Skin repair peptide and application thereof

Abstract

The application belongs to the technical field of protein peptides, and discloses a skin repair peptide AF-16 and application thereof. The skin repair peptide comprises giant salamander hydrolyzed collagen peptide and a transdermal peptide. The skin repair peptide is obtained by modifying the giant salamander hydrolyzed collagen peptide obtained by hydrolyzing giant salamander tissue by the transdermal peptide. The skin repair peptide can promote fibroblast proliferation, promote fibroblast scratch repair activity, and promote collagen synthesis. The skin repair peptide can be used in the improvement or treatment of skin damage repair.

Description

Technical Field

[0001] The present invention relates to the technical field of protein peptides, and in particular to a skin repairing peptide and applications thereof. Background Art

[0002] The skin is the first barrier between the body and the outside world, maintaining a stable internal environment and preventing the invasion of microorganisms, chemicals, and the like. Various types of trauma can cause skin defects, such as external trauma. Another example is the loss of collagen with aging and the effects of ultraviolet rays, which reduces the amount of collagen and glycosaminoglycans synthesized by fibroblasts. This leads to the breakage of collagen peptide bonds that support the skin and skin damage. In the case of skin damage, active peptides play a unique and important physiological role, participating in the process of skin repair. They can not only promote the proliferation of skin cells, but also provide nutrition to the skin, delay skin aging, and promote the repair of skin wounds. Currently, active peptides are widely added to skin care products as one of the active raw materials for skin repair. However, these peptides in the prior art are difficult to synthesize and have limited activity. Therefore, the development of ultrashort peptides that promote skin repair with short sequences, simple synthesis, and high activity has become one of the key directions in the fields of skin repair, regenerative medicine, and beauty. Summary of the Invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a skin repair peptide and its application.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides a skin repair peptide comprising giant salamander hydrolyzed collagen peptide and / or transdermal peptide.

[0006] The skin-repairing peptides of the present invention can promote fibroblast proliferation, fibroblast scratch repair activity, collagen synthesis, and elastin synthesis. They can be used to improve or treat skin damage repair. The skin-repairing peptides of the present invention contain transdermal short peptides that promote transdermal absorption, are highly effective in trace amounts, and offer guaranteed safety. They are suitable for use in skin-repairing cosmetics or pharmaceuticals to improve or treat skin damage repair.

[0007] As a preferred embodiment of the skin repair peptide of the present invention, the amino acid sequence of the giant salamander hydrolyzed collagen peptide is GLYYF.

[0008] As a preferred embodiment of the skin repair peptide of the present invention, the amino acid sequence of the transdermal peptide is any one of ACSSSPSKHCG, CYGRKKRRQRRRC, RGDFK, LCLR, RPARPAR, VVVR, CTWLKY, and TWLKYH.

[0009] Preferably, the amino acid sequence of the transdermal peptide is ACSSSPSKHCG.

[0010] Furthermore, the amino acid sequence of the skin repair peptide is ACSSSPSKHCGGLYYF.

[0011] In a second aspect, the present invention provides a method for preparing the hydrolyzed giant salamander collagen peptide, comprising the steps of: obtaining freshly cultured giant salamander muscle tissue, grinding it into a homogenate, and then adding trypsin, neutral protease, alkaline protease, and pepsin for enzymatic hydrolysis; centrifuging the mixture, collecting the supernatant, and separating and purifying the mixture using a liquid chromatograph, and collecting the eluate to obtain the peptide.

[0012] As a preferred embodiment of the preparation method of the present invention, the enzymatic hydrolysis is performed using 80U-120U of trypsin, 80U-120U of neutral protease, 80U-120U of alkaline protease, and 80U-120U of pepsin for 4-6 hours.

[0013] As a preferred embodiment of the preparation method of the present invention, the chromatographic filler used in the liquid chromatography separation and purification is UniSil 10-120C18, the detection wavelength is 220 nm, the flow rate is 0.5 ml / min, the mobile phase A is acetonitrile and 0.05% trifluoroacetic acid, and the mobile phase B is purified water and 0.05% trifluoroacetic acid.

[0014] In a third aspect, the present invention applies the skin repair peptide in cosmetics or medicines for skin repair.

[0015] In a fourth aspect, the present invention provides a cosmetic or medicine containing the skin repair peptide.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The skin-repairing peptides of the present invention can promote fibroblast proliferation, fibroblast scratch repair activity, collagen synthesis, and elastin synthesis. They can be used to improve or treat skin damage repair. The skin-repairing peptides of the present invention contain transdermal short peptides that promote transdermal absorption, are highly effective in trace amounts, and offer guaranteed safety. They are suitable for use in skin-repairing cosmetics or pharmaceuticals to improve or treat skin damage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a statistical diagram of the migration rate of cells with different components in the scratch test;

[0019] Figure 2 This is a statistical graph of the cytotoxicity of synthetic peptides detected by CCK-8;

[0020] Figure 3This is a statistical chart of the content of synthetic peptides in in vitro transdermal testing;

[0021] Figure 4 Electron micrograph of the scratch repair ability of the synthetic peptide tested in a cell scratch experiment;

[0022] Figure 5 This is a statistical chart showing the effects of synthetic peptides on the expression levels of CollagenⅠ, CollagenⅢ, Elastic, and MMP-9 genes. DETAILED DESCRIPTION

[0023] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The term "peptide" refers to a linear molecule formed by amino acid residues linked to each other by peptide bonds. Peptides can be synthesized by chemical synthesis methods known in the art, in particular solid phase synthesis techniques or liquid phase synthesis techniques.

[0025] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0026] Example 1: Isolation and identification of giant salamander polypeptides

[0027] (1) Separation

[0028] Freshly farmed giant salamanders were obtained, and muscle tissue was ground into a homogenate. 10 g of the homogenate was then digested with 100 U of trypsin, 100 U of neutral protease, 100 U of alkaline protease, and 100 U of pepsin for 5 hours. The mixture was centrifuged at 12,000 rpm for 30 minutes, and the supernatant was collected. Separation and purification were performed using a Unique AutoPrep high-pressure preparative liquid chromatograph using UniSil 10-120C18 as the chromatographic packing, a detection wavelength of 220 nm, a flow rate of 0.5 ml / min, and mobile phase A: acetonitrile (0.05% trifluoroacetic acid), mobile phase B: purified water (0.05% trifluoroacetic acid). Gradient elution (0% to 100% A) was used for 20 minutes. The column temperature was 25°C, and the injection volume was 100 mL. The eluate was collected according to the peak. The eluates were labeled P1, P2, P3, and P4, respectively. An ultramicro spectrophotometer was used with a detection wavelength of 280 nm to quantitatively analyze the different components, including P1 1.51μg / μL, P2 1.75μg / μL, P3 3.25μg / μL, and P4 2.57μg / μL.

[0029] The efficacy of different components P1, P2, P3, and P4 were screened by human skin fibroblast scratch test. Human skin fibroblasts were cultured at 2×10 5 Cells were seeded at a density of 100 μg / mL in a 6-well plate and cultured in an incubator for 24 hours. A straight horizontal line was drawn with a pipette tip, and the cells were washed three times with PBS. The crossed cells were removed and serum-free culture medium was added. A blank control group and a test group were set up, and the different components P1, P2, P3, and P4 were diluted to 0.1 μg / μL. The cells were placed in a 37°C, 5% CO2 incubator and observed and photographed at the 24-hour time point. The results are shown in Figure 2. Figure 1 As shown, component P4 has better scratch repair ability compared with other test groups.

[0030] (2) Identification

[0031] The method for identifying the polypeptide sequence of component P4 is as follows:

[0032] Edman degradation-based peptide N-terminal sequencing analysis: Fraction P4 was analyzed using the automated amino acid sequencer PPSQ-33A. The following steps were performed: After buffer replacement, 50 μL of sample was transferred to an ultrafiltration tube. 200 μL of 8 mol / L Urea solution was added. The sample was centrifuged at 12,000 rcf at 4°C for 10 min, and the process was repeated. 100 μL of ddH2O was added, and the sample was centrifuged at 12,000 rcf at 4°C for 10 min, and the process was repeated. For on-board analysis, the replaced sample solution was dripped onto the membrane and placed in the reactor. After assembling the reactor, it was positioned in a fixed position on the instrument. The PPSQ-30Analysis software was used to set the sample name, sample number, number of test cycles, and select the method file. After completing the settings, the test was started. For data processing, the raw data and chromatograms generated by the PPSQ-33A were identified using PPSQ-30DataProcessing software, and the corresponding peaks were exported. The N-terminal sequence of the test product is: H-Gly-Leu-Tyr-Tyr-Phe-OH, and it is named GF-5.

[0033] Example 2: Transdermal modification of giant salamander polypeptide

[0034] In order to make the screened polypeptide GF-5 have better transdermal absorption performance when used in the skin, a variety of transdermal peptides were used to modify it. After analysis of polypeptide hydrophobicity, polypeptide isoelectric point, structural stability, etc., the transdermal peptides ACSSSPSKHCG, CYGRKKRRQRRRC, RGDFK, LCLR, RPARPAR, VVVR, CTWLKY, and TWLKYH matched the polypeptide GF-5 with good stability and a suitable isoelectric point.

[0035] (1) AF-16 (ACSSSPSKHCGGLYYF, transdermal peptide N-terminal modification), GG-16 (GLYYFACSSSPSKHCG, transdermal peptide C-terminal modification), GF-5 (GLYYF), AG-11 (ACSSSPSKHCG), CF-18 (CYGRKKRRQRRRCGLYYF), RF-10 (RGDFKGLYYF), LF-9 (LCLRGLYYF), RF-12 (RPARPARGLYYF), VF-9 (VVVRGLYYF), CF-11 (CTWLKYGLYYF), and TF-11 (TWLKYHGLYYF) were synthesized by peptide solid-phase synthesis.

[0036] The specific method is as follows:

[0037] use Peptide synthesis was performed using an X high-throughput peptide synthesizer. Amino acid sequences were entered in the editing window from N-terminus to C-terminus. The starting materials used were Fmoc-Ala-Wang resin, Fmoc-Cys(Me)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Ac)-OH, Fmoc-His(Bom)-OH, Fmoc-Glu-OH, Fmoc-Leu-OH, Fmoc-Tyr-OH, Fmoc-Phe-OH, Fmoc-Arg-OH, Fmoc-Glu-OH, Fmoc-Val-OH, Fmoc-Thr(HPO3Bzl)-OH, and Fmoc-Trp(2-Me)-OH. The following protocol was used: resin was fully activated in DMF for 30–60 min, followed by deprotection twice (10 min and 30 min) in 20% piperidine in DMF. The resin was washed three times with 15 mL of isopropanol and DMF. The condensation agent DIC / HOBt (DIC 170 mmol, HOBt 100 mmol) was used to start the synthesis according to the input sequence.

[0038] (2) Purification of synthetic peptides

[0039] The synthetic peptides were separated and purified using a Unique AutoPrep high-pressure preparative liquid chromatograph using UniSil 10-120C18 as the chromatographic medium, a detection wavelength of 220 nm, a flow rate of 0.5 ml / min, mobile phase A consisting of acetonitrile (0.05% trifluoroacetic acid) and mobile phase B consisting of purified water (0.05% trifluoroacetic acid). A gradient elution (0% to 100% A) was used over 20 min. The column temperature was 25°C, and the sample was automatically injected, and the eluted peak was collected.

[0040] (3) Purity testing

[0041] High performance liquid chromatography (HPLC) was performed on an Agilent 1260 instrument equipped with a quaternary pump, an autosampler, and a VWD detector. The chromatographic column was a C18 column (4.6 mm × 250 mm, 5 μm). The mobile phases A and B were 0.1% TFA in water and acetonitrile, respectively. The elution procedure was as follows: gradient elution (see Table 1 ). The column temperature was 30°C. The injection volume was 20 μL. The flow rate was 1.0 mL / min. The detector was a VWD detector with a detection wavelength of 220 nm.

[0042] Table 1 Gradient elution program

[0043] time A / ％ B / ％ 0min 90 10 20min 10 90 20.1min 90 10 25min 90 10

[0044] The purity of the synthesized peptides was tested and is shown in Table 2.

[0045] Table 2 Purity of synthetic peptides

[0046]

[0047]

[0048] Test Example 1: Cytotoxicity Test

[0049] The human fibroblast cell line HSF was used to assess the cytotoxicity of the test substances using CCK-8. Normal mammalian cells cultured in vitro continuously divide and proliferate. Toxic substances, regardless of their site of action or mechanism of action, will interfere with this process, leading to a decrease in cell growth rate and number. The WST-8 in the CCK-8 assay, oxidized and reduced by intracellular dehydrogenases, produces an orange-yellow formazan that can be dissolved in the culture medium. The amount of formazan produced is proportional to the number of viable cells. Cell viability was then calculated using a microplate reader at 450 nm.

[0050] After HSF cell counting, dilute and add the cell suspension to a 96-well microplate for plating; 24±1h after plating, the cell density reaches 40% to 60%. Use a spray gun to add the synthetic polypeptide prepared in Example 2 to make its final concentration the set concentration, set a solvent control, and set no less than 3 parallel test wells. After 48h of sample addition, remove the waste liquid, add DMEM containing 10% CCK-8, and culture for 1 to 1.5h before detection by microplate reader. The microplate reader is set to: medium speed vibration for 10 seconds, OD450nm for reading. Export the file after the instrument completes the reading. The formula for calculating cell viability in the toxicity test of the test substance is as follows:

[0051]

[0052] Here are the results:

[0053] Depend on Figure 2It can be seen that AF-16 improves HSF cell survival at a concentration range of 0.1-1.56 mg / ml, and its effect on promoting HSF cell proliferation is more significant than that of GF-5. Compared with GG-16, GG-16 is a peptide modified at the C-terminus with the transdermal peptide ACSSSPSKHCG, which exhibits cytotoxicity to HSF cells at a concentration range of 0.78-1.56 mg / ml.

[0054] Test Example 2: In vitro permeation test

[0055] Before the transdermal test, mice were taken, their hair was shaved with an electric razor, and they were killed. The abdominal skin was then taken and repeatedly rinsed with saline. The skin was then placed on a glass plate, the fat layer was carefully peeled off, and the transdermal test was immediately performed. A modified Franz two-chamber osmotic diffusion apparatus (diffusion area of ​​3.14 cm) was used. 2 , the diffusion cell volume is 15 mL). The treated ex vivo mouse skin is placed at the junction of the horizontal diffusion cells, with the stratum corneum facing the supply cell and fixed with a spring clip. The synthetic polypeptide prepared in Example 2 is added to the supply cell. Physiological saline is added to the receiving cell, and constant electromagnetic stirring (200 r / min) is applied. The water bath temperature of the diffusion cell interlayer is maintained at 32°C. At the predetermined sampling time (2h, 4h, 6h, 8h, 10h, 12h, 24h), 1 mL of sample is taken from the receiving cell, and an equal amount of physiological saline is added. The content of the synthetic polypeptide is detected by HPLC.

[0056] The HPLC detection method is as follows:

[0057] High performance liquid chromatography (HPLC) was performed on an Agilent 1260 instrument equipped with a quaternary pump, an autosampler, and a VWD detector. Peptides (purity ≥ 98%), acetonitrile (chromatographic grade, Roan Reagent Company), trifluoroacetic acid / TFA (analytical grade, Macklin Reagent) and ultrapure water were used in the experiments.

[0058] C18 chromatographic column (4.6 mm × 250 mm, 5 μm); mobile phase A (0.1% TFA water), mobile phase B (acetonitrile); gradient elution, elution program see Table 3; column temperature: 30°C; injection volume: 20 μL; flow rate: 1.0 mL / min; detector: VWD detector, detection wavelength 220 nm.

[0059] Table 3 Gradient elution program

[0060] time A / ％ B / ％ 0min 90 10 20min 10 90 20.1min 90 10 25min 90 10

[0061] Record the peak area integral value, calculate the content of the synthetic peptide, and enter it into the formula to calculate the cumulative permeation amount (Qn, μg / cm 2 ). The formula is as follows:

[0062] Qn=(Cn*V+∑Ci*Vi) / A

[0063] Where Cn is the concentration of the aspirated solution at the nth time point (mg / L); Ci is the concentration of the aspirated solution at the n-1th time point (mg / L); V is the total volume of the receiving solution, i.e., 15 mL; Vi is the volume of the liquid aspirated each time, i.e., 1 mL; A is the area of ​​the receiving pool, i.e., 3.14 cm 2 .

[0064] The experimental results are as follows Figure 3 As shown in the results, compared with other transdermal short peptides, AF-16 (ACSSSPSKHCGGLYYF) has better transdermal performance and can promote skin absorption.

[0065] Experimental Example 3: Cell scratch test

[0066] The effects of AF-16 (ACSSSPSKHCGGLYYF), GF-5 (GLYYF), and AG-11 (ACSSSPSKHCG) on scratch repair were detected by human skin fibroblast scratch test. Human skin fibroblasts were cultured at a rate of 2×10 5 Cells were seeded at a density of 100 cells / ml in a 6-well plate and cultured in an incubator for 24 hours. A horizontal line was drawn straight with a pipette tip, and the cells were washed three times with PBS. The drawn cells were removed and serum-free culture medium was added. A blank control group and a test group were set up respectively. The test group included AF-16 (ACSSSPSKHCGGLYYF), GF-5 (GLYYF), and AG-11 (ACSSSPSKHCG) at a concentration of 0.39 μg / μL. The cells were placed in a 37°C, 5% CO2 incubator and observed and photographed at the 24-hour culture time point.

[0067] The results are as follows Figure 4 As shown, AF-16 has better scratch repair ability than other test groups.

[0068] Experimental Example 4: Gene Expression Detection

[0069] The effects of synthetic peptides on the gene expression of CollagenⅠ (type Ⅰ collagen), CollagenⅢ (type Ⅲ collagen), Elastic (elastin), and MMP-9 (matrix metalloproteinase-9) in human fibroblasts were detected.

[0070] RT-PCR was used to detect the gene transcription level in HSF cells. HSF cells in the logarithmic growth phase were cultured at 3×10 5HSF cells were seeded at 100 μg / well in a six-well plate. After 24 hours of cell attachment, 0.39 mg / mL AF-16 (ACSSSPSKHCGGLYYF), 0.39 mg / mL GF-5 (GLYYF), and 0.39 mg / mL AG-11 (ACSSSPSKHCG) were added to the cells for 24 hours. Total RNA was extracted using the TRIZOL method. 1 μg of RNA was reverse transcribed using the EasyScript 5*all-in (100 runs) AE341-02 kit. PCR reactions were performed using the cDNA synthesized by reverse transcription as a template. The reaction system is shown in Table 4.

[0071] Table 4 Reaction system

[0072] Component Volume Template* Variable Forward Primer (10μM) 0.4 μl Reverse Primer (10μM) 0.4 μl 2×TransStart Green qPCR SuperMix 10 μl Passive Reference Dye(50×)(optional) 0.4 μl Nuclease-free Water Variable Total volume 20 μl

[0073] The primer sequences are shown in Table 5. The reaction conditions were: pre-deformation at 94°C for 30 s, denaturation at 94°C for 5 s, annealing at 55°C for 30 s, and extension at 72°C for 10 s, for a total of 40 cycles.

[0074] Table 4 Reaction system

[0075]

[0076]

[0077] Taking GAPDH as the reference gene, the calculation was performed using 2^-ΔΔct:, and the results are as follows Figure 5 AF-16 can promote the expression of CollagenⅠ, CollagenⅢ, and Elastic genes and inhibit the expression of MMP-9 gene, thereby promoting the production of extracellular matrix and inhibiting the degradation of extracellular matrix.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A skin repair peptide, characterized in that The amino acid sequence of the skin repair peptide is ACSSSPSKHCGGLYYF.

2. A method for preparing the skin repair peptide according to claim 1, characterized in that: The following steps are involved: The muscle tissue of freshly cultured giant salamanders was ground into a homogenate, and then trypsin, neutral protease, alkaline protease and pepsin were added for enzymatic hydrolysis; the supernatant was collected by centrifugation, and the supernatant was separated and purified by liquid chromatography, and the eluate was collected to obtain the product; The enzymatic hydrolysis is performed using 80U-120U of trypsin, 80U-120U of neutral protease, 80U-120U of alkaline protease, and 80U-120U of pepsin for 4-6 hours; The chromatographic filler used in the liquid chromatography separation and purification was UniSil 10-120 C18, the detection wavelength was 220 nm, the flow rate was 0.5 ml / min, the mobile phase A was acetonitrile and 0.05% trifluoroacetic acid, and the mobile phase B was purified water and 0.05% trifluoroacetic acid.

3. Use of the skin repair peptide according to claim 1 in cosmetics or medicines for skin repair.

4. A cosmetic or medicine containing the skin repair peptide according to claim 1.

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