Acetyl hexapeptide as well as preparation method and application thereof

Abstract

The invention relates to acetyl hexapeptide as well as a preparation method and application thereof. The acetyl hexapeptide comprises hexapeptide of which the amino acid sequence is EMEQRR. The acetyl hexapeptide disclosed by the invention not only can play an anti-aging effect at low concentration, but also shows better safety and higher bioavailability.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and cosmetics, specifically relating to a class of acetyl hexapeptides, their preparation methods, and uses. Background Technology

[0002] Peptides, as a class of signaling molecules, are widely used in skincare products. One type of botulinum toxin-like peptide, such as acetyl hexapeptide-8, is widely used in anti-aging products as a safe and effective peptide. This type of peptide can mimic the six N-terminal amino acid oligopeptides of the SNAP-25 protein, acting on the neuromuscular junction. By competing with SNAP-25 for its site in the vesicle-melting complex, it affects the formation of the vesicle-melting complex, preventing the effective release of neurotransmitters from vesicles and reducing wrinkles caused by facial muscle contractions, resulting in a significant wrinkle-reducing effect. Although this peptide is widely used in skincare products, its effective concentration remains relatively high. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention designs and synthesizes an acetyl hexapeptide to improve bioavailability and reduce its effective concentration.

[0004] According to one aspect of the present invention, an acetyl hexapeptide is provided, the acetyl hexapeptide comprising the amino acid sequence EMEQRR, namely arginine (Arg)-arginine (Arg)-glutamine (Gln)-glutamic acid (Glu)-methionine (Met)-glutamic acid (Glu).

[0005] In some embodiments, the N-terminus of the hexapeptide is acetylated.

[0006] In some embodiments, the acetyl hexapeptide has the following structure: .

[0007] According to another aspect of the present invention, a method for preparing the acetyl hexapeptide of the present invention is provided, the method comprising performing a condensation reaction of a hexapeptide with the amino acid sequence EMEQRR and acetic acid.

[0008] In some embodiments, the molar ratio of the acetic acid to the hexapeptide is 1:(1.0-1.5), for example, 1:0.5, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any value between them.

[0009] In some embodiments, the condensation reaction uses 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) as condensing agents.

[0010] In some embodiments, the molar ratio of acetic acid to NHS is 1:(1.0-1.5), for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any value between them.

[0011] In some embodiments, the molar ratio of acetic acid to EDC is 1:(1.0-1.5), for example, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5 or any value between them.

[0012] In some embodiments, the condensation reaction time is 0.1 h to 24 h, for example, 0.1 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 10 h, 15 h, 20 h, 24 h, or any value between them. In some preferred embodiments, the condensation reaction time is 0.1 h to 4 h.

[0013] In some embodiments, the temperature of the condensation reaction is 0-80°C, for example, 0°C, 5°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C or any value between them.

[0014] In some embodiments, the condensation reaction uses TFA, anisole, ethylene dithiol, phenol, and water as cleavage agents.

[0015] In some embodiments, the volume ratio of TFA, anisole, ethylene dithiol, phenol, and water is (80:1:1:0.5:0.5) to (90:10:10:8:2). In some specific embodiments, the volume ratio of TFA, anisole, ethylene dithiol, phenol, and water is 85:5:5:4:1.

[0016] In some embodiments, the hexapeptide is prepared using a solid-phase synthesis method. In some embodiments, the preparation method of the hexapeptide includes: linking amino acids to an insoluble resin, then performing a coupling reaction and a deprotection reaction to link the amino acids, and finally cleaving the hexapeptide molecule from the resin. In some embodiments, the insoluble resin is a Wang resin.

[0017] In some embodiments, the hexapeptide can be prepared by biosynthesis. To this end, the present invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding the hexapeptide. The present invention also provides an expression vector comprising the nucleic acid molecule described herein. The present invention also provides a host cell comprising the nucleic acid molecule described herein or the expression vector described herein.

[0018] According to another aspect of the present invention, the use of the acetyl hexapeptide described herein or the acetyl hexapeptide prepared by the preparation method thereof in the field of skin care products is provided.

[0019] According to another aspect of the present invention, a skin care product is provided, the skin care product comprising an active ingredient and the acetyl hexapeptide described in the present invention or the acetyl hexapeptide prepared by the preparation method described in the present invention.

[0020] In some embodiments, the acetyl hexapeptide is present in the skin care product at a mass content of 0.05-1%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 1%, or any value between them.

[0021] In some embodiments, the active ingredient is a skin-care active ingredient, such as a whitening or acne-reducing ingredient, including but not limited to: tranexamic acid, niacin, niacinamide, salicylic acid, etc.

[0022] In some embodiments, the skin care product may be a skin care lotion, cream, mask, etc.

[0023] The acetyl hexapeptide of the present invention not only exerts anti-aging effects at low concentrations, but also demonstrates better safety and higher bioavailability. Attached Figure Description

[0024] Figure 1 The infrared spectrum of acetyl hexapeptide-13 is shown.

[0025] Figure 2 The NMR spectrum of acetyl hexapeptide-13 is shown.

[0026] Figure 3 The results show a comparison of transdermal fluorescence images of acetyl hexapeptide-13 (AHP-13) and acetyl hexapeptide-8 (AHP-8).

[0027] Figure 4 Showing Figure 3 Quantitative statistical results of fluorescence images.

[0028] Figure 5 The image shows a frozen section (collagen) of the skin after treatment with the serum prepared in Example 3.

[0029] Figure 6 Showing Figure 5 Quantitative statistical results of total collagen in frozen section images.

[0030] Figure 7 Images of frozen sections of skin treated with the emulsion prepared in Example 4 are shown.

[0031] Figure 8Showing Figure 7 Quantitative statistical results of collagen I in frozen section images.

[0032] Figure 9 Images of frozen sections of skin treated with the emulsion prepared in Example 4 are shown.

[0033] Figure 10 Showing Figure 7 Quantitative statistical results of collagen III in frozen section images. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0035] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the reagents used in the following embodiments are conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or by existing methods; unless otherwise specified, the reagent dosages are those used in routine experimental operations; unless otherwise specified, the experimental methods are conventional methods.

[0036] In this document, the amino acid sequences of the peptides described herein are as follows: R represents arginine (Arg), Q represents glutamine (Gln), E represents glutamic acid (Glu), and M represents methionine (Met). Unless otherwise stated, the amino acids (residues) mentioned herein may be D-type or L-type. Unless otherwise stated, the amino acid sequences mentioned herein are in the N-terminal to C-terminal order from left to right.

[0037] The acetyl hexapeptide-8 (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Ac-EEMQRR-NH2) used in this article is also called acereline.

[0038] Example 1 Acetyl hexapeptide-13 was prepared according to the following steps: (1) Preparation of polypeptides Preparation of Fmoc-Arg-Wang resin: Weigh 5 g of Wang resin with a substitution degree of 0.85 mM / g and add it to a solid-phase reaction column. Wash twice with 10 mL of N,N-dimethylformamide (DMF), then add 35 mL of DMF to swell for 30 min. Filter the filtrate and dissolve 6.49 g of Fmoc-Glu-OH and 1.38 g of 1-hydroxybenzotriazole (HOBt) in 30 mL of DMF. Add 1.58 mL of N,N'-diisopropylcarbodiimide (DIC) under ice bath conditions and activate by standing in the dark for 5 min. Add the above solution to the solid-phase reaction column containing the fully swollen resin, add 0.11 g of dimethylaminopyridine (DMAP), and stir under nitrogen protection for 3 h. Monitor the reaction completion using the ninhydrin colorimetric method. Remove the solvent, wash three times each with DMF and dichloromethane (DCM), add 50 mL of blocking buffer (acetic anhydride-pyridine, volume ratio 7:3), and block for 6 h. After removing the liquid, the resin was washed four times with DCM, shrunk with methanol (MeOH), and vacuum dried to obtain 7.56 g of Fmoc-Arg-Wang resin. The degree of substitution was 0.32 mM / g, the resin weight gain was 1.07 g, and the yield was 90.42%.

[0039] Peptide chain elongation: Weigh 5.0 g of Fmoc-Arg-Wang resin and add it to a solid-phase reaction column. Wash twice with 10 mL of DMF to remove the solvent, then add 35 mL of DMF and allow it to swell for 30 min. Wash twice with DMF, add a piperidine-DMF mixed solution (volume ratio 1:3), and stir for 15 min. Monitor the reaction completion using the ninhydrin colorimetric method. Wash 5 times each with DMF and DCM, dissolve 2.06 g of Fmoc-Arg-OH and 0.52 g of HOBt in DMF, add 0.59 mL of DIC under ice bath conditions, stir for 5 min in the dark, and then add it to the solid-phase reaction column where the solvent has been removed. Add 0.04 g of DMAP, and stir under nitrogen protection for 3 h. Monitor the reaction completion using the ninhydrin colorimetric method. Remove the solvent and wash 5 times with DMF to obtain Fmoc-Met-Glu-Wang resin. Following the above coupling method, add the corresponding Fmoc protected amino acids according to the peptide sequence "EMEQRR" to sequentially condense and couple to extend the peptide chain. After the final coupling reaction, the resin was washed four times each with DCM, DMF, and MeOH, and then dried under vacuum to constant weight to obtain 6.23 g of fully protected polypeptide fragment X-Wang resin. The resin weight gain was 1.23 g, and the yield was 76.32%.

[0040] Repeat the above steps to obtain Wang-EMEQRR resin, with a resin weight gain of 1.2g and a yield of 75.1%.

[0041] (2) Acetic acid and polypeptide undergo a condensation reaction. Weigh 0.3 g of acetic acid and dissolve it in 15 mL of DMF. Under ice bath conditions, add 0.22 g of EDC and 0.16 g of NHS, stir for 2 h, then add 1.0 g of the Wang-EMEQRR resin prepared above, and continue the reaction overnight. After filtering out the DMF, wash five more times with DMF to obtain the Wang-EMEQRR-Ac resin.

[0042] The prepared Wang-EMEQRR-Ac resin was transferred to a 500 mL three-necked flask, and 250 mL of freshly prepared cleavage reagent TFA: benzyl sulfide (PhSMe): ethylene dithiol (EDT): phenol (PhOH): H2O (volume ratio 85:5:5:4:1) was added. The mixture was reacted at 25°C for 3 h. The mixture was filtered, and the filtrate was poured into 1 L of ice-cold diethyl ether for precipitation. The precipitate was then allowed to stand overnight at 0°C. The filtrate was then filtered separately and precipitated with 30 mL of ice-cold diethyl ether. 5), Ethyl acetate (30 mL) 5) The filter cake was washed and freeze-dried to obtain acetyl hexapeptide (EMEQRR-Ac) as shown in Formula I (named acetyl hexapeptide-13 in this application). The total yield of crude peptide was 25.65%, and the purity of crude peptide was 52.35% as determined by analytical HPLC.

[0043] The structural characterization of the acetyl hexapeptide-13 prepared in this embodiment is as follows: Figure 1 and Figure 2 As shown in the figure, it can be seen that the present invention has successfully prepared EMEQRR-Ac with the following structure.

[0044]

[0045] Example 2. Transdermal absorption assay of acetyl hexapeptide (a) Preparation of fluorescently labeled acetyl hexapeptide-13 0.83 g of acetyl hexapeptide-13 prepared in Example 1 was dissolved in a 0.1 M pH 5.5 buffer solution. Then, under ice bath conditions, 0.22 g of EDC, 0.16 g of NHS and 0.1 g of the fluorescent probe rhodamine were added. After reacting for 6 h, unreacted rhodamine was removed by dialysis. After freeze-drying, fluorescently labeled acetyl hexapeptide-13 was obtained.

[0046] (b) Preparation of fluorescently labeled acetyl hexapeptide-8 0.83 g of acetyl hexapeptide-8 was dissolved in 0.1 M pH 5.5 buffer solution. Then, under ice bath conditions, 0.22 g of EDC, 0.16 g of NHS and 0.1 g of the fluorescent probe rhodamine were added. After reacting for 6 h, unreacted rhodamine was removed by dialysis. After freeze-drying, fluorescently labeled acetyl hexapeptide-8 was obtained.

[0047] (c) Transdermal assay method: Fluorescently labeled acetyl hexapeptide-8 or acetyl hexapeptide-13 were added to the blank emulsion at a mass concentration of 0.1%. Live Kunming mice (KM mice) were used as the research subjects. Three mice were applied to the skin of each group. At 2 and 8 hours later, the mice were euthanized simultaneously by cervical dislocation, and approximately 0.5 cm of the central area of ​​the treated area was excised. 0.5 cm pieces of skin tissue were rinsed with PBS to remove residual drugs and fixed in formalin for 24 hours. After fixation, the skin tissue was rinsed, dehydrated, cleared, paraffin-embedded, and then sectioned to a thickness of 4–6 mm. The sections were then dewaxed, dehydrated, cleared, unstained, and unmounted. The sections were then scanned.

[0048] The composition of the blank emulsion is shown in Table 1 below:

[0049] Transdermal absorption images of acetyl hexapeptide-13 and acetyl hexapeptide-8 are shown below. Figures 3-4 As shown in the figure. The results indicate that the acetyl hexapeptide-13 of this application has a significantly improved transdermal absorption capacity compared to acetyl hexapeptide-8.

[0050] Example 3. Preparation of an essence containing acetyl hexapeptide-13 0.1 g of acetyl hexapeptide-13 prepared in Example 1 was dissolved in 5 mL of aqueous solution and used as the active ingredient.

[0051] 1. Sodium hyaluronate 0.15g, glycerin 50g, EMT10 0.5g, jojoba wax PEG-120 ester 4g, PEG20-methyl dextran 4g, tremella polysaccharide 3g, p-hydroxyacetophenone 3g, deionized water 920.35g, heated to 85℃, mixed and homogenized to obtain a mixture; 2. After the temperature drops to 45℃, add acetyl hexapeptide-13 to the above solution and stir until fully dissolved; 3. Add 10g of 1,2-pentanediol to the above solution, stir thoroughly to obtain essence a, cool to room temperature, discharge and let stand.

[0052] Comparative Example 1. Preparation of an essence containing acetyl hexapeptide-8 1. Sodium hyaluronate 0.15g, glycerin 50g, EMT10 0.5g, jojoba wax PEG-120 ester 4g, PEG20-methyl dextran 4g, tremella polysaccharide 3g, p-hydroxyacetophenone 3g, deionized water 920.35g, heated to 85℃, mixed and homogenized to obtain a mixture; 2. After the temperature drops to 45℃, add acetyl hexapeptide-8 to the above solution and stir until fully dissolved; 3. Add 10g of 1,2-pentanediol to the above solution, stir thoroughly to obtain essence b, cool to room temperature, discharge and let stand.

[0053] Example 4. Preparation of an emulsion containing acetyl hexapeptide-13 1. Dissolve 0.2 g of acetyl hexapeptide-13 prepared in Example 1 in 5 mL of aqueous solution to obtain functionalized acetyl hexapeptide-13.

[0054] 2. Heat 20g of emulsifier G66, 15g of Dow Corning silicone oil PMX-200 5cst, and 20g of XF49-811 to 85℃ to obtain mixture A; 3. Place 20g of butanediol, 50g of glycerol, 0.1g of sodium hyaluronate, 0.2g of erythritol, 3g of p-hydroxyacetophenone, and 856.7g of deionized water into a mixing pot and stir to dissolve while heating to 85-95℃ to ensure complete dissolution. Keep warm for 15-20 minutes to obtain mixture B. 4. Add mixture A to mixture B and stir for 40 minutes to obtain mixture C; 5. Cool mixture C to 40-45℃, add 5 mL of acetyl hexapeptide-13 prepared in step 1, stir to dissolve, and obtain mixture D; 6. While maintaining a temperature of 40-45℃, add 10g of 1,2-diol to mixture D, stir thoroughly to obtain emulsion c, cool to room temperature, discharge and let stand.

[0055] Comparative Example 2. Preparation of an emulsion containing acetyl hexapeptide-8 1. Dissolve 0.2g of acetyl hexapeptide-8 in 5mL of aqueous solution to obtain functionalized acetyl hexapeptide-8.

[0056] 2. Heat 20g of emulsifier G66, 15g of Dow Corning silicone oil PMX-200 5cst, and 20g of XF49-811 to 85 degrees Celsius to obtain mixture A; 3. Place 20g of butanediol, 50g of glycerol, 0.1g of sodium hyaluronate, 0.2g of erythritol, 3g of p-hydroxyacetophenone, and 856.7g of deionized water into a mixing pot and stir to dissolve while heating to 85-95℃ to ensure complete dissolution. Keep warm for 15-20 minutes to obtain mixture B. 4. Add mixture A to mixture B and stir for 40 minutes to obtain mixture C; 5. Cool mixture C to 40-45℃, add 5 mL of acetyl hexapeptide-8 prepared in step 1, stir to dissolve, and obtain mixture D; 6. While maintaining a temperature of 40-45℃, add 10g of 1,2-diol to mixture D, stir thoroughly to obtain emulsion d, cool to room temperature, discharge and let stand.

[0057] Application Example 1: Effects of polypeptide derivatives on human keratinocytes (1) Cell culture and treatment Human keratinocyte cell line HaCaT cells (Beijing Union Medical College Cell Resource Center) adhered and grew on 1640 medium containing 10% fetal bovine serum, incubated at 37°C (5% CO2, 95% air), with the medium changed every other day. Acetyl hexapeptide-13 and acetyl hexapeptide-8 were dissolved in dimethyl sulfoxide at a concentration of 0.01M. HaCaT cells in logarithmic growth phase were cultured for 36 h in 1640 medium containing 1 μg / mL and 5 μg / mL of acetyl hexapeptide-13 (Example 1), acetyl hexapeptide-8, and acetyl hexapeptide-free medium (control group), respectively, and total RNA was extracted from the cells.

[0058] (2) Extraction and reverse transcription of total RNA from cells RNA extraction was performed using the Fast200 kit: 500 μL of RA2 solution was added to the cells and thoroughly mixed by inverting for 1 min. The mixture was then centrifuged for 1 min. 500 μL of washing buffer was added to the inner tube, and the mixture was centrifuged for 1 min. This process was repeated once. 25 μL of elution buffer was added to the center of the membrane to obtain total RNA. The RNA was reverse transcribed into cDNA according to the instructions of the Takara reverse transcription PCR kit and stored at -70°C for later use.

[0059] (3) Real-time quantitative PCR The reverse-transcribed cDNA was amplified by PCR to obtain the target gene fragment. Reaction conditions: 95°C for 30 s pre-denaturation, 95°C for 5 s, annealing at 60°C for 30 s, for a total of 40 cycles. Products were identified by 2% agarose gel electrophoresis. Three duplicates were prepared for each reaction. GAPDH was used as an internal control. The expression level of the target gene was calculated using the formula ΔΔCT = [CT(target gene) - CT(GAPDH)]drug-treated group - [CT(target gene) - CT(GAPDH)]control group.

[0060] (4) Detect the expression of related genes The S100A7A protein plays a potential role in skin homeostasis, epidermal differentiation, and inflammation, highlighting its importance in skin health and disease. KRT14 protein is one of the signals for epidermal wound healing in activated keratinocytes. RARG protein is a receptor for retinoic acid (RA) and plays an important role in cell growth, differentiation, and apoptosis. CXCL3 protein is a major regulator of skin immune and inflammatory responses. SERRINB1 protein plays a crucial role in preventing tissue damage and inflammatory responses. HAS3 protein, or hyaluronic acid-binding protein 3, is a widely expressed protein on the cell surface that interacts with hyaluronic acid (HA) molecules and plays an important role in cell adhesion, migration, proliferation, and differentiation. TGM1 protein maintains skin integrity and function by regulating the aggregation of keratinocyte cytoskeleton proteins, forming a stable intercellular matrix structure.

[0061] The expression of relevant genes in cells after different treatments is shown in Table 2 below.

[0062] Table 2

[0063] As shown in Table 2, after treatment with acetyl hexapeptide-13 of the present invention, S100A7A (which plays a potential role in epidermal differentiation and inflammation, indicating its key role in promoting complex processes of skin homeostasis and immune response), KRT14 (an intermediate filament protein expressed in epidermal cells, which, together with keratin 5, participates in maintaining the structure and function of skin cells), RARG (this gene plays a role in various physiological and pathological processes by regulating biological processes such as cell growth and differentiation), and CXCL3 (an important chemokine that plays a role in the body's immune defense, inflammatory response, tissue repair, and tumor progression) are all enhanced. The gene expression levels of acetyl hexapeptide-13 of the present invention were increased, while the gene expression level of HAS3 (an enzyme molecule involved in hyaluronic acid synthesis that plays an important role in epithelial formation, wound healing, and tumor cell invasion and proliferation) was decreased. This indicates that acetyl hexapeptide-13 of the present invention can delay the rate of skin aging.

[0064] Application Example 2: Safety Testing of Peptide Derivatives (1) Chicken embryo chorioallantoic membrane vascular test The chorioallantoic membrane (CAM) is a respiratory membrane surrounding the chicken embryo. This experiment utilizes the intact, clear, and transparent chorioallantoic vascular system of mid-stage hatched chicken embryos. A certain amount of the test substance is directly exposed to the chorioallantoic membrane. After a period of time, changes in chorioallantoic membrane toxicity indicators (such as hemorrhage, coagulation, and angiolysis) are observed. These indicators reflect changes in the morphology, color, and permeability of blood vessels and vascular networks, as well as phenomena such as chorioallantoic membrane protein denaturation and the degree of damage. These indicators are then combined to obtain a score used to assess the eye irritation of the test substance.

[0065] Experimental Methods: The test was conducted according to SN / T2329-2009 "Eye Irritation / Corrosivity Test of Chicken Embryo Villi Allantoic Membrane in Cosmetics". The evaluation criteria for the chicken embryo villonoalanine membrane irritation scoring method are shown in Table 3 below.

[0066] Table 3 Evaluation of the results of the chicken embryo chorioallantoic membrane stimulation scoring method

[0067] The experimental results of the toxic effects of the acetyl hexapeptide-13 sample from Example 1 on the chorioallantoic membrane of chicken embryos are shown in Table 4 below.

[0068] Table 4

[0069] The results in Table 4 show that the acetyl hexapeptide-13 prepared in Example 1 is non-irritating to the skin.

[0070] (2) Human skin patch test (closed type) Experimental basis: Cosmetic Safety Technical Specifications (2015 Edition) Experimental Methods: Using qualified patch testing equipment, a closed patch test was conducted. 0.02g-0.025g of the test substance was placed inside the testing equipment, and a hypoallergenic adhesive tape was applied to the flexor aspect of the subject's forearm. The test substance was removed after 24 hours. Skin reactions were observed at 0.5, 24, and 48 hours after removal, and the results were recorded according to the skin reaction grading standards in the *Cosmetic Safety Technical Specifications* (2015 edition). The skin reaction grading standards for the closed patch test are shown in Table 5 below.

[0071] Table 5. Grading Criteria for Skin Reactions in Closed Patch Tests

[0072] The test results showed that no adverse skin reactions occurred with the acetyl hexapeptide-13 prepared in Example 1. The specific test results are shown in Table 6. Table 6

[0073] Application Example 3: Application of peptide derivatives in aging mouse models (1) The effect of the serum in Example 3 on promoting the production of total collagen in the skin Eighty 12-month-old Babl / c mice were randomly divided into four groups: an aging model group (D-Gal), an acetyl hexapeptide-8 treatment group (AHP-8 group, administered at doses of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively), an acetyl hexapeptide-13 treatment group (AHP-13 group, administered at doses of 50, 100, and 200 μg / mL, respectively), and a control group (Control group), with 10 mice in each group. The aging model group received a subcutaneous injection of 5% D-galactose (1 g / kg) into the neck and back of the mice daily for 42 consecutive days. The acetyl hexapeptide-8 treatment group received D-galactose at regular intervals similar to the aging model group, and simultaneously received essence b prepared in Comparative Example 1. The acetyl hexapeptide-13 treatment group received daily skin application of essence a prepared in Example 3, similar to the aging model group. After 4 weeks of treatment, skin samples were taken from the same location, processed, and the total collagen content in the skin was measured using the Masson staining method.

[0074] The steps for detecting total collagen in tissues using Masson staining are as follows: 1. Preparation of tissue specimens Skin tissue was removed from the drug administration site using a scalpel. The removed tissue block was then cleaned with physiological saline, placed in a 10% formalin solution, dehydrated, embedded in paraffin, and prepared into tissue sections with a thickness of 0.4 mm. The sections were baked and stored at -20°C.

[0075] 2. Masson staining method Masson staining was performed using the Masson trichrome staining kit (Nanjing Beyotime). The specific steps are as follows: (1) First, according to the kit instructions, place the slide in Bouin solution and mordant it in a 37°C incubator for two hours. Then, wash the wax slide with water to remove the yellow color; (2) stain with azurite blue and Mayer hematoxylin respectively, and wash with water after three minutes each; (3) differentiate with acidic ethanol differentiation solution for 5 seconds, rinse with running water for 10 minutes, then stain with Ponceau S and fuchsin staining solution for 10 minutes, and rinse slightly with deionized water; (4) treat with phosphomolybdic acid solution for 5 minutes, pour off the liquid on the slide, the slide does not need to be washed with water, directly drop the slide with aniline blue staining solution, stain for 5 minutes, and then treat with weak acid solution for 2 minutes; (5) treat with 95% ethanol for 10 seconds, 100% ethanol I for 10 seconds, 100% ethanol II for 10 seconds, 100% ethanol III for 10 seconds, xylene I for 2 minutes, xylene II for 2 minutes, and xylene III for 2 minutes; (6) place the treated slide in a ventilated and dry place for 5 minutes, and mount with neutral resin; (7) observe and photograph under a microscope.

[0076] The effects of the serums in Example 3 and Comparative Example 1 on total collagen production in the skin are as follows: Figure 5 and Figure 6 As shown in the figure, the results indicate that acetyl hexapeptide-13 treatment significantly increased the collagen content in the skin compared to acetyl hexapeptide-8. Furthermore, the change in skin collagen content was dose-dependent with the amount of acetyl hexapeptide-13 added.

[0077] (2) The promoting effect of the emulsion in Example 4 on the production of type I and type III collagen Eighty 12-month-old Babl / c mice were randomly divided into four groups: an aging model group (D-Gal), an acetyl hexapeptide-8 treatment group (AHP-8 group, administered at doses of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively), an acetyl hexapeptide-13 treatment group (AHP-13 group, administered at doses of 50, 100, and 200 μg / mL, respectively), and a blank control group (Control group), with 10 mice in each group. The aging model group received a subcutaneous injection of 5% D-galactose 1 g / kg daily in the neck and back for 42 consecutive days. The acetyl hexapeptide-8 treatment group received D-galactose regularly as in the aging model group, and simultaneously received emulsion d prepared in Comparative Example 2. The acetyl hexapeptide-13 treatment group received D-galactose applied to the skin regularly as in the aging model group, and simultaneously received emulsion c prepared in Example 4. After 4 weeks of treatment, skin samples were taken from the same area, processed, and then immunostained to test the content of collagen I and collagen III in the skin.

[0078] The steps for detecting collagen in tissues using immunohistoscopy are as follows: 1. Preparation of tissue specimens Skin tissue was removed from the drug administration site using a scalpel. The removed tissue block was then cleaned with physiological saline, placed in a 10% formalin solution, dehydrated, embedded in paraffin, and prepared into tissue sections with a thickness of 0.4 mm. The sections were baked and stored at -20°C.

[0079] 2. Immunohistochemical detection of type I and type III collagen expression After dewaxing and dehydrating the preserved tissue sections, proceed with the following steps: (1) Heat antigen retrieval (heat to 98°C with citrate buffer) for 6 min, repeat retrieval after 8-10 min interval, and allow to cool naturally; (2) Rinse with PBS solution for 5 min × 3 times; (3) Soak the slide with 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase activity; (4) Rinse with PBS solution for 5 min × 3 times; (5) Block the antigen with goat serum for 20 min, absorb excess liquid with filter paper, and do not wash with water; (6) Add an appropriately diluted (1:100) primary antibody (anti-mouse type I and III collagen polyclonal antibody; Sigmaaldrich, catalog number: AB765P; C7805) and incubate overnight at 4°C; (7) Rinse with PBS solution for 5 min × 3 times. (8) Add biotinylated secondary antibody (ThermoFisher, catalog number: B2763) working solution and incubate at 37°C for 20 min; (9) Rinse with PBS solution for 5 min × 3 times; (10) After treatment with horseradish-labeled streptavidin, incubate at 37°C for 20 min; (11) Rinse with PBS solution for 5 min × 3 times; (12) DAB color development: Take 850 μL of distilled water, add one drop each of solution A, B, and C from the DAB color development kit to the slide under the dark, mix well, add to the slide, develop color for 1 min, and rinse with tap water for 1 min; (13) Counterstain with hematoxylin for 5 min, dehydrate with gradient alcohol, clear with xylene, dry and then mount with resin, take pictures under a microscope, and perform statistical analysis using Image J.

[0080] The effects of the emulsions from Example 4 and Comparative Example 2 on the production of collagen I in the skin are as follows: Figures 7-8 As shown, the results of the effect on collagen III production are as follows: Figures 9-10 As shown in the figure, the results indicate that acetyl hexapeptide-13 treatment significantly increased the levels of collagen I and collagen III in the skin compared to acetyl hexapeptide-8. Furthermore, the changes in the levels of collagen I and collagen III in the skin were dose-dependent with the amount of acetyl hexapeptide-13 added.

[0081] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. An acetyl hexapeptide, characterized in that, The acetyl hexapeptide comprises a hexapeptide with the amino acid sequence EMEQRR.

2. The acetyl hexapeptide according to claim 1, characterized in that, The N-terminus of the acetyl hexapeptide is acetylated.

3. The acetyl hexapeptide according to claim 1, characterized in that, The acetyl hexapeptide has the following structure: 。 4. A method for preparing an acetyl hexapeptide according to any one of claims 1-3, characterized in that, The preparation method includes a condensation reaction of a hexapeptide with the amino acid sequence EMEQRR and acetic acid.

5. The preparation method according to claim 4, characterized in that, The hexapeptide was prepared by solid-phase synthesis; and / or The molar ratio of acetic acid to the hexapeptide is 1:(0.5-1.5).

6. The preparation method according to claim 4, characterized in that, The condensation reaction uses 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) as condensing agents; and / or The condensation reaction uses trifluoroacetic acid (TFA), anisole, ethylene dithiol, phenol, and water as cleavage agents; and / or, The condensation reaction takes 0.1 h to 24 h; and / or The condensation reaction is carried out at a temperature of 0-80℃.

7. The preparation method according to claim 6, characterized in that, The molar ratio of acetic acid to NHS is 1:(1.0-1.5); and / or The molar ratio of acetic acid to EDC is 1:(1.0-1.5); and / or The volume ratio of TFA, anisole, ethylene dithiol, phenol and water is (80:1:1:0.5:0.5) - (90:10:10:8:2).

8. Use of the acetyl hexapeptide according to any one of claims 1-3 or the acetyl hexapeptide prepared by the preparation method according to any one of claims 4-7 in the field of skin care products.

9. A skincare product, characterized in that, The skincare product includes the active ingredient and the acetyl hexapeptide of any one of claims 1-3 or the acetyl hexapeptide prepared by the preparation method of any one of claims 4-7.

10. The skincare product according to claim 9, characterized in that, The acetyl hexapeptide is present in the skincare product at a mass content of 0.05-1%; and / or The active ingredients include one or more of tranexamic acid, nicotinic acid, nicotinamide, or salicylic acid; The skincare products include toners, lotions, creams, or masks.

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