiments, the concentration of the cyclic peptide composition is between 0.00000001% (by weight) and 20% (by weight) relative to the total weight of the cosmetic; in some embodiments, the concentration of the cyclic peptide composition is between 0.000001% (by weight) and 15% (by weight) relative to the total weight of the cosmetic; in some embodiments, the concentration of the cyclic peptide composition is between 0.0001% (by weight) and 10% (by weight) relative to the total weight of the cosmetic; in some embodiments, the concentration of the cyclic peptide composition is between 0.0001% (by weight) and 5% (by weight) relative to the total weight of the cosmetic.

[0015] In some embodiments, the dosage form of the cosmetic includes ointment, cream, emulsion, aqueous solution, oil, gel, powder, tablet, mud, patch, film, aerosol, spray, lyophilized preparation or nano-preparation.

[0016] Another aspect of this disclosure provides the use of the above-described cyclic peptide composition in the preparation of compositions for oil control, acne treatment, repair, firming, wrinkle reduction, or anti-aging.

[0017] In another aspect of this disclosure, there is provided the use of the above-described cyclic peptide composition in the preparation of a composition for reducing skin sebum synthesis or secretion; or in the preparation of a composition for promoting collagen synthesis; or in the preparation of a composition for increasing skin elasticity and / or improving skin firmness; or in the preparation of a composition for repairing the skin barrier.

[0018] Another aspect of this disclosure provides the use of the above-described cyclic peptide composition in the preparation of cosmetics.

[0019] Another aspect of this disclosure provides a cosmetic method for non-therapeutic purposes, the method comprising applying the above-described cyclic peptide composition or the above-described cosmetic to the skin.

[0020] Another aspect of this disclosure provides a cosmetic method for controlling oil, removing acne, repairing, firming, reducing wrinkles, or delaying aging, the method comprising using the above-described cyclic peptide composition or the above-described cosmetic on the skin, the method being for non-therapeutic purposes.

[0021] In this disclosure, the term "skin" should be understood as comprising its multiple layers, from the uppermost layer or stratum corneum to the lowermost layer or subcutaneous tissue, including both ends. These layers are composed of different types of cells, such as keratinocytes, fibroblasts, melanocytes, and / or adipocytes. In this disclosure, the term "skin" includes the scalp.

[0022] The term "skin care" refers to the maintenance and care of the skin to improve its condition, making it delicate, smooth, soft, and healthy.

[0023] This disclosure has the following advantages and effects: The cyclic peptide composition disclosed herein produces an unexpected synergistic effect, which can significantly reduce the synthesis or secretion of sebum in the skin, effectively promote the synthesis of collagen, and has the effects of oil control, acne removal, repair, firming, wrinkle reduction or delaying aging. Detailed Implementation

[0024] To make the objects, features, and advantages of this disclosure more apparent and understandable, the disclosure will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of this disclosure. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without inventive effort are within the scope of protection of the appended claims.

[0025] Unless otherwise specified, all experimental reagents and materials used in this disclosure are commercially available.

[0026] In this paper, Arenariphilin H can be prepared without doubt according to methods known from the literature. Arenariphilin H can be prepared according to the literature of Jia AQ et al. (New cyclopeptides in Arenaria oreophila (Caryophyllaceae)[J]. Journal of Asian Natural Products Research. 9:6, 569-574) The known methods for obtaining from plants Arenaria oreophila It is obtained by extraction and separation. In addition, those skilled in the art can also obtain Ganoderma lucidum cyclic peptide H by conventional solid-phase synthesis methods.

[0027] The compounding ratios of each cyclic peptide in the compositions of this disclosure are as follows: Composition s-μ-conospiropeptide CnIIIC (hereinafter referred to as cyclic peptide A) Cyclic tetrapeptide-24-aminocyclohexanecarbamate (hereinafter referred to as cyclic peptide B) Snow Ganoderma Lucidum Cyclic Peptide H (hereinafter referred to as Cyclic Peptide C) Composition 1 3 9 / Composition 2 3 6 / Composition 3 3 3 / Composition 4 3 1.5 / Composition 5 3 1 / Composition 6 3 / 3 Composition 7 / 3 3 Note: When the concentration of the composition is 10 ppm, such as cyclic peptide A:cyclic peptide B = 3:3, it means that cyclic peptide A is 5 ppm and cyclic peptide B is 5 ppm. " / " indicates that it does not exist.

[0028] Example 1: Oil Content Test 1.1 Reagents and Materials 0.25% trypsin digestion solution (prepared by mixing 0.25g trypsin with 100mL water), PBS, complete culture medium, FFA (prepared by mixing linoleic acid and palmitic acid in a molar ratio of 1:1), and Oil Red O staining kit.

[0029] 1.2 Instruments Constant temperature CO2 incubator, clean bench, microplate reader.

[0030] 1.3 Cell lines Human sebaceous gland cells SZ-95.

[0031] 1.4 Samples to be tested and grouping 1.4.1 Sample to be tested Compositions 1, 2, 3, 4, 5, 6, 7, cyclic peptide A, cyclic peptide B, and cyclic peptide C were tested at a concentration of 10 ppm.

[0032] 1.4.2 Grouping Experimental group: Sample to be tested, FFA; Blank control group: PBS; Model groups: PBS, FFA.

[0033] 1.5 Experimental Methods Take one flask of SZ-95 cells in good exponential growth phase, add 0.25% trypsin digestion solution, digest to detach the adherent cells, and count (1~4)×10⁻⁶ cells. 5 Cells were cultured at a concentration of [number] cells / mL to prepare a cell suspension. An appropriate amount of the cell suspension was seeded into a 12-well plate containing complete culture medium and incubated in a CO2 incubator for 24 h. Except for the blank control group which received PBS, each well in the other wells was treated with FFA to achieve a final concentration of 225 μmol / L. Simultaneously with cell modeling induction, the experimental groups received the corresponding test samples, while the model group received the same amount of PBS. All wells were incubated in a CO2 incubator for 48 h. The culture medium was then discarded, and staining was performed according to the Oil Red O staining kit instructions.

[0034] Discard the solution in the well plate, add isopropanol to dissolve the Oil Red O staining solution, and measure the absorbance at 492 nm using a microplate reader. Calculate the relative content of lipid secretion from the cells based on the absorbance values.

[0035] 1.6 Experimental Results Oil Red O is a fat-soluble dye that is highly soluble in fat. Its staining principle is that Oil Red O specifically adsorbs onto neutral triglycerides, lipids, and lipoproteins in tissues and cells, thus staining the fat. Free-floxacin (FFA) is an inducer; under FFA stimulation, SZ-95 cells secrete large amounts of oil that can be stained by Oil Red O. This experiment used test samples to treat FFA-induced SZ-95 cells, and by detecting the amount of oil produced by SZ-95 cells, determined whether the test sample could inhibit the secretion of oil by human sebaceous gland cells.

[0036] The effects of the test samples on lipid secretion in SZ-95 cells are shown in Table 1.

[0037] Table 1. Relative lipid secretion of SZ-95 cells (Mean ± SD) Group Relative sebum secretion (%) Blank control group 46.10±3.60 Model group <![CDATA[100.00±6.43 ### ]]> Composition 1 34.99±1.51*** Composition 2 45.58±1.45*** Composition 3 42.31±3.01*** Composition 4 43.43±1.99*** Composition 5 54.89±4.47*** Composition 6 76.68±7.65** Composition 7 68.87±7.85** Cyclic Peptide A 75.45±8.04*** Cyclic peptide B 73.21±7.24** Cyclic Peptide C 82.29±2.38** Note: Compared with the blank control group ### P <0.001; compared to the model group, ** P <0.01, *** P <0.001.

[0038] Experimental results showed that compositions 1-7, cyclic peptide A, cyclic peptide B, and cyclic peptide C all significantly inhibited the synthesis and secretion of sebum by sebaceous gland cells, thus slowing down sebum deposition. Compared to cyclic peptide A and cyclic peptide B, compositions 1-4 showed a more significant effect in inhibiting sebum secretion, demonstrating that cyclic peptide A and cyclic peptide B produced a synergistic effect after combination, further enhancing their inhibitory effect on sebum secretion. Compared to compositions 1-4, composition 5 showed a weaker effect in inhibiting sebum secretion. Compared to cyclic peptide A and cyclic peptide C, composition 6 did not show a more significant effect in inhibiting sebum secretion, indicating that the combination of cyclic peptide A and cyclic peptide C failed to produce a synergistic effect. Compared to cyclic peptide B and cyclic peptide C, composition 7 also did not show a more significant effect in inhibiting sebum secretion. These results demonstrate that not all cyclic peptides with sebum-inhibiting effects can produce a synergistic effect after combination. The cyclic peptide composition disclosed herein has a synergistic effect, which can be used to reduce the synthesis or secretion of sebum in the skin, improve problems such as excessive oil production and water-oil imbalance in the skin, and can also reduce the occurrence of pimples or acne, and help the skin recover after pimples or acne occur. Therefore, the cyclic peptide composition disclosed herein has the functions of oil control, acne removal, and repair.

[0039] Example 2: Collagen III Content Test 2.1 Reagents and Materials PBS, complete culture medium, trypsin, RIPA lysis buffer, collagen III ELISA kit, BCA protein kit.

[0040] 2.2 Instruments CO2 incubator, clean bench, microplate reader, vortex mixer.

[0041] 2.3 Cell lines Human skin fibroblasts (HSF).

[0042] 2.4 Sample to be tested Experimental group: Composition 1, Composition 2, Composition 5, Composition 7, Cyclic peptide A, Cyclic peptide B and Cyclic peptide C, dissolved in PBS, and tested at a concentration of 10 ppm.

[0043] Control group: PBS.

[0044] UV group: UV radiation, PBS.

[0045] 2.5 Experimental Methods HSF cells in the exponential growth phase were taken and digested with 0.25% trypsin solution to detach the adherent cells. The cells were then counted (1–4) × 10⁻⁶. 5 Cells were prepared at a density of 10 cells / mL to form a cell suspension. The cells were then seeded into 12-well plates and incubated in a CO2 incubator for 24 hours.

[0046] The complete culture medium in the wells was aspirated. The experimental and UV groups were added to PBS and exposed to UVB lamps for 15 min. The blank control group was added to complete culture medium and was not exposed to UVB lamps.

[0047] After 15 minutes of irradiation, the PBS solution was aspirated. The experimental groups were given complete culture medium and the test samples, respectively, while the UV group was given complete culture medium and PBS. The experimental groups, UV groups, and blank control group were all cultured for 48 hours.

[0048] After culture, cells were collected, centrifuged to obtain the cell pellet, and RIPA lysis buffer was added. The pellet was vortexed three times (30 s / time, 3 min interval) and centrifuged at 12000 rpm for 10 min. The supernatant was collected, and the total protein concentration in the supernatant was measured and quantified using a BCA protein quantification kit. The collagen III ELISA was performed according to the instructions, and the OD value of each well was measured to calculate the relative content of collagen III.

[0049] 2.6 Experimental Results Collagen III is a major component of the microcollagen layer, a tissue structure located between the epidermis and dermis. It is crucial for supporting the epidermis and is the first step in preventing skin sagging. Furthermore, collagen III can repair damaged collagen I and stimulate its regeneration. Therefore, promoting collagen III synthesis is significant for firming and repairing damaged skin. In environments with excessive UV exposure, collagenase activity increases significantly, and collagen synthesis is inhibited. This experiment used test samples to treat cells exposed to UV radiation and measured the collagen III content in the corresponding cells to determine whether the cyclic peptide composition disclosed herein can promote collagen III synthesis.

[0050] The results of the test samples' effects on collagen III synthesis are shown in Table 2.

[0051] Table 2. Results of the effect of the test samples on collagen III synthesis (Mean±SD) Test group / sample Relative content of collagen III (%) Blank control group 166.75±15.61 UV group 100.00±8.42 Composition 1 168.77±25.91 Composition 2 196.70±30.48 Composition 5 126.76±13.75 Composition 7 102.13±19.35 Cyclic Peptide A 99.40±5.76 Cyclic peptide B 138.81±32.32 Cyclic Peptide C 133.33±61.38

[0052] The results showed that, compared with the blank control group, the collagen III content in the UV group was significantly reduced after UV light irradiation. Compared with the UV group, cyclic peptide A did not show a collagen-promoting effect, while cyclic peptides B and C could both promote collagen production to some extent. Compositions 1, 2, and 5 all showed collagen-promoting effects, while composition 7 did not. Furthermore, compared with cyclic peptides A and B, compositions 1 and 2 showed more significant collagen-promoting effects, producing a synergistic effect. Although composition 5 had a better collagen-promoting effect than cyclic peptide A, it did not show a better effect than cyclic peptide B and did not meet the criteria for a synergistic effect. Therefore, it was concluded that cyclic peptides A and B did not produce a synergistic effect at a concentration ratio of 3:1. Compared with cyclic peptides B and C, composition 7 had a worse collagen-promoting effect, exhibiting an antagonistic effect. The above results demonstrate that not all combinations of cyclic peptides that promote collagen production can exert a synergistic effect; some cyclic peptide compositions may even exhibit antagonistic effects. The compositions disclosed herein produce a synergistic effect, significantly promoting collagen synthesis and can be used to increase skin elasticity, improve skin firmness, reduce skin sagging, decrease wrinkles, repair the skin barrier, and delay aging. Therefore, the cyclic peptide compositions disclosed herein have firming, repairing, and anti-aging effects.

[0053] In this disclosure, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0054] While specific embodiments of this disclosure have been described for illustrative purposes, various modifications or alterations can be made by those skilled in the art without departing from the spirit and scope of this disclosure. All such modifications or alterations should fall within the scope of the appended claims.

Claims

1. A cyclic peptide composition, characterized in that, Including s-μ-conospiropeptide CnIIIC and cyclic tetrapeptide-24-aminocyclohexanecarboxylate.

2. The cyclic peptide composition according to claim 1, characterized in that, The concentration ratio of the s-μ-conospiropeptide CnIIIC to the cyclic tetrapeptide-24-aminocyclohexanecarboxylate is 3:(1.5~9).

3. The cyclic peptide composition according to claim 1, characterized in that, The concentration ratio of the s-μ-conospiropeptide CnIIIC to the cyclic tetrapeptide-24-aminocyclohexanecarboxylate is 3:(6~9).

4. A cosmetic product, characterized in that, The composition comprises an effective amount of the cyclic peptide composition according to any one of claims 1-3, and at least one additional ingredient.

5. The cosmetic product according to claim 4, characterized in that, The dosage forms of the cosmetics include ointments, creams, emulsions, liquids, oils, gels, powders, tablets, muds, patches, films, aerosols, sprays, freeze-dried preparations, or nano-preparations.

6. Use of the cyclic peptide composition according to any one of claims 1-3 in the preparation of compositions for oil control, acne treatment, repair, firming, wrinkle reduction or anti-aging.

7. Use of the cyclic peptide composition according to any one of claims 1-3 in the preparation of a composition for reducing skin sebum synthesis or secretion; or in the preparation of a composition for promoting collagen synthesis; or in the preparation of a composition for increasing skin elasticity and / or improving skin firmness; or in the preparation of a composition for repairing the skin barrier.

8. Use of the cyclic peptide composition according to any one of claims 1-3 in the preparation of cosmetics.

9. A non-therapeutic cosmetic method, characterized in that, The method includes using the cyclic peptide composition of any one of claims 1-3, or the cosmetic of claim 4 or 5, on the skin.

10. A beauty method for controlling oil, removing acne, repairing, firming, reducing wrinkles, or delaying aging, characterized in that, The method comprises using the cyclic peptide composition of any one of claims 1-3 or the cosmetic of claim 4 or 5 on the skin, the method being non-therapeutic.