Anti-inflammatory anti-aging skin care polypeptides, methods of synthesis and use thereof

Abstract

This invention discloses the structure, synthesis method and application of a group of anti-inflammatory and anti-aging skin care peptides. The sequences of the anti-inflammatory and anti-aging active peptides provided by this invention are: Lys-Asp-Glu-Lys-Glu-Lys-Leu-Leu-Lys-Glu, Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp, Lys-Asp-Glu-Lys-Glu-Lys-Ala-Leu-Lys-Glu, Arg-Lys-Glu-Tyr-Glu-Leu-Glu-Lys-Gln-Lys, Tyr-Leu-Glu-Val-Phe-Ser-Met-Ala-Arg-Asp-Glu-Asp, Leu-Glu-Val-Phe-Ser-Ala-Leu-Arg-Asp-Glu-Asp, Tyr-Leu-Glu-Val-Phe-Ser-Met-Leu-Arg-As The ten linear peptides p-Glu-Asp, Lys-Asp-Glu-Lys-Glu-Ala-Leu-Leu-Lys-Glu, Asn-Thr-Asp-Phe-Gly-Asp-Glu-Phe-Tyr-Ser-Ala-Phe-Ile, and Tyr-Glu-Leu-Glu-Lys-Gln-Lys-Lys-Leu-Glu exhibit excellent anti-inflammatory and anti-aging properties at a concentration of 0.05%. The anti-inflammatory and anti-aging peptides prepared in this invention show excellent effects when applied to serums or face creams.

Description

Technical Field

[0001] This invention relates to a group of peptides and their synthesis methods and applications, belonging to the field of cosmetic technology, and particularly to a group of anti-inflammatory and anti-aging skin care peptides with excellent performance, their synthesis methods and applications. Background Technology

[0002] As the largest immune organ in the human body, the skin's barrier function is closely related to inflammation regulation. External stimuli (ultraviolet radiation, pollutants, pathogens) are significant factors accelerating the skin's natural aging process. Prolonged exposure to UVB and atmospheric pollutants leads to excessive production of reactive oxygen species (ROS) on the skin. These substances damage the skin's antioxidant defense system, causing oxidative stress. Oxidative stress results in uncontrolled melanin synthesis by melanocytes, reduced hyaluronic acid production by keratinocytes, and decreased ECM secretion from dermal fibroblasts, such as type I and III collagen and elastin, ultimately leading to macroscopic signs of skin aging.

[0003] Endogenous stress can activate the TLR4 / NF-κB pathway in keratinocytes, promoting the release of pro-inflammatory factors such as IL-1β and TNF-α.

[0004] While existing technologies have developed peptides with certain anti-inflammatory or anti-aging functions, they often focus on inhibiting single inflammatory factors, resulting in limited functionality. Furthermore, the inherent contradiction between collagen stimulation and inflammation (e.g., modifications such as palmitoylation and PEGylation used to enhance collagen regeneration may upregulate inflammatory factor signaling) hinders their application in cosmetic products. Therefore, it is necessary to develop peptide raw materials with advantages in cost, efficacy, and stability in cosmetic formulations to provide new solutions for skin inflammation and aging problems. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide a group of ten anti-inflammatory and anti-aging linear peptides, their synthesis methods and applications, which are stable in cosmetic formulations and provide new solutions for skin inflammation and aging problems.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a group of ten linear peptides with excellent anti-inflammatory and anti-aging skin-care activity, wherein the peptides are:

[0008] SEQ-1: Lys-Asp-Glu-Lys-Glu-Lys-Leu-Leu-Lys-Glu;

[0009] The structural formula of SEQ-1 is shown in Formula I:

[0010]

[0011] SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp; The structural formula of SEQ-2 is as shown in Formula II:

[0012]

[0013] SEQ-3: Lys-Asp-Glu-Lys-Glu-Lys-Ala-Leu-Lys-Glu; The structural formula of SEQ-3 is as shown in Formula III:

[0014]

[0015] SEQ-4: Arg-Lys-Glu-Tyr-Glu-Leu-Glu-Lys-Gln-Lys; The structural formula of SEQ-4 is shown in Formula IV:

[0016]

[0017] SEQ-5: Tyr-Leu-Glu-Val-Phe-Ser-Met-Ala-Arg-Asp-Glu-Asp; The structural formula of SEQ-5 is shown in formula V:

[0018]

[0019] SEQ-6: Leu-Glu-Val-Phe-Ser-Ala-Leu-Arg-Asp-Glu-Asp; The structural formula of SEQ-6 is shown in Formula VI:

[0020]

[0021] SEQ-7: Tyr-Leu-Glu-Val-Phe-Ser-Met-Leu-Arg-Asp-Glu-Asp; The structural formula of SEQ-7 is as shown in formula VII:

[0022]

[0023] SEQ-8: Lys-Asp-Glu-Lys-Glu-Ala-Leu-Leu-Lys-Glu;

[0024] The structural formula of SEQ-8 ​​is as shown in formula VIII:

[0025]

[0026] SEQ-9: Asn-Thr-Asp-Phe-Gly-Asp-Glu-Phe-Tyr-Ser-Ala-Phe-Ile; The structural formula of SEQ-9 is shown in formula IX:

[0027]

[0028] SEQ-10: Tyr-Glu-Leu-Glu-Lys-Gln-Lys-Lys-Leu-Glu;

[0029] The structural formula of SEQ-10 is shown in Formula X:

[0030]

[0031] Secondly, the present invention provides a method for synthesizing anti-inflammatory and anti-aging skin-care linear peptides, the steps of which include:

[0032] Step 1: Compound Synthesis

[0033] 1) Place 2-Chlorotrityl Chloride Resin into a reaction tube, add DMF (15 ml / g), and shake for 60 min;

[0034] 2) Filter the solvent through a sand filter, add 3 times the molar excess of Fmoc, then add 10 times the molar excess of DIEA, and finally add DMF to dissolve. Shake for 30 min; cap with methanol and let stand for 30 min.

[0035] 3) Remove DMF, add 20% pyridinium DMF solution (15 ml / g), 5 min, remove again, add 20% pyridinium DMF solution (15 ml / g), 15 min;

[0036] 4) Remove the piperidine solution, take a dozen or so resin grains, wash them three times with ethanol, add 2-3 drops of Kaiser reagent, heat at 105℃-110℃ for 5 minutes, and a deep blue color indicates a positive reaction.

[0037] 5) DMF (10ml / g) twice, methanol (10ml / g) twice, DMF (10ml / g) twice;

[0038] 6) Add 3 times molar excess of Fmoc, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, and finally add DMF to dissolve. Shake for 45 minutes.

[0039] 7) Take a dozen or so resin grains, wash them three times with ethanol, add 2-3 drops of Kaiser's reagent, heat at 105℃-110℃ for 5 minutes, and the colorless reaction indicates a negative reaction.

[0040] 8) DMF (10ml / g) once, methanol (10ml / g) twice, DMF (10ml / g) twice;

[0041] 9) Repeat steps three to eight, linking amino acids in the sequence from right to left until the Fmoc protecting group of the last amino acid is removed;

[0042] 10) DMF (10ml / g) twice, DCM (10ml / g) three times, methanol (10ml / g) four times, then vacuum dry for 10 minutes;

[0043] 11) Prepare the cutting fluid (10ml / g): TFA 95%; water 2%; EDT 2%; TIS 1%, cut for 180min;

[0044] 12) Dry the lysis buffer as much as possible with nitrogen gas, precipitate diethyl ether, remove the supernatant by centrifugation, wash the precipitate with diethyl ether six times, and then evaporate to dryness at room temperature;

[0045] Step 2: Compound purification:

[0046] 1) Take a small amount of crude product and dissolve it in H2O / ACN.

[0047] 2) Take a small amount of sample and analyze it on an HPLC analyzer to determine the elution time of the target peak.

[0048] 3) Prepare the solution using a C18 reversed-phase chromatography system: Wavelength: 220 nm; Flow Rate: 15 ml / min; Inj. Vol: 20 mL Column; Temp: 25 °C; Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution.

[0049] 4) Take a small amount of the target peak solution in a 1.5ml centrifuge tube for mass spectrometry confirmation and purity detection.

[0050] 5) After lyophilizing the qualified target peak solution, dissolve it again with H2O / ACN and perform salt conversion treatment on an HPLC instrument. Perform a second lyophilization of the salt-converted solution to obtain the final product.

[0051] Thirdly, this invention provides a method for applying anti-inflammatory and anti-aging skincare linear peptides in cosmetics:

[0052] The anti-inflammatory and anti-aging skincare linear polypeptide provided by this invention can be used in cosmetics in forms including but not limited to: serums, creams, masks, etc. The method of using the anti-inflammatory and anti-aging skincare linear polypeptide in cosmetics is to disperse the polypeptide in solvents such as glycerin, propylene glycol, and butylene glycol and then add it to the cosmetic product.

[0053] Fourthly, this invention provides the application of anti-inflammatory and anti-aging skincare linear peptides in cosmetic essences:

[0054] The anti-inflammatory and anti-aging essence provided by this invention contains the following percentage components:

[0055] Phase A: water balance, EDTA-2Na 0.1%, carbomer 0.2%, xanthan gum 0.05%;

[0056] Phase B: p-hydroxyacetophenone 0.2%, phenoxyethanol 0.2%, ethylhexylglycerin 0.1%;

[0057] Phase C: 2% propylene glycol, linear peptides SEQ1-10 - 0.001% - 0.1%.

[0058] The steps for preparing the essence are as follows:

[0059] Add phase A sequentially to the reaction vessel, stir to disperse, heat to 80℃ and stir for 15-30 minutes;

[0060] After phase A is completely dispersed, add phase B, stir and cool to below 40°C;

[0061] Add the well-mixed C phase and stir to disperse evenly to obtain the final soothing and anti-aging essence.

[0062] Fourthly, this invention provides the application of an anti-inflammatory and anti-aging linear skincare polypeptide in the preparation of anti-inflammatory and / or anti-aging cosmetics, wherein the amino acid sequence of the polypeptide is as follows:

[0063] SEQ-1: Lys-Asp-Glu-Lys-Glu-Lys-Leu-Leu-Lys-Glu;

[0064] SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp;

[0065] SEQ-3: Lys-Asp-Glu-Lys-Glu-Lys-Ala-Leu-Lys-Glu;

[0066] SEQ-4: Arg-Lys-Glu-Tyr-Glu-Leu-Glu-Lys-Gln-Lys;

[0067] SEQ-5: Tyr-Leu-Glu-Val-Phe-Ser-Met-Ala-Arg-Asp-Glu-Asp;

[0068] SEQ-6: Leu-Glu-Val-Phe-Ser-Ala-Leu-Arg-Asp-Glu-Asp;

[0069] SEQ-7: Tyr-Leu-Glu-Val-Phe-Ser-Met-Leu-Arg-Asp-Glu-Asp;

[0070] SEQ-8: Lys-Asp-Glu-Lys-Glu-Ala-Leu-Leu-Lys-Glu;

[0071] SEQ-9: Asn-Thr-Asp-Phe-Gly-Asp-Glu-Phe-Tyr-Ser-Ala-Phe-Ile;

[0072] SEQ-10: Tyr-Glu-Leu-Glu-Lys-Gln-Lys-Lys-Leu-Glu.

[0073] By employing the above technical solution, the present invention has the following technical effects:

[0074] 1) This invention designs and develops a group of anti-inflammatory and anti-aging skincare peptides with excellent performance, including:

[0075] SEQ-1: Lys-Asp-Glu-Lys-Glu-Lys-Leu-Leu-Lys-Gl,

[0076] SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp,

[0077] SEQ-3: Lys-Asp-Glu-Lys-Glu-Lys-Ala-Leu-Lys-Glu,

[0078] SEQ-4: Arg-Lys-Glu-Tyr-Glu-Leu-Glu-Lys-Gln-Lys,

[0079] SEQ-5: Tyr-Leu-Glu-Val-Phe-Ser-Met-Ala-Arg-Asp-Glu-Asp,

[0080] SEQ-6: Leu-Glu-Val-Phe-Ser-Ala-Leu-Arg-Asp-Glu-Asp,

[0081] SEQ-7: Tyr-Leu-Glu-Val-Phe-Ser-Met-Leu-Arg-Asp-Glu-Asp,

[0082] SEQ-8: Lys-Asp-Glu-Lys-Glu-Ala-Leu-Leu-Lys-Glu,

[0083] SEQ-9: Asn-Thr-Asp-Phe-Gly-Asp-Glu-Phe-Tyr-Ser-Ala-Phe-Ile,

[0084] SEQ-10: Tyr-Glu-Leu-Glu-Lys-Gln-Lys-Lys-Leu-Glu;

[0085] 2) This invention provides a method for synthesizing anti-inflammatory and anti-aging skin care peptides, which can efficiently and in large quantities prepare high-purity active peptides;

[0086] 3) The anti-inflammatory and anti-aging skin care peptides designed and developed in this invention have excellent effects in inhibiting IL-1β inflammatory factors and TNF-α inflammatory factors and promoting collagen production, which can soothe itchy and red skin and reduce skin texture and facial wrinkles.

[0087] 4) This invention provides the application of anti-inflammatory and anti-aging skin care peptides in cosmetics, such as soothing and anti-aging essences. Attached Figure Description

[0088] Figure 1 The HPLC spectrum of SEQ-1;

[0089] Figure 2 The HPLC spectrum of SEQ-2;

[0090] Figure 3 The HPLC spectrum of SEQ-3;

[0091] Figure 4 The HPLC spectrum of SEQ-4;

[0092] Figure 5 The HPLC spectrum of SEQ-5;

[0093] Figure 6 The HPLC spectrum of SEQ-6;

[0094] Figure 7 The HPLC spectrum of SEQ-7;

[0095] Figure 8 The HPLC spectrum of SEQ-8;

[0096] Figure 9 The HPLC spectrum of SEQ-9;

[0097] Figure 10 The HPLC spectrum of SEQ-10;

[0098] Figure 11 A to F represent the results of HSF cell anti-aging activity assays performed using SEQ-1 to SEQ-6.

[0099] Figure 12 A to D are the results of HSF cell anti-aging activity assays performed using SEQ-7 to 10.

[0100] Figure 13 A to D are the microscopic examination results of the HSF cell anti-aging experiment in the blank model group before staining and the blank model group after staining.

[0101] Figure 14 A to D are the microscopic examination results of HSF cell anti-aging experiments performed on SEQ-1 to SEQ-4;

[0102] Figure 15 A to D are the microscopic examination results of HSF cell anti-aging experiments using SEQ-5 to 8;

[0103] Figure 16 A to B are the microscopic examination results of HSF cell anti-aging experiments using SEQ-9 to 10. Detailed Implementation

[0104] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below.

[0105] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0106] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0107] Numerous specific details are set forth in the following description to provide a full understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and similar modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0108] Examples 1-10

[0109] This invention provides a set of ten linear peptides, with the following sequences and structures:

[0110] SEQ-1: Lys-Asp-Glu-Lys-Glu-Lys-Leu-Leu-Lys-Glu

[0111] The structural formula of SEQ-1 is shown in Formula I:

[0112]

[0113] The HPLC chromatogram of SEQ-1 is as follows: Figure 1 As shown.

[0114] SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp

[0115] The structural formula of SEQ-2 is as shown in Formula II:

[0116]

[0117] The HPLC chromatogram of SEQ-2 is as follows Figure 2 As shown.

[0118] SEQ-3: Lys-Asp-Glu-Lys-Glu-Lys-Ala-Leu-Lys-Glu; The structural formula of SEQ-3 is as shown in Formula III:

[0119]

[0120] The HPLC chromatogram of SEQ-3 is as follows: Figure 3 As shown.

[0121] SEQ-4: Arg-Lys-Glu-Tyr-Glu-Leu-Glu-Lys-Gln-Lys; The structural formula of SEQ-4 is shown in Formula IV:

[0122]

[0123] The HPLC chromatogram of SEQ-4 is as follows Figure 4 As shown.

[0124] SEQ-5: Tyr-Leu-Glu-Val-Phe-Ser-Met-Ala-Arg-Asp-Glu-Asp; The structural formula of SEQ-5 is shown in formula V:

[0125]

[0126] The HPLC chromatogram of SEQ-5 is as follows: Figure 5 As shown.

[0127] SEQ-6: Leu-Glu-Val-Phe-Ser-Ala-Leu-Arg-Asp-Glu-Asp; The structural formula of SEQ-6 is shown in Formula VI:

[0128]

[0129] The HPLC chromatogram of SEQ-6 is as follows Figure 6 As shown.

[0130] SEQ-7: Tyr-Leu-Glu-Val-Phe-Ser-Met-Leu-Arg-Asp-Glu-Asp; The structural formula of SEQ-7 is as shown in formula VII:

[0131]

[0132] The HPLC chromatogram of SEQ-7 is as follows: Figure 7 As shown.

[0133] SEQ-8: Lys-Asp-Glu-Lys-Glu-Ala-Leu-Leu-Lys-Glu;

[0134] The structural formula of SEQ-8 ​​is as shown in formula VIII:

[0135]

[0136] The HPLC chromatogram of SEQ-8 ​​is as follows: Figure 8 As shown.

[0137] SEQ-9: Asn-Thr-Asp-Phe-Gly-Asp-Glu-Phe-Tyr-Ser-Ala-Phe-Ile; The structural formula of SEQ-9 is shown in formula IX:

[0138]

[0139] The HPLC chromatogram of SEQ-9 is as follows: Figure 9 As shown.

[0140] SEQ-10: Tyr-Glu-Leu-Glu-Lys-Gln-Lys-Lys-Leu-Glu; The structural formula of SEQ-10 is shown in formula X:

[0141]

[0142] The HPLC chromatogram of SEQ-10 is as follows: Figure 10 As shown.

[0143] The linear peptide is prepared by the following steps:

[0144] Step 1: Compound Synthesis

[0145] 1) Place 2-Chlorotrityl Chloride Resin into a reaction tube, add DMF (15 ml / g), and shake for 60 min;

[0146] 2) Filter the solvent through a sand filter, add 3 times the molar excess of Fmoc, then add 10 times the molar excess of DIEA, and finally add DMF to dissolve. Shake for 30 min; cap with methanol and let stand for 30 min.

[0147] 3) Remove DMF, add 20% pyridinium DMF solution (15 ml / g), 5 min, remove again, add 20% pyridinium DMF solution (15 ml / g), 15 min;

[0148] 4) Remove the piperidine solution, take a dozen or so resin grains, wash three times with ethanol, and add 2-3 drops of Kaiser's reagent.

[0149] Heat at 105℃-110℃ for 5 minutes; a deep blue color indicates a positive reaction.

[0150] 5) DMF (10ml / g) twice, methanol (10ml / g) twice, DMF (10ml / g) twice;

[0151] 6) Add 3 times molar excess of Fmoc, 3 times molar excess of HBTU, then add 10 times molar excess of DIEA, and finally add DMF to dissolve. Shake for 45 minutes.

[0152] 7) Take a dozen or so resin grains, wash them three times with ethanol, add 2-3 drops of Kaiser's reagent, heat at 105℃-110℃ for 5 minutes, and a colorless reaction indicates a negative reaction;

[0153] 8) DMF (10ml / g) once, methanol (10ml / g) twice, DMF (10ml / g) twice;

[0154] 9) Repeat steps three to eight, linking amino acids in the sequence from right to left until the Fmoc protecting group of the last amino acid is removed;

[0155] 10) DMF (10ml / g) twice, DCM (10ml / g) three times, methanol (10ml / g) four times, then vacuum dry for 10 minutes;

[0156] 11) Prepare the cutting fluid (10ml / g): TFA 95%; water 2%; EDT 2%; TIS 1%, cut for 180min;

[0157] 12) Dry the lysis buffer as much as possible with nitrogen gas, precipitate diethyl ether, remove the supernatant by centrifugation, wash the precipitate with diethyl ether six times, and then evaporate to dryness at room temperature;

[0158] Step 2: Compound purification:

[0159] 1) Take a small amount of crude product and dissolve it in H2O / ACN.

[0160] 2) Take a small amount of sample and analyze it on an HPLC analyzer to determine the elution time of the target peak.

[0161] 3) Prepare the solution using a C18 reversed-phase chromatography system: Wavelength: 220 nm; Flow Rate: 15 ml / min; Inj. Vol: 20 mL Column; Temp: 25 °C; Buffer A: 0.1% TFA in water; Buffer B: 0.1% TFA in Acetonitrile; collect the target peak solution.

[0162] 4) Take a small amount of the target peak solution in a 1.5ml centrifuge tube for mass spectrometry confirmation and purity detection.

[0163] 5) After lyophilizing the qualified target peak solution, dissolve it again with H2O / ACN and perform salt conversion treatment on an HPLC instrument. Perform a second lyophilization of the salt-converted solution to obtain the final product.

[0164] This invention provides a method for applying anti-inflammatory and anti-aging linear peptides in cosmetics:

[0165] The anti-inflammatory and anti-aging peptides provided by this invention can be used in cosmetics in various forms, including but not limited to: serums, creams, and masks. The linear peptides are used in cosmetics by dispersing them in solvents such as glycerin, propylene glycol, and butylene glycol before adding them to the cosmetic products.

[0166] Application Examples 1-10 and Comparative Examples 1-3

[0167] The preparation method of the product containing the anti-inflammatory and anti-aging skin care peptides of this invention is as follows:

[0168] Preparation of an extract containing anti-inflammatory and anti-aging linear peptides:

[0169] The essences used in Examples 1-10 and Comparative Examples 1-3 were prepared according to the weight percentages added as shown in Table 1, and the steps are as follows:

[0170] 1) Add phase A sequentially to the reaction vessel, stir to disperse, heat to 80℃ and keep stirring for 15-30 minutes;

[0171] 2) After phase A is completely dispersed, add phase B, stir and cool to below 40℃;

[0172] 3) Add the well-mixed C phase and stir to disperse evenly to obtain the final essence.

[0173] Table 1 Application Examples 1-10 and Comparative Examples 1-3 of the Anti-inflammatory and Anti-aging Essence

[0174]

[0175]

[0176] Effect Test Example 1

[0177] Human skin fibroblast HSF experiment

[0178] Test methods for the effect of type I collagen on cell viability promotion rate:

[0179] Human skin fibroblasts (HSFs) were cultured in DMEM complete medium. When the confluence rate reached 80%, the old medium was discarded, and medium containing 0.001% linear peptide was added for cell culture. After 24 hours of culture, the supernatant was aspirated, centrifuged at 10,000 rpm for 15 minutes to remove cell debris and exosomes. The supernatant of the centrifuged medium was used to detect the content of type I collagen. The promotion rate of type I collagen concentration was calculated using the formula [(drug-treated histone concentration - blank histone concentration) / blank histone concentration]. The results are shown in Table 2.

[0180] Table 2. Sample list and type I collagen promotion rate (%)

[0181]

[0182]

[0183] Previous studies have shown that palmitoyl tripeptide-38 has a significant anti-aging effect, therefore palmitoyl tripeptide-8 was selected as a positive control in this efficacy test.

[0184] As can be seen from Table 2, under the condition of adding 10 PPM, the linear peptide of the present invention can significantly promote the content of type I collagen in human skin fibroblasts. This indicates that the linear peptide of the present invention can significantly promote collagen production and inhibit aging, and enhance the expression of skin ECM.

[0185] Effect Test Example 2

[0186] Human skin fibroblast HSF experiment

[0187] HSF cell senescence model testing methods:

[0188] The detection of senescence-related β-galactosidase is a classic and effective method for detecting cellular senescence. X-Gal (5-bromo-4-chloro-3-indole-β-D-galactosidase) is a substrate for β-galactosidase. X-Gal itself is colorless, but after hydrolysis by β-galactosidase, it produces galactose and the blue 5-bromo-4-chloro-indigo. Utilizing this characteristic of X-Gal, the activity of β-galactosidase can be indirectly measured. The lysosomal contents in senescent cells typically increase, leading to elevated activity of the lysosomal enzyme β-galactosidase. Therefore, X-Gal can be used as a substrate to detect cellular senescence. Senescent cells turn blue after staining, while non-senescent cells remain colorless. By staining human skin fibroblasts cultured with different anti-aging peptides for β-galactosidase, their anti-aging activity can be determined. The remaining cells were added to CCK8 reagent and incubated at 37 degrees Celsius for 4 hours for cell viability testing. The cell viability enhancement results were processed in the same way as the collagen results.

[0189] After testing the anti-aging activity of ten linear peptides in HSF cells, the CCK-8 cell proliferation results were as follows: Figure 11-12 The results showed that linear peptides 1-10 all had good anti-aging effects at concentrations above 12.5 PPM, with linear peptides 2-10 showing a particularly significant proliferative effect at a concentration of 3.25 PPM.

[0190] The results of the β-galactosidase staining experiment are as follows: Figure 13-16 As shown. Figure 13 The staining results of the blank model group before and after β-galactosidase staining are shown. Figure 14 The staining results for the linear peptide 1-4β-galactosidase assay; Figure 15 The staining results for the linear peptide 5-8β-galactosidase assay; Figure 16 The staining results for the β-galactosidase experiment of linear peptides 9-10 are shown. Based on the staining area, the staining area of ​​linear peptides 1-10 is significantly smaller than that of the blank model group after staining, indicating that linear peptides 1-10 all have good anti-aging effects.

[0191] The results showed that all ten linear peptides could significantly improve cell aging, indicating that the ten linear peptides have excellent anti-aging ability.

[0192] Effect Test Example 3

[0193] Human immortalized keratinocyte HACAT experiment

[0194] HACAT cell inflammatory factor (TNF-α) inhibition assay method:

[0195] Tumor necrosis factor-α (TNF-α) is a multifunctional pro-inflammatory cytokine that plays a crucial role in inflammation, immune regulation, and apoptosis, and is therefore frequently used as a means of assessing inflammation suppression. ELISA is the most commonly used method for quantitative detection of TNF-α. Commercially available ELISA kits, such as sandwich ELISA kits, are typically used to detect TNF-α in serum, plasma, or cell culture supernatants. This allows for qualitative or quantitative detection of inflammatory cytokine levels in models, thereby determining its anti-inflammatory activity.

[0196] Human immortalized keratinocytes (HACAT) were cultured in DMEM complete medium. When the confluence rate reached 80%, the old medium was discarded, and medium containing 0.001% linear peptide was added for cell culture. After 24 hours of culture, lipopolysaccharide (LPS) was added to induce inflammation. After another 24 hours of culture, the supernatant was aspirated, centrifuged at 10,000 rpm for 15 minutes to remove cell debris and exosomes. The supernatant of the centrifuged medium was used to detect the TNF-α content. The TNF-α inflammatory factor production rate was calculated using the formula: [(drug group factor concentration - blank group factor concentration) / blank group factor concentration].

[0197] The results of the assay for inhibiting TNF-α inflammatory factors in HACAT cells using ten linear peptides are shown in Table 3.

[0198] Table 3. List of samples to be tested and TNF-α factor rate (%)

[0199]

[0200]

[0201] The results in Table 3 show that the TNF-α inflammatory factor production rate of the ten linear peptides was lower than that of the positive control group dexamethasone, thus indicating that all ten linear peptides have significant TNF-α inhibitory function.

[0202] Effect Test Example 4

[0203] Human immortalized keratinocyte HACAT experiment

[0204] HACAT cell inflammatory factor (IL-1β) inhibition assay method:

[0205] IL-1β is a major pro-inflammatory cytokine released by immune cells (such as macrophages and monocytes) during infection, injury, or immune activation. As a key mediator in immune and inflammatory responses, it is often used as an indicator of inflammation suppression. ELISA is a commonly used method for the quantitative detection of IL-1β. A sandwich ELISA method is typically used, in which a specific anti-IL-1β antibody is coated onto a microplate to capture IL-1β in the sample. The bound IL-1β is then detected by an enzyme-labeled secondary antibody, and quantification is achieved by measuring the color reaction, thereby determining its anti-inflammatory activity.

[0206] Human immortalized keratinocytes (HACAT) were cultured in DMEM complete medium. When the confluence rate reached 80%, the old medium was discarded, and medium containing 0.001% linear peptide was added for cell culture. After 24 hours of culture, lipopolysaccharide (LPS) was added to induce an inflammation model. After another 24 hours of culture, the supernatant was aspirated, centrifuged at 10,000 rpm for 15 minutes to remove cell debris and exosomes. The supernatant of the centrifuged medium was used to detect the IL-1β content. The IL-1β inflammatory factor production rate was calculated using the formula: [(drug group factor concentration - blank group factor concentration) / blank group factor concentration].

[0207] The results of the assay for inhibiting IL-1β inflammatory factors in HACAT cells using ten linear peptides are shown in Table 4.

[0208] Table 4. List of samples to be tested and IL-1β factor rate (%)

[0209]

[0210]

[0211] The results in Table 4 show that the IL-1β inflammatory factor production rate of the ten linear peptides was lower than that of the positive control group dipotassium glycyrrhizinate. Therefore, it can be concluded that all ten linear peptides have significant IL-1β inhibitory function.

[0212] Effect Test Example 5

[0213] Molecular docking verification

[0214] Molecular docking is a technique that uses computational simulations to predict the binding modes and affinities between small molecules (ligands) and biomolecules (receptors, such as protein channel switches). Its core objective is to find the optimal binding conformation of the ligand in the receptor's active pocket and to assess the strength of their interaction.

[0215] Binding energy is the energy released when a ligand binds to a receptor, usually expressed as ΔG (unit: kcal / mol). A negative ΔG value indicates that the binding process occurs spontaneously, and the larger the negative value (generally, -8 kcal / mol or -5 kcal / mol is considered to indicate that the ligand has an advantage in affinity to the target and stronger biological activity), the more stable the binding.

[0216] Target selection:

[0217] Anti-inflammatory target: NF-κB p65 (PDB ID: 1NFK)

[0218] Anti-aging target: TGF-β receptor I (TβR-I, PDB ID: 3KFD)

[0219] Connection method:

[0220] Software: AutoDock Vina 1.2.3, Force Field: AMBER14.

[0221] Parameters: Search space covers active pocket, exhaustiveness = 32.

[0222] The docking results of ten linear peptides and their binding energies ΔG are shown in Table 5.

[0223] Table 5. Binding energies of ten linear peptides to molecular docking.

[0224]

[0225]

[0226] The results in Table 5 show that the binding energies of the ten linear peptides are all much less than -8 kcal / mol, thus concluding that all ten linear forms have strong biological activity.

[0227] Effect Test Example 6

[0228] Method for testing the soothing and anti-aging effects of serums:

[0229] 1) Volunteers aged 18-40 were divided into 23 groups, with 6 people in each group (3 men and 3 women);

[0230] 2) Cleanse the subject's face with the same cleanser in a constant temperature and humidity environment;

[0231] 3) In groups 1 to 13, blank control samples were applied to the left side of the face and forehead, and samples from Application Examples 1 to 10 and Comparative Examples 1 to 3 were applied to the right side of the face and forehead, respectively. The sample amount was 2 ± 0.2 g.

[0232] 4) Use the VISIA facial analyzer to measure facial wrinkles at 12 hours and 24 hours, the area of ​​the red zone, and three sets of data for each symmetrical area, and calculate the average value.

[0233] 5) The anti-aging effect of the serum is evaluated based on the percentile score (the percentile score describes the skin characteristics score of the test subject compared with others of the same age, gender and skin type, the higher the score, the better) given by the VISIA facial analyzer and the improvement value (score after improvement - score before improvement).

[0234] The test results of the soothing and anti-aging serum are shown in Table 6:

[0235] Table 6. Test results of soothing and anti-aging serums (number of participants)

[0236]

[0237]

[0238] The results in Table 6 show that in the 12-hour test for groups 1-10, the percentage of participants in the 10-20 range of facial and forehead texture improvement and red area improvement values ​​was ≥50% (average 66.7%). In the 24-hour test, the percentage of participants in the 10-20 range of facial and forehead texture improvement and red area improvement values ​​was ≥83.3% (average 89.2%). This indicates that applications 1-10 have excellent soothing and anti-aging effects. In Table 6, the texture improvement values ​​of most participants in groups 11-13 were less than 10%, and the difference between the 12-hour and 24-hour improvement rates was ≤33.3%. This indicates that the control group also has some soothing and anti-aging effects, but the effects are relatively weak.

[0239] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. The application of a polypeptide in the preparation of cosmetics, characterized in that, The amino acid sequence of the polypeptide is as follows: SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp.

2. The application of the polypeptide as described in claim 1 in the preparation of cosmetics, characterized in that: The cosmetic applications include serums, creams, and masks.

3. The application of the polypeptide as described in claim 2 in the preparation of cosmetics, characterized in that: The method for preparing cosmetics involves dispersing the polypeptide in glycerol, propylene glycol, and butylene glycol solvents and then adding it to the cosmetic product.

4. The application of the polypeptide as described in claim 3 in the preparation of cosmetics, characterized in that: The cosmetic product is in the form of a serum, and the serum contains the following components by weight percentage: Phase A: Water balance, EDTA-2Na 0.1%, Carbomer 0.2%, Xanthan gum 0.05%; Phase B: p-hydroxyacetophenone 0.2%, phenoxyethanol 0.2%, ethylhexylglycerin 0.1%; Phase C: Propylene glycol 2%, polypeptide SEQ-2: Tyr-Leu-Glu-Val-Phe-Ala-Met-Leu-Arg-Asp-Glu-Asp 0.001%-0.1%.

5. The application of the polypeptide as described in claim 4 in the preparation of cosmetics, characterized in that, The steps for preparing the essence are as follows: Add phase A sequentially to the reaction vessel, stir to disperse, heat to 80℃ and stir for 15-30 minutes; After phase A is completely dispersed, add phase B, stir and cool to below 40°C; Add the well-mixed C phase and stir to disperse evenly to obtain the essence.

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