A polypeptide having anti-wrinkle and oil control efficacy

Abstract

This invention discloses a polypeptide with anti-wrinkle and oil-controlling effects, belonging to the field of cosmetic polypeptide technology. The polypeptide is heptapeptide-6, with the amino acid sequence Asn-Asp-Glu-Gly-Leu-Tyr-Leu. At concentrations of 0.001 mg / mL to 0.1 mg / mL, heptapeptide-6 significantly promotes the synthesis of type I collagen and elastin by human skin fibroblasts, exhibiting excellent anti-wrinkle and firming activity. At concentrations of 1 ppm to 50 ppm, it significantly inhibits lipid accumulation in *C. elegans* and lipid expression in zebrafish, with a maximum lipid inhibition rate of 24.38%, and also demonstrates clear oil-controlling activity. This invention's polypeptide has a single structure, overcoming the shortcomings of existing skincare polypeptides with single efficacy and complex formulations. It features stable processing, high safety, and is suitable as a core active ingredient in anti-wrinkle and oil-controlling cosmetics, providing a new avenue for the development and application of multifunctional skincare polypeptides.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic technology, specifically relating to a polypeptide with anti-wrinkle and oil-controlling effects. Background Technology

[0002] The core mechanism of skin wrinkle formation is the decline in the activity of human dermal fibroblasts, which leads to a reduction in the synthesis of extracellular matrix components such as type I collagen and elastin, resulting in decreased skin elasticity, loose texture, and the appearance of fine lines and wrinkles. Abnormal sebum secretion originates from sebaceous gland lipid metabolism disorder. Excessive lipid accumulation causes problems such as shine, enlarged pores, and acne, which are the main pain points of oily and combination skin.

[0003] Currently, most commercially available and research-developed peptide cosmetic ingredients focus on a single efficacy: anti-wrinkle peptides (such as palmitoyl tripeptide-5, palmitoyl tripeptide-1, and acetyl hexapeptide-8) can only promote collagen and elastin synthesis, without lipid regulation or oil control effects; oil-controlling peptides (such as dipeptide-15) can only inhibit sebaceous gland lipid metabolism and reduce oil accumulation, without promoting collagen synthesis or improving wrinkles. Products on the market that combine anti-wrinkle and oil-controlling effects mostly use multi-ingredient compounding, which has drawbacks such as poor ingredient compatibility, complex formulation systems, insufficient stability, and uncontrollable efficacy. There is currently no single-structure peptide that simultaneously possesses highly effective in vitro anti-wrinkle and firming and oil-controlling activities.

[0004] With consumers' increasing demand for efficient, simple, and gentle skincare ingredients, developing a novel polypeptide with a single molecular structure, low concentration, high cell safety, and significant anti-wrinkle and oil-control effects can effectively solve the problems of single efficacy, complicated compounding, and poor stability in existing technologies. This will meet the development needs of high-end cosmetics for multifunctional active ingredients and has significant market value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a new use for heptapeptide-6 in the preparation of cosmetics with dual functions of anti-wrinkle and oil control, so as to solve the technical problems of existing skin care peptides having single efficacy, complicated compounding, and poor stability, and to realize that a single peptide raw material can simultaneously meet the application needs of skin anti-wrinkle and firming and oil regulation.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: The application of heptapeptide-6 in the preparation of peptide cosmetics: The active ingredient in peptide cosmetics is heptapeptide-6, whose amino acid sequence is Asn-Asp-Glu-Gly-Leu-Tyr-Leu; Peptide cosmetics have anti-wrinkle, firming and oil-controlling effects. Preferably, the peptide cosmetic is used to improve at least one of the following: fine lines, sagging, oiliness, and enlarged pores.

[0007] Preferably, the polypeptide cosmetic is at least one of serum, toner, lotion, cream, freeze-dried powder, and mask.

[0008] Preferably, the anti-wrinkle and firming effect is achieved by promoting the synthesis of type I collagen and elastin in human skin fibroblasts.

[0009] Preferably, the oil-controlling effect is to inhibit abnormal lipid accumulation and lipid expression.

[0010] This invention is the first to discover that heptapeptide-6 (Asn-Asp-Glu-Gly-Leu-Tyr-Leu) possesses dual bioactivity, combining anti-wrinkle and firming effects with oil control and sebum inhibition. This breakthrough overcomes the existing technological limitations of treating this peptide merely as a conventional structural peptide with no clearly documented skincare efficacy, constituting a novel, non-obvious use. Compared to traditional anti-wrinkle peptides that only promote collagen synthesis without oil control, and traditional oil-controlling peptides that only regulate lipid metabolism without anti-wrinkle effects, this invention's heptapeptide-6, with its single molecular structure, can simultaneously achieve significant enhancement of type I collagen and elastin, as well as highly efficient inhibition of abnormal lipid accumulation and expression at low concentrations. It achieves both anti-wrinkle and oil-control effects without the need for multi-component formulations, fundamentally solving industry pain points such as poor compatibility, insufficient stability, uncontrollable efficacy, and complex formulations inherent in compound formulations.

[0011] Meanwhile, heptapeptide-6 exhibits no cytotoxicity to human skin fibroblasts across a wide concentration range, demonstrating excellent biocompatibility. The preparation process utilizes a mature solid-phase synthesis route, facilitating large-scale production and quality control. It provides the cosmetics industry with a multifunctional active ingredient that is structurally well-defined, has clear efficacy, is safe and gentle, and has stable processing. This fills the global technological gap in single peptides possessing both highly effective anti-wrinkle and oil-controlling effects, demonstrating outstanding novelty, creativity, and industrial application value.

[0012] A method for preparing heptapeptide-6 includes the following steps: A solid-phase Fmoc strategy was employed for synthesis. Using Rink Amide resin as the solid support, the peptides were swollen with DMF and deprotected with a deprotection solution. DIC and HOBt were used as coupling agents to sequentially couple the corresponding protected amino acids from the C-terminus to the N-terminus, yielding a fully protected peptide resin. The resin was cleaved using a TFA / TIS / deionized water mixed cleavage solution. After precipitation with methyl tert-butyl ether, centrifugation, washing, and vacuum drying, crude heptapeptide-6 was obtained. The crude peptide was purified by reversed-phase high-performance liquid chromatography, concentrated, sterilized, and freeze-dried to obtain the final heptapeptide-6 product. Protected amino acids include Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH.

[0013] Preferably, the degree of substitution of the Rink Amide resin is 0.5~0.7 mmol / g.

[0014] Preferably, the deprotection solution comprises piperidine and DMF, wherein the volume ratio of piperidine to DMF is 1:4~6.

[0015] Preferably, the molar ratio of DIC to HOBt in the coupling agent is 0.5~1.5:1.

[0016] Preferably, during each coupling step, the molar ratio of the protected amino acid to the Rink Amide resin is 1.5 to 2.5:1.

[0017] Preferably, the volume ratio of TFA to TIS in the mixed cutting fluid is 93~97:3, and the volume ratio of TIS to deionized water is 2~1.2:1.

[0018] This invention also provides a method for preparing heptapeptide-6, comprising the steps of preparing a fully protected peptide resin, preparing a crude peptide, and preparing a purified polypeptide: Step 1: Preparation of fully protected peptide resin: Place Rink Amide resin in a solid-phase reactor, add DMF and stir to swell for 20-40 min, add deprotection solution to remove Fmoc protecting groups for 20-30 min; use DIC and HOBt as coupling agents, sequentially couple the protected amino acids from C-terminus to N-terminus, single-step coupling at room temperature for 1-2 h, after each coupling step is completed, wash with DMF, and check the reaction is complete with ninhydrin before proceeding to the next step; after all amino acids are coupled, remove the terminal Fmoc protecting groups, shrink with methanol, and vacuum dry to obtain fully protected peptide resin.

[0019] Preferably, the degree of substitution of the Rink Amide resin is 0.5~0.7 mmol / g.

[0020] Preferably, the deprotection solution comprises piperidine and DMF, wherein the volume ratio of piperidine to DMF is 1:4~6.

[0021] Preferably, the molar ratio of DIC to HOBt in the coupling agent is 0.5~1.5:1.

[0022] Preferably, the volume-to-mass ratio of DMF for swelling to resin is 5-15 mL: 1 g.

[0023] Preferably, the protected amino acids include Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH.

[0024] Preferably, the molar ratio of the protected amino acid to the resin is 1.5~2.5:1.

[0025] Preferably, the volume-to-mass ratio of DMF for washing to resin is 6-10 mL: 1 g.

[0026] Preferably, the volume-to-mass ratio of methanol to resin is 6-10 mL: 1 g.

[0027] Step 2: Preparation of crude peptide: Using a mixed cutting solution, stir and cut the fully protected peptide resin at room temperature for 2-3 hours. Filter to remove the resin and collect the cutting solution. Slowly add the cutting solution dropwise to methyl tert-butyl ether pre-cooled to -25~-15℃ until it settles completely. Collect the solid product by centrifugation, wash it 2-4 times with cold methyl tert-butyl ether, and vacuum dry it for 10-14 hours to obtain the crude peptide.

[0028] Preferably, the volume-to-mass ratio of the cutting fluid to the fully protected peptide resin is 8-12 mL: 1 g.

[0029] Preferably, the mixed cutting fluid includes TFA, TIS and deionized water, with the volume ratio of TFA to TIS being 93~97:3 and the volume ratio of TIS to deionized water being 2~1.2:1.

[0030] Step 3: Preparation of purified peptides: The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC). A mobile phase A was prepared using 0.08%–0.12% TFA aqueous solution and a mobile phase B was prepared using 0.08%–0.12% TFA acetonitrile solution. The target fraction was collected by gradient elution, concentrated under reduced pressure, filtered through a 0.20–0.24 μm filter membrane, and freeze-dried to obtain the purified peptides.

[0031] Preferably, the gradient elution includes: 0~5 min, 5%~10% B; 5~25 min, 10%~80% B; 25~30 min, 80%~95% B.

[0032] This invention discovers a novel function of heptapeptide-6: simultaneously promoting collagen and elastin synthesis and inhibiting abnormal lipid accumulation. This breakthrough overcomes the existing understanding that this peptide is only used as a conventional peptide and also overcomes the inherent limitations of traditional anti-wrinkle peptides, such as lacking oil-controlling properties and oil-controlling peptides lacking anti-wrinkle effects. Therefore, it offers the following advantages: heptapeptide-6 can simultaneously achieve excellent in vitro anti-wrinkle and firming activity and oil-controlling activity at low concentrations; it exhibits high cellular safety, requires no compounding, simplifies the formulation, and provides stable effects, offering a new technical pathway for the development of multifunctional cosmetic raw materials. Therefore, this invention represents a novel and inventive application of a known peptide in anti-wrinkle and oil-controlling cosmetics. Attached Figure Description

[0033] Figure 1 This is a schematic diagram showing the results of the type I collagen enhancement rate of heptapeptide-6.

[0034] Figure 2 This is a schematic diagram showing the elastin enhancement rate of heptapeptide-6. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The concepts involved in this application will first be described with reference to the accompanying drawings. It should be noted that the following descriptions of various concepts are only for the purpose of making the content of this application easier to understand and do not constitute a limitation on the scope of protection of this application; furthermore, the embodiments and features in the embodiments of this application can be combined with each other unless otherwise specified. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0037] The Chinese meanings of the abbreviations used in this invention are shown in Table 1 below.

[0038] Table 1. Chinese meanings of abbreviations

[0039] Example 1: This example provides a method for preparing a polypeptide. The preparation steps mainly include the preparation of a fully protected peptide resin, a crude peptide, a purified polypeptide, and a polypeptide sample solution. The specific preparation steps are as follows. The polypeptide is heptapeptide-6, and the amino acid sequence of heptapeptide-6 is shown in SEQ ID NO.1, which is N-terminus → C-terminus, H-Asn-Asp-Glu-Gly-Leu-Tyr-Leu-NH2.

[0040] Step 1: Preparation of fully protected peptide resin: Rink Amide resin was placed in a solid-phase reactor, DMF was added and stirred to swell for 30 min, and deprotection solution was added to remove the Fmoc protecting group for 25 min; DIC and HOBt were used as coupling agents to couple the protected amino acids sequentially from the C-terminus to the N-terminus, and single-step coupling was carried out at room temperature for 1.5 h. After each coupling step was completed, the amino acids were washed with DMF and the reaction was detected by ninhydrin before proceeding to the next step; after all amino acids were coupled, the terminal Fmoc protecting group was removed, methanol was used for shrinkage, and the mixture was dried under vacuum to obtain the fully protected peptide resin. The Rink Amide resin has a degree of substitution of 0.6 mmol / g. The deprotection solution includes piperidine and DMF in a volume ratio of 1:5. The coupling agent has a molar ratio of DIC to HOBt of 1:1. The volume-to-mass ratio of DMF to resin for swelling is 10 mL:1 g. The protected amino acids include Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH, with a molar ratio of protected amino acids to resin of 2.0:1. The volume-to-mass ratio of DMF to resin for washing is 8 mL:1 g, and the volume-to-mass ratio of methanol to resin is 8 mL:1 g.

[0041] Step 2: Preparation of crude peptide: Using a mixed cleavage buffer, the fully protected peptide resin was stirred and cleaved at room temperature for 2.5 h. The resin was removed by filtration, and the cleavage buffer was collected. The cleavage buffer was slowly added dropwise to methyl tert-butyl ether pre-cooled to -20℃ until it settled completely. The solid product was collected by centrifugation, washed three times with cold methyl tert-butyl ether, and vacuum dried for 12 h to obtain the crude peptide. The volume-to-mass ratio of cleavage buffer to fully protected peptide resin was 10 mL:1 g. The mixed cleavage buffer included TFA, TIS, and deionized water, with a TFA to TIS volume ratio of 95:3 and a TIS to deionized water volume ratio of 3:2.

[0042] Step 3: Preparation of purified peptides: The crude peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC). A gradient elution was performed using 0.1% TFA aqueous solution as mobile phase A and 0.1% TFA acetonitrile solution as mobile phase B. The target fraction was collected, concentrated under reduced pressure, filtered through a 0.22 μm filter membrane, and freeze-dried to obtain the purified peptides. The gradient elution consisted of 0–5 min, 5%–10% B; 5–25 min, 10%–80% B; and 25–30 min, 80%–95% B.

[0043] Step 4: Preparation of Peptide Sample Solutions: The peptide sample solutions include cell safety test sample solutions, anti-wrinkle activity test sample solutions, and oil control activity test sample solutions. The purified peptides are added to PBS buffer to prepare sample solutions with concentrations of 0.008 mg / mL, 0.016 mg / mL, 0.031 mg / mL, 0.063 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively. These solutions are then filtered through a 0.22 μm aqueous filter membrane for sterilization to obtain the cell safety test sample solution. The purified peptides are then used to prepare working solutions with cell culture medium at concentrations of 0.001 mg / mL, 0.01 mg / mL, and 0.1 mg / mL. These solutions are then filtered through a 0.22 μm filter membrane to obtain the anti-wrinkle activity test sample solution. The purified peptides are then used to prepare 1 ppm, 10 ppm, and 50 ppm sample solutions with NGM medium, and 10 ppm and 20 ppm sample solutions with standard dilution water to obtain the oil control activity test sample solution.

[0044] Comparative Example 1: The only difference between this comparative example and Example 1 is that the polypeptide in Comparative Example 1 is palmitoyl tripeptide-5, and the amino acid sequence of palmitoyl tripeptide-5 is N-terminus → C-terminus, Pal-Gly-Gln-Pro-NH2; in the preparation steps of the fully protected peptide resin, the protected amino acids in Comparative Example 1 include Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, and Fmoc-Gly-OH.

[0045] Comparative Example 2: The only difference between this comparative example and Example 1 is that the polypeptide in Comparative Example 2 is palmitoyl tripeptide-1, and the amino acid sequence of palmitoyl tripeptide-1 is N-terminus → C-terminus, Pal-Lys-Val-Lys-NH2; in the preparation steps of the fully protected peptide resin, the protected amino acids in Comparative Example 2 include Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, and Fmoc-Lys(Boc)-OH.

[0046] Comparative Example 3: The only difference between this comparative example and Example 1 is that the polypeptide in Comparative Example 3 is acetyl hexapeptide-8. The amino acid sequence of acetyl hexapeptide-8 is shown in SEQ ID NO.2, which is N-terminus → C-terminus, Ac-Glu-Met-Gln-Lys-Leu-Met-NH2. In the preparation steps of the fully protected peptide resin, the protected amino acids in Comparative Example 3 include Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Met-OH, and Fmoc-Glu(OtBu)-OH.

[0047] Comparative Example 4: The only difference between this comparative example and Example 1 is that the polypeptide in Comparative Example 4 is dipeptide-15, and the amino acid sequence of dipeptide-15 is N-terminus → C-terminus, H-Val-Trp-NH2; in the preparation steps of the fully protected peptide resin, the protected amino acids in Comparative Example 4 include Fmoc-Trp(Boc)-OH and Fmoc-Val-OH.

[0048] Experimental Example 1: Cellular safety assay of heptapeptide-6.

[0049] Test samples: Cell safety test sample solutions prepared in Example 1 and Comparative Examples 1-4.

[0050] Test method: A zeroing group, a blank control group (PBS), a positive control group (DMSO), and a sample group (Example 1, Comparative Examples 1-4) were set up. Human skin fibroblasts were seeded at 8×10³ cells / well in 96-well plates and cultured overnight at 37°C and 5% CO2. Sample solution was added and cultured for another 24 hours. MTT reagent was added and incubated in the dark for 2 hours. After discarding the supernatant, DMSO was added, and the OD value was measured at 490 nm to calculate the cell viability.

[0051] This study systematically evaluated the safe concentration range of heptapeptide-6 on human skin fibroblasts using the MTT assay. The results showed that within a wide concentration range of 0.008 mg / mL to 1 mg / mL, the cell viability of each heptapeptide-6 concentration group remained between 92.71% and 110.06%, with no statistically significant difference compared to the blank control group. Furthermore, no significant inhibition of cell proliferation or toxic damage was observed. These results fully demonstrate that heptapeptide-6, within the commonly used concentration range for cosmetic additives, has no significant cytotoxicity to human skin fibroblasts, exhibits excellent biocompatibility, and meets the core requirements for the safety of skin cells in cosmetic active ingredients. This provides a reliable safety basis for its subsequent practical application in anti-wrinkle and oil-control cosmetics.

[0052] Experimental Example 2: Anti-wrinkle and firming activity test of heptapeptide-6.

[0053] Test samples: Anti-wrinkle activity test sample solutions prepared in Example 1 and Comparative Examples 1-4.

[0054] Test methods: Blank control group (BC, no irradiation), negative control group (NC, UVA irradiation), positive control group (PC, 100 μg / mL VC + 7 μg / mL VE), and sample groups (Example 1, Comparative Examples 1-4, 0.001 mg / mL, 0.01 mg / mL, 0.1 mg / mL); A human skin fibroblast photoaging model was established using UVA 9 J / cm² irradiation; the experiment included a blank control group, a negative control group, a positive control group, the experimental group of Example 1, and the control groups of Comparative Examples 1-4; after cells were cultured for 24 h after drug administration, the supernatant was collected, and the content of type I collagen and elastin was measured using an ELISA kit, and the corresponding increase rate was calculated.

[0055] The results of the anti-wrinkle and firming activity test of heptapeptide-6 are shown in Table 2. The results of the type I collagen lifting rate of heptapeptide-6 are shown in Table 2. Figure 1 As shown, the elastin enhancement rate of heptapeptide-6 is as follows: Figure 2 As shown.

[0056] Table 2. Results of anti-wrinkle and firming activity test of heptapeptide-6.

[0057]

[0058] This experiment used UVA photoaging of human skin fibroblasts as a model to verify the anti-wrinkle and firming effects of heptapeptide-6. The positive control group consisted of 100 μg / mL vitamin C combined with 7 μg / mL vitamin E. The final measured increase in type I collagen reached 705.8%, a high value derived from the use of 9 J / cm² in this experiment. 2 The UVA-constructed photoaging human skin fibroblast model has a unique computational logic that prevents direct comparison with data from normal cells without UV irradiation. Data shows that heptapeptide-6, at three test concentrations of 0.001 mg / mL, 0.01 mg / mL, and 0.1 mg / mL, significantly enhanced the synthesis levels of type I collagen and elastin in photoaged cells. The increases in type I collagen were 441.28%, 465.03%, and 639.18%, respectively, while the increases in elastin were 34.2%, 49.0%, and 70.1%, respectively. Furthermore, the overall effect showed a clear concentration-dependent enhancement trend.

[0059] Comparative Example 1 was palmitoyl tripeptide-5, a peptide with a long, hydrophobic palmitoyl chain. This peptide exhibited a bell-shaped dose-response effect, with low concentrations promoting elastin synthesis and high concentrations causing activity to decline, unlike the monotonically increasing dose-response relationship of ordinary hydrophilic peptides. At low concentrations of 0.001 mg / mL and 0.01 mg / mL, the peptide bound to the human skin fibroblast membrane in small amounts and activated the TGF-β / Smad pathway, stably upregulating elastin expression. The replicate data showed low dispersion, and statistical analysis revealed a significant difference compared to the negative control group. Therefore, 27.1% and 35.0% of the elastin were measured. The increase in elastin concentration was observed at 0.1 mg / mL. Excess palmitoyl chains inserted extensively into the phospholipid bilayer of the cell membrane, disrupting membrane fluidity and receptor conformation, blocking the signaling pathway promoting matrix synthesis, and simultaneously inducing mild ROS accumulation in cells. This upregulated MMP-12 elastase accelerated elastin degradation. Under the dual effects of decreased synthesis and increased degradation, elastin content significantly decreased. Furthermore, individual differences in cell response increased at this concentration, and the dispersion of ELISA replicate data significantly increased. After analysis of variance, the mean of this group showed no statistically significant difference from the UVA photoaging negative control group; therefore, ns was used. Comparative Example 2 was palmitoyl tripeptide-1, whose overall effect on promoting type I collagen and elastin was weaker than heptapeptide-6. The highest collagen increase rate across the entire concentration range was only 361.2%, while the elastin increase was less than 16%, indicating overall low activity. Comparative Example 3 was acetyl hexapeptide-8, whose core target is neurotransmitter release. It had almost no effect on promoting collagen and elastin synthesis, and the increase in collagen and elastin was very low at all concentrations, with no statistically significant difference in most groups. Comparative Example 4 was dipeptide-15, which only had oil-controlling activity. It had no significant effect on promoting collagen and elastin synthesis in photoaged fibroblasts, and the collagen and elastin indicators did not increase significantly in most concentration groups.

[0060] Compared with mainstream commercially available peptides such as palmitoyl tripeptide-5, palmitoyl tripeptide-1, acetyl hexapeptide-8, and dipeptide-15, heptapeptide-6 is comparable to high-quality anti-wrinkle peptides in promoting collagen synthesis, and also performs better in improving elastin. In contrast, control peptides either only promote collagen production or have no obvious anti-wrinkle effect. This fully demonstrates that heptapeptide-6 has highly efficient and stable in vitro anti-wrinkle and firming activity, and can effectively improve skin sagging, fine lines, and wrinkles caused by the loss of collagen and elastin.

[0061] Experimental Example 3: In vitro oil-controlling activity test of heptapeptide-6 in Caenorhabditis elegans.

[0062] Test samples: Oil-controlling activity test sample solutions prepared in Example 1 and Comparative Examples 1-4.

[0063] Test methods: Blank control group (BC), positive control group (PC, orlistat), and sample groups (Example 1, Comparative Examples 1-4, 1ppm, 10ppm, 50ppm); synchronized L4 stage Caenorhabditis elegans were placed in drug-containing culture medium for intervention and culture for 3 days; the experiment set up blank control group, positive control group, heptapeptide-6 test group and comparative control group; after Oil Red O staining, the relative intensity of lipids in nematodes was quantified using ImageJ software, and the lipid inhibition rate was calculated.

[0064] The results of the in vitro oil-controlling activity test of heptapeptide-6 in Caenorhabditis elegans are shown in Table 3.

[0065] Table 3. Results of in vitro oil-controlling activity test of heptapeptide-6 in Caenorhabditis elegans.

[0066]

[0067] This experiment used a *C. elegans* model to evaluate the lipid regulation and oil-controlling ability of heptapeptide-6. The results showed that heptapeptide-6 inhibited lipid accumulation in nematodes at concentrations of 1 ppm, 10 ppm, and 50 ppm by 9.35%, 19.01%, and 24.38%, respectively, with the inhibitory effect gradually increasing with concentration, exhibiting a good concentration-dependent relationship. Orlistat, a pharmaceutical lipase inhibitor, was used as a reference for system effectiveness in this experiment, verifying that the nematode lipid staining evaluation system could achieve a highly efficient lipid-inhibiting effect. This control design was sufficiently reasonable. Meanwhile, the results of Comparative Studies 1-4 showed that traditional anti-wrinkle peptides palmitoyl tripeptide-5, palmitoyl tripeptide-1, and acetyl hexapeptide-8 had no lipid-inhibiting effect within this concentration range. Only the oil-controlling peptide dipeptide-15 showed a weak oil-controlling effect, with a maximum inhibition rate of only 14.4%, far lower than the 24.38% of heptapeptide-6. These results directly demonstrate that heptapeptide-6 can effectively inhibit excessive lipid accumulation in the body, and its oil-controlling activity is significantly better than that of existing single oil-controlling peptides, providing solid in vitro data support for its regulation of skin sebum secretion and improvement of oily shine and enlarged pores.

[0068] Experimental Example 4: In vivo oil-controlling activity test of heptapeptide-6 in zebrafish.

[0069] Test samples: Oil-controlling activity test sample solutions prepared in Example 1 and Comparative Examples 1-4.

[0070] Test methods: Blank control group (standard dilution water), model control group (fructose + egg yolk powder), and sample group (Example 1, Comparative Examples 1-4, 10 ppm, 20 ppm); a zebrafish lipid accumulation model was established using fructose + egg yolk powder; the experiment included a blank control group, a model control group, a heptapeptide-6 experimental group, and a comparative control group; after 48 h of sample treatment, 5 dpf zebrafish were euthanized in ice water, fixed, and stained with Oil Red O. The relative lipid expression levels in zebrafish were statistically analyzed using ImageJ software.

[0071] The results of the in vivo oil-controlling activity test of heptapeptide-6 in zebrafish are shown in Table 4.

[0072] Table 4. Results of in vivo oil-controlling activity test of heptapeptide-6 in zebrafish.

[0073]

[0074] This experiment further validated the in vivo oil-controlling effect of heptapeptide-6 using a high-fat-induced zebrafish lipid accumulation model. Data showed that compared with the model control group, 10 ppm and 20 ppm of heptapeptide-6 reduced the relative lipid expression level in zebrafish by 6.70% and 10.40%, respectively, with highly significant differences. This indicates that heptapeptide-6 can effectively inhibit abnormal lipid deposition and expression at the in vivo level. In a simultaneous control experiment, commercially available anti-wrinkle peptides showed no in vivo lipid regulation effect, and dipeptide-15 only showed a weak effect at high concentrations, with its overall oil-controlling ability far inferior to that of heptapeptide-6. Combined with the results of the nematode model experiment, the dual biological models jointly confirmed that heptapeptide-6 possesses stable and reliable in vitro and in vivo oil-controlling activity, improving skin oiliness from the perspective of lipid metabolism. It is a high-quality peptide raw material with both anti-wrinkle and oil-controlling effects.

[0075] The embodiments and / or implementation methods described above are merely preferred embodiments and / or implementation methods for implementing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any way. Any person skilled in the art can make some modifications or alterations to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as the technology or embodiments that are substantially the same as the present invention.

[0076] This document uses specific examples to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the methods and core ideas of this application. The above descriptions are only preferred embodiments of this application. It should be noted that due to the limitations of written expression, while there are objectively infinite specific structures, those skilled in the art can make several improvements, modifications, or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner. These improvements, modifications, changes, or combinations, or the direct application of the inventive concept and technical solution to other situations without modification, should all be considered within the scope of protection of this application.

Claims

1. The application of heptapeptide-6 in the preparation of polypeptide cosmetics, characterized by: The active ingredient in the peptide cosmetic is heptapeptide-6, and the amino acid sequence of heptapeptide-6 is Asn-Asp-Glu-Gly-Leu-Tyr-Leu; the peptide cosmetic has anti-wrinkle, firming, and oil-controlling effects.

2. The application according to claim 1, characterized in that: The peptide cosmetic is used to improve at least one of the following: fine lines, sagging, oily skin, and enlarged pores.

3. The application according to claim 1, characterized in that: The anti-wrinkle and firming effects are achieved by promoting the synthesis of type I collagen and elastin in human skin fibroblasts.

4. The application according to claim 1, characterized in that: The oil-controlling effect is achieved by inhibiting abnormal lipid accumulation and lipid expression.

5. A method for preparing heptapeptide-6, characterized in that: Includes the following steps, A solid-phase Fmoc strategy was employed for synthesis. Using Rink Amide resin as the solid support, the peptides were swollen with DMF and deprotected with a deprotection solution. DIC and HOBt were used as coupling agents to sequentially couple the corresponding protected amino acids from the C-terminus to the N-terminus, yielding a fully protected peptide resin. The resin was cleaved using a TFA / TIS / deionized water mixed cleavage solution. After precipitation with methyl tert-butyl ether, centrifugation, washing, and vacuum drying, crude heptapeptide-6 was obtained. The crude peptide was purified by reversed-phase high-performance liquid chromatography, concentrated, sterilized, and freeze-dried to obtain the final heptapeptide-6 product. The protected amino acids include Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Asp(OtBu)-OH, and Fmoc-Asn(Trt)-OH.

6. The preparation method according to claim 5, characterized in that: The degree of substitution of the Rink Amide resin is 0.5~0.7 mmol / g.

7. The preparation method according to claim 5, characterized in that: The deprotection solution comprises piperidine and DMF, with a volume ratio of piperidine to DMF of 1:4~6.

8. The preparation method according to claim 5, characterized in that: The molar ratio of DIC to HOBt in the coupling agent is 0.5~1.5:

1.

9. The preparation method according to claim 5, characterized in that: During each coupling step, the molar ratio of the protected amino acid to Rink Amide resin is 1.5~2.5:

1.

10. The preparation method according to claim 5, characterized in that: The volume ratio of TFA to TIS in the mixed cutting fluid is 93~97:3, and the volume ratio of TIS to deionized water is 2~1.2:1.

Images