Preparation method and application of andrias davidianus hydrolyzed collagen active peptide

Abstract

The present application relates to the technical field of bioactive peptides, and specifically discloses a preparation method and application of giant salamander hydrolyzed collagen active peptide. The amino acid sequence of the giant salamander hydrolyzed collagen active peptide is SPPRARV. The single polypeptide in giant salamander peptide powder is separated and identified by combining gel chromatography column and preparative chromatography for the first time, and the giant salamander hydrolyzed collagen active peptide with the amino acid sequence of SPPRARV is successfully obtained. The polypeptide is modified by using molecular docking technology, and the obtained modified peptide has good transdermal ability and cell repair ability, can promote cell scratch repair and cell proliferation, promote the increase of type I collagen content, and is suitable for skin repair and anti-aging medical and beauty products or skin damage repair drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioactive peptides, and particularly relates to a preparation method and application of giant salamander hydrolyzed collagen active peptide. BACKGROUND

[0002] Giant salamander (Andrias davidianus) belongs to Hynobiidae and is distributed in Asia, including Chinese giant salamander and Japanese giant salamander. Chinese giant salamander is commonly known as baby fish due to its baby crying sound. Giant salamander is the largest amphibian in the world.

[0003] Giant salamander is a kind of organism with excellent regenerative ability. Zhang Hongxing research team of Shaanxi Institute of Zoology found that baby fish can regenerate after amputation. These phenomena can provide very important information for the field of regenerative medicine and tissue engineering. These organisms help us understand tissue regeneration or other complex diseases so as to design drugs for treatment.

[0004] The main stages of limb regeneration after amputation can be summarized as wound healing, blastema formation, blastema formation and final cell differentiation to replace all lost cell types. Successful scarless wound healing is essential for the progress of regeneration. In the wound healing stage, cells from the distal epithelial cells commonly known as wound epithelium migrate to close the wound. During the whole migration process, cells differentially regulate gene expression and may begin to express and / or secrete proteins that activate surrounding cells to form blastema. Earlier studies have shown that covering the wound with unwounded skin will inhibit the regeneration process, which indicates that the changes in gene expression in response to injury are essential for the progress of the scarless regeneration process. The wound epithelium formed during the regeneration process is considered a special structure because when keratinocytes begin to migrate, they begin to produce their own ECM, mainly composed of laminin, collagen type IV, collagen type XII, MMP3 and MMP9 (Campbell & Crews, 2008). During the re-epithelialization process at the wound site, other key processes are advancing in the stump tissue, such as tissue lysis and ECM remodeling of the residual wounded tissue, such as dermis, muscle, adhesion, etc.

[0005] It is of great significance to excavate and have relevant polypeptides from giant salamander tissues to play tissue regeneration function. At present, the preparation methods of giant salamander polypeptides mainly include acid hydrolysis, alkali hydrolysis and enzymatic hydrolysis. At present, the preparation of giant salamander collagen peptides pays more attention to the molecular weight of the hydrolysis product peptides, so as to obtain collagen peptides with smaller molecular weight and better absorption of the body, and less attention is paid to specific functional active peptides. For example, patent application CN105018555A discloses a preparation method of giant salamander skin collagen peptide. The giant salamander skin is used as raw material, after defatting, removing black skin and impurities and high pressure treatment, alkali protease is added for enzymatic hydrolysis, activated carbon is used for decolorization, centrifugation, filter membrane microfiltration, ultrafiltration membrane ultrafiltration, nanofiltration membrane nanofiltration to obtain filtrate, the filtrate is concentrated and freeze-dried to obtain giant salamander skin collagen peptide powder. The prepared collagen peptide has small molecular weight, and more than 90% of the molecular weight is less than 1000 daltons. The enzymatic hydrolysate is mainly mixed polypeptide, and the specific sequence is unknown, and the specific effect is also unknown. SUMMARY

[0006] The purpose of the present application is to overcome the shortcomings of the prior art and provide a preparation method and application of giant salamander hydrolyzed collagen active peptide.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0008] The first purpose is to provide a giant salamander hydrolyzed collagen active peptide, and the amino acid sequence of the giant salamander hydrolyzed collagen active peptide is SPPRARV (as shown in SEQ ID NO: 1).

[0009] The giant salamander hydrolyzed collagen active peptide obtained by separation and purification can promote cell scratch repair and cell proliferation, and is applied to related products, which is beneficial to the repair of skin damage.

[0010] The second purpose is to provide a preparation method of giant salamander hydrolyzed collagen active peptide, which comprises the following steps:

[0011] 1) Dissolve the giant salamander peptide powder with a solvent to obtain a giant salamander peptide solution;

[0012] 2) Sample the giant salamander peptide solution to a gel chromatography column, use dextran gel G15 as a separation chromatography medium, balance, elute and collect sample peaks;

[0013] 3) Chromatographically purify the sample collected in step 2), collect the sample in sections, then detect the molecular weight and analyze the sequence to obtain the giant salamander hydrolyzed collagen active peptide, and the amino acid sequence of the giant salamander hydrolyzed collagen active peptide is SPPRARV.

[0014] The application first separates and identifies a single polypeptide in giant salamander peptide powder by combining a gel chromatography column and a preparative chromatography, and successfully obtains a giant salamander hydrolyzed collagen active peptide with an amino acid sequence of SPPRARV.

[0015] As a preferred embodiment of the preparation method of the giant salamander hydrolyzed collagen active peptide, in step 3), the sample collected in step 2) is subjected to chromatographic purification, the mobile phase A is water and TFA with a concentration of 0.01% to 0.1%, the mobile phase B is acetonitrile and TFA with a concentration of 0.01% to 0.1%, the flow rate is 0.5 to 1 ml / min, and the detection wavelength is 220 nm.

[0016] Preferably, the mobile phase A is water and TFA with a concentration of 0.05%, and the mobile phase B is acetonitrile and TFA with a concentration of 0.05%.

[0017] As a preferred embodiment of the preparation method of the giant salamander hydrolyzed collagen active peptide, in step 3), the sample is subjected to molecular weight detection by using an AB SCIEX TripleTOF 5600 mass spectrometer, and subjected to sequence analysis by using a Shimadzu full-automatic protein polypeptide sequencer (PPSQ-33A).

[0018] The third object of the application provides a modified peptide, which is obtained by modifying the above-mentioned giant salamander hydrolyzed collagen active peptide by one of the following transdermal peptides: ACSSSPSKHCG, RGDFK, LCLE and CTWLKY.

[0019] Preferably, the amino acid sequence of the modified peptide is LCLESPPRARV.

[0020] The application adopts molecular simulation docking technology to modify the peptide, and the modified polypeptide has good transdermal absorption capacity and cell repair capacity, can promote cell scratch repair and cell proliferation, promote the increase of type I collagen content, and is suitable for skin repair and anti-aging medical and beauty products or skin damage repair drugs.

[0021] The fourth object of the application provides a solid-phase synthesis method of a modified peptide, which comprises the following steps:

[0022] 1) Fmoc-L-Leu-Wang Resin as solid phase synthesis carrier, 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, Fmoc-Trp(2-Me)-OH as synthesis raw materials, Fmoc-L-Leu-Wang Resin was weighed and soaked in DMF solution, and piperidine-containing DMF solution was added for deprotection; Fmoc-Cys(Trt)-OH was added, and condensing agent DIC / HOBt was added for reaction;

[0023] 2) According to step 1), the sequence C-L-E-S-P-P-R-A-R-V was sequentially condensed to obtain Fmoc-condensed peptide-L-Leu-Wang Resin; finally, a mixed cutting agent was prepared by using TFA, water, EDT and TLS, and the polypeptide LCLESPPRARV was cut from Fmoc-L-Leu-Wang Resin;

[0024] 3) The solution collected in step 2) was subjected to chromatographic purification, mobile phase A was water and TFA with a concentration of 0.01% to 0.1%, mobile phase B was acetonitrile and TFA with a concentration of 0.01% to 0.1%, flow rate was 0.5 to 1 ml / min, detection wavelength was 220 nm, and the modified peptide was collected.

[0025] The fifth object of the present application provides the use of the modified peptide in the preparation of a product having any one of the following functions:

[0026] (a) promoting fibroblast proliferation;

[0027] (b) promoting fibroblast scratch repair activity;

[0028] (c) promoting collagen synthesis.

[0029] As a preferred embodiment of the use of the present application, the product is a pharmaceutical product or a cosmetic product.

[0030] The sixth object of the present application provides a cosmetic composition comprising the above-mentioned giant salamander hydrolyzed collagen active peptide or modified peptide, and a cosmetically acceptable carrier thereof.

[0031] The weight ratio of the Andrias koreanum hydrolyzed collagen active peptide or modified peptide to the cosmetically acceptable carrier is (1-1000):(1-1000). For example, the weight ratio is 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0032] In the above cosmetic, the content of the Andrias koreanum hydrolyzed collagen active peptide or modified peptide added is a cosmetically effective amount, and the term "cosmetically effective amount" means an amount sufficient to achieve the efficacy of the cosmetic composition.

[0033] The cosmetic composition can be prepared in any dosage form conventionally prepared in the art, such as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a cleanser containing a surfactant, an oil, a foundation, an emulsion foundation, a wax foundation, and a spray, but is not limited thereto. For example, it can be prepared as a flexible emulsion, a nourishing emulsion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a lyophilized powder.

[0034] The seventh object, the present application provides a pharmaceutical composition comprising the above-mentioned Andrias koreanum hydrolyzed collagen active peptide or modified peptide, and a pharmaceutically acceptable carrier thereof; the weight ratio of the Andrias koreanum hydrolyzed collagen active peptide or modified peptide to the pharmaceutically acceptable carrier is (1-1000):(1-1000). For example, the weight ratio is 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2, but is not limited thereto.

[0035] The pharmaceutically acceptable carrier is a carrier commonly used in formulations, including at least one of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline, cellulose, polyvinylpyrrolidone, cellulose, water, sugar syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0036] The seventh object of the present application provides the use of the modified peptide in the preparation of a transdermal absorption promoting preparation.

[0037] Compared with the prior art, the present application has the following beneficial effects:

[0038] The present application provides a preparation method and application of giant salamander hydrolyzed collagen active peptide. For the first time, a gel chromatography column and a preparative chromatography are combined to separate and identify a single polypeptide in giant salamander peptide powder, and a giant salamander hydrolyzed collagen active peptide with an amino acid sequence of SPPRARV is successfully obtained. The polypeptide is modified by using molecular docking technology, and the modified peptide has good transdermal ability and cell repair ability, can promote cell scratch repair and cell proliferation, and promote the increase of type I collagen content. It is suitable for skin repair and anti-aging medical and beauty products or skin damage repair drugs. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a cell viability diagram of P1, P2, P3 and P4;

[0040] Figure 2 is a diagram of the influence of different concentrations of modified peptide LV-11 on 3t3-l1 cell proliferation in Example 3;

[0041] Figure 3 is a diagram of the influence of different concentrations of modified peptide AV-18 on 3t3-l1 cell proliferation in Example 3;

[0042] Figure 4 is a diagram of the influence of different concentrations of modified peptide SV-7 on 3t3-l1 cell proliferation in Example 3;

[0043] Figure 5 is a diagram of the influence of different concentrations of modified peptide CV-13 on 3t3-l1 cell proliferation in Example 3;

[0044] Figure 6 is a diagram of the influence of different concentrations of modified peptide RV-12 on 3t3-l1 cell proliferation in Example 3;

[0045] Figure 7 is a diagram of the in vitro transdermal test results of modified peptides LV-11, AV-18, SV-7, CV-13 and RV-12;

[0046] Figure 8 is a diagram of the results of modified peptide SV-7 and modified peptide LV-11 on scratch repair;

[0047] Figure 9 is a diagram of the detection results of different gene expression amounts of modified peptide SV-7 and modified peptide LV-11. DETAILED DESCRIPTION

[0048] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described below in conjunction with the drawings and specific embodiments.

[0049] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are commercially available unless otherwise specified.

[0050] Example 1, Isolation and screening of giant salamander hydrolyzed collagen active peptide

[0051] The specific steps include:

[0052] 1. Giant salamander hydrolyzed collagen active peptide separation: Freshly bred giant salamander was taken, the muscle part tissue was ground into homogenate, 10 g of homogenate was taken, and trypsin 100 U, neutral protease 100 U, alkaline protease 100 U, and pepsin 100 U were added for enzymolysis for 5 hours. Centrifugation at 12000 rpm for 30 min, and taking the supernatant. Unique AutoPrep high-pressure preparative liquid chromatograph was used for separation and purification, the chromatographic packing material 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, the mobile phase B was pure water and 0.05% trifluoroacetic acid, and gradient elution was used. Elution with 0%-100% of the mobile phase A for 20 min, column temperature 25°C, automatic sampling, sample volume 100 ml, and the eluate was collected according to different peaks. Different components were labeled as P1, P2, P3, and P4. An ultramicro spectrophotometer was used for quantitative analysis of different components, in which P1 was 1.51 μg / μL, P2 was 1.75 μg / μL, P3 was 3.25 μg / μL, and P4 was 2.57 μg / μL.

[0053] 2. Polypeptide screening: Mouse embryonic fibroblast 3t3-l1 cells were used, CCK-8 was used to detect the cell viability of the test substance, and after cell counting, it was diluted to 1×10 5 6 / ml, and the cell suspension was added to the 96-well plate for plating operation. After 24±1 h, the cell density reached 40-60%, 1 μg / ml of P1, P2, P3, and P4 was added using a row gun, a solvent control was set, and the test hole was not less than 3 in parallel. After 48 h of detection, the waste liquid was removed, 10% CCK-8-containing DMEM was added, and incubation was performed for 1-1.5 h. Enzyme-labeled instrument detection: medium shaking for 10 seconds, OD450nm reading. After the instrument completed the reading, the file was exported. The cell viability was calculated, and the results are shown in the following table. The P3 component has a strong effect on promoting cell viability. Figure 1

[0054] ​3. Polypeptide sequence identification: Polypeptide N-terminal sequencing analysis based on Edman degradation method: The component P3 was analyzed by using an amino acid automatic sequencer PPSQ33A, and the specific steps were as follows: replace the buffer, take 50 μl sample into an ultrafiltration tube, add 8 mol / L urea solution 200 μl, centrifuge at 12000 rcf speed for 10 min at 4℃, and repeat the operation once. Add 100 μl ddH2O, centrifuge at 12000 rcf speed for 10 min at 4℃, and repeat the operation once. Machine detection, drop the replaced sample solution on the membrane, place it in the reactor, assemble the reactor, and place it in the instrument fixed position. Set the sample name, sample number, test cycle number, and method file by using the software PPSQ-30Analysis, and start testing after setting. Data processing, the raw data and spectrum generated by PPSQ-33A are recognized by PPSQ-30DataProcessing software, and the corresponding spectrum is exported. The N-terminal sequence of the test product is: SPPRARV (as shown in SEQ ID NO: 1), which is named SV-7 (Amphibian hydrolyzed collagen active peptide).

[0055] Example 2, solid phase synthesis method of amphibian hydrolyzed collagen active peptide

[0056] In order to make the screened amphibian hydrolyzed collagen active peptide have better transdermal absorption performance in the application of skin, various transdermal peptides are used for modification. Through analysis of polypeptide hydrophobicity, polypeptide isoelectric point, and structural stability, etc., among them, the transdermal peptides ACSSSPSKHCG, RGDFK, LCLE, CTWLKY, etc. are more matched with the polypeptide SV-7, and have better stability and suitable isoelectric point.

[0057] The following examples respectively synthesize AV-18 (ACSSSPSKHCGSPPRARV, as shown in SEQ ID NO: 2), SV-7 (SPPRARV), RV-12 (RGDFKSPPRARV, as shown in SEQ ID NO: 3), LV-11 (LCLESPPRARV, as shown in SEQ ID NO: 4), CV-13 (CTWLKYSPPRARV, as shown in SEQ ID NO: 5) and other modified peptides.

[0058] The specific steps include:

[0059] 1. Use The modified peptide was synthesized by X high-throughput polypeptide synthesizer. The amino acid single letter was inputted from N terminal to C terminal in the editing window according to the sequence of the modified peptide. The synthesis raw 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, Fmoc-Trp(2-Me)-OH.

[0060] The setting program was as follows: 30-60 min in DMF solution, the resin was fully activated, and 20% piperidine in DMF solution was added for deprotection twice (10 min, 30 min). The resin Fmoc-Ala-Wang resin was washed with 15 ml of isopropanol and DMF solution three times in turn. The condensing agent DIC / HOBt (DIC 170 mmol, HOBt 100 mmol) was added to the reaction system, and the synthesis was started according to the input sequence.

[0061] 2. Purification of the synthesized polypeptide: the synthesized modified peptide was separated and purified by Unique AutoPrep high-pressure preparative liquid chromatograph, the chromatographic packing 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, the mobile phase B was purified water and 0.05% trifluoroacetic acid, and gradient elution was adopted. Gradient elution was adopted with 10%-90% of the mobile phase A for 20 min, the column temperature was 25°C, automatic sampling was adopted, and the elution peak was collected.

[0062] Purity detection:

[0063] High performance liquid chromatograph (Agilent 1260 with four-pump, automatic sampler and VWD detector); the chromatographic column was a C18 chromatographic column (4.6 mm x 250 mm, 5 μm); the mobile phase A (0.1% TFA + water), the mobile phase B (acetonitrile); gradient elution, the elution program was shown in Table 1; the column temperature: 30°C; the injection amount: 20 μL; the flow rate: 1.0 mL / min; the detector: VWD detector, the detection wavelength: 220 nm.

[0064] Table 1 Gradient elution program

[0065]

[0066]

[0067] The purity of the synthesized modified peptides was as shown in Table 2 below:

[0068] Table 2 Purity of the synthesized modified peptides

[0069] Synthetic modified peptides Purity AV-18 (ACSSSPSKHCGSPPRARV) 99.8％ SV-7 (SPPRARV) 99.5％ RV-12 (RGDFKSPPRARV) 99.4％ LV-11 (LCLESPPRARV) 99.5％ CV-13 (CTWLKYSPPRARV) 98.8％

[0070] Example 3, cytotoxicity test

[0071] Cytotoxicity test: mouse embryonic fibroblast 3t3-l1 cells were used, and CCK-8 was used to detect the cytotoxicity of the test substances.

[0072] In vitro cultured normal mammalian cells are constantly dividing and proliferating, and toxic substances, regardless of their action site and mechanism, will interfere with the division and proliferation process of the cells, resulting in a decrease in cell growth rate and a decrease in cell number. The orange yellow formazan generated after the WST-8 in the CCK-8 detection reagent is oxidized and reduced by the dehydrogenase in the cells can be dissolved in the culture medium, and the amount of formazan generated is proportional to the number of living cells. Finally, the cell survival rate was calculated after reading the results at 450 nm by a microplate reader.

[0073] Mouse embryonic fibroblast 3t3-l1 cells were counted and diluted to 1×10 5 After 24±1h, the cell density reached 40-60%, the test substances (LV-11, AV-18, SV-7, CV-13, RV-12) were added using a row gun, and the final concentration was set to the set concentration. The solvent control was set, and the test wells were not less than 3 in parallel. After 48h of detection, the waste liquid was removed, 10% CCK-8 was added to DMEM, and the cells were cultured for 1-1.5h. The microplate reader was used for detection. The microplate reader was set to: moderate shaking for 10 seconds, OD450nm for reading. After the instrument completed the reading, the file was exported. Calculation. Cell survival rate calculation in the toxicity test of the test substance:

[0074]

[0075] The results are as follows:

[0076] From Figures 2-6 It can be seen that the modified peptide LV-11 has the effect of increasing the cell survival rate of 3t3-l1 cells at a concentration range of 0.10-30μM, and has higher activity compared with AV-18 (ACSSSPSKHCGSPPRARV), SV-7 (SPPRARV), RV-12 (RGDFKSPPRARV), and CV-13 (CTWLKYSPPRARV).

[0077] Example 4, in vitro transdermal test

[0078] Specific steps:

[0079] Before the transdermal experiment, the mouse was taken, the fur was shaved off with an electric shaver, and then the mouse was sacrificed. Then the abdominal skin was washed repeatedly with normal saline, and then it was placed on a glass plate. The fat layer was carefully peeled off, and the transdermal test was immediately performed. A modified Franz double-chamber permeation and diffusion device (diffusion area of 3.14 cm 2 , diffusion cell volume of 15 ml) was used. The treated mouse skin in vitro was placed at the junction of the horizontal diffusion cell, with the horny layer facing the supply pool, and was fixed with a spring clip. The corresponding test sample was added to the supply pool. Normal saline was added to the receiving pool, and constant speed electromagnetic stirring (200 r / min) was performed. The temperature of the diffusion cell sandwich water bath was maintained at 32℃. At the predetermined sampling time (2h, 4h, 6h, 8h, 10h, 12h, 24h), 1ml was taken from the receiving pool, and an equal amount of normal saline was added. HPLC content detection was performed. The HPLC detection method is as follows:

[0080] High performance liquid chromatograph (Agilent 1260 with four-pump, automatic sampler and VWD detector); the above prepared modified peptide (purity ≥98%), acetonitrile (chromatographic pure, Ron Reagent Company), trifluoroacetic acid / TFA (analytical pure, Macklin Reagent), and experimental water were ultrapure water.

[0081] C18 chromatographic column (4.6mm×250mm, 5μm); mobile phase A (0.1% TFA + water), mobile phase B (acetonitrile); gradient elution, elution program as shown in Table 3; column temperature: 30℃; sample size: 20μL; flow rate: 1.0mL / min; detector: VWD detector, detection wavelength 220nm.

[0082] Table 3 Gradient elution program

[0083]

[0084] The above prepared modified peptides LV-11, AV-18, SV-7, CV-13, and RV-12 were detected according to the above method. The peak area integral value was recorded, the content of the synthesized exosome was calculated, and the cumulative permeation amount (Q n , μg / cm 2 ) was calculated by formula. The formula is as follows:

[0085] Qn=(Cn*V+∑Ci*Vi) / A

[0086] Note: Cn is the concentration of the n-th time point (mg / L); Ci is the concentration of the n-1-th time point (mg / L); V is the total volume of the receiving liquid, i.e. 15ml; Vi is the volume of each suction liquid, i.e. 1ml; A is the area of the receiving pool, i.e. 3.14cm2 .

[0087] Reference Figure 7 , the experimental results show that LV-11 has better transdermal performance than other modified peptides, and can promote skin absorption.

[0088] Example 5, cell scratch test

[0089] Specifically, the following steps are included: the effects of SV-7 (SPPRARV) and LV-11 (LCLESPPRARV) on scratch repair are detected by 3t3-l1 cell scratch test, 3t3-l1 cells are inoculated in a 6-well plate at a cell density of 2*10 5 / ml, and cultured in an incubator for 24h; a straight line is drawn with a gun head, and the cells are washed with PBS for 3 times to remove the cells scratched, and serum-free medium is added; a blank control group and a test group are set up, the concentrations of SV-7 (SPPRARV) and LV-11 (LCLESPPRARV) are 1ug / ul, and they are placed in a 37℃, 5% CO2 incubator for 24h, and then observed and photographed at a time point.

[0090] The results are shown in Figure 8 , LV-11 has better scratch repair ability than other test groups.

[0091] Example 6, gene expression detection

[0092] This embodiment relates to the detection of Collagen I (type I collagen), Collagen III (type III collagen), Elastic (elastin), and MMP-9 (matrix metalloproteinase-9) gene expression.

[0093] The RT-PCR method is used to detect the gene transcription level in HSF cells, and 3t3-l1 cells in the logarithmic growth phase are inoculated in a six-well plate at 3*10 5 cells per well, and after the cells are attached for 24h, 0.39mg / mL SV-7 (SPPRARV) and 0.39mg / mL LV-11 (LCLESPPRARV) are used to act on HSF cells for 24h. The total RNA of the cells is extracted by the TRIZOL method. 1ug of RNA is used for reverse transcription by using the EasyScript5*all-in (100 runs) AE341-02 kit. The cDNA synthesized by reverse transcription is used as a template for PCR reaction, and the reaction system is as follows:

[0094] Table 4 reaction system

[0095]

[0096]

[0097] The primer sequence design is shown in Table 5. The reaction condition is: 94℃ pre-denaturation for 30s, 94℃ denaturation for 5s; 55℃ annealing for 30s; 72℃ extension for 10s, 40 cycles in total.

[0098] Table 5 Primer sequence

[0099]

[0100] The calculation is performed by using 2^-ΔΔct with GAPDH as the reference gene, and the result is as follows Figure 9 LV-11 can promote the expression of Collagen I, Collagen III and Elastic genes, and inhibit the expression of MMP-9 gene. Thus, the production of extracellular matrix is promoted, and the degradation of extracellular matrix is inhibited.

[0101] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced equivalently without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A Megalobranchia hydrolyzed collagen active peptide, characterized in that, The amino acid sequence of the giant salamander hydrolyzed collagen active peptide is SPPRARV.

2. A method for preparing a Megalobranch hydrolyzed collagen active peptide, characterized by, The method comprises the following steps: 1) dissolving the giant salamander peptide powder with a solvent to obtain a giant salamander peptide solution; 2) loading the giant salamander peptide solution into a gel chromatography column, using dextran gel G15 as a separation chromatography medium, balancing, eluting, and collecting sample peaks; 3) chromatographically purifying the sample collected in step 2), collecting samples in sections, and then detecting the molecular weight and analyzing the sequence to obtain the giant salamander hydrolyzed collagen active peptide, the amino acid sequence of which is SPPRARV; The method for preparing the giant salamander peptide powder comprises the following steps: Freshly cultured giant salamanders are taken, and muscle parts are taken and ground into homogenate, and the homogenate is taken and subjected to trypsin, neutral protease, alkaline protease, and pepsin enzymolysis, and the supernatant is taken after centrifugation.

3. The production method according to claim 2, wherein In step 3), the sample collected in step 2) is chromatographically purified, the mobile phase A is water and TFA with a concentration of 0.01% to 0.1%, the mobile phase B is acetonitrile and TFA with a concentration of 0.01% to 0.1%, the flow rate is 0.5 to 1 ml / min, and the detection wavelength is 220 nm.

4. The production method according to claim 3, wherein The mobile phase A is water and TFA with a concentration of 0.05%, and the mobile phase B is acetonitrile and TFA with a concentration of 0.05%.

5. A modified peptide, characterized in that, The giant salamander hydrolyzed collagen active peptide as claimed in claim 1 is modified by one of the following transdermal peptides: ACSSSPSKHCG, RGDFK, LCLE, and CTWLKY, and the amino acid sequence of the modified peptide is one of the following: ACSSSPSKHCGSPPRARV, RGDFKSPPRARV, LCLESPPRARV, and CTWLKYSPPRARV.

6. A method for solid phase synthesis of a modified peptide according to claim 5, characterized in that, The method comprises the following steps: 1) using Fmoc-L-Leu-Wang Resin as a solid-phase synthesis carrier, using 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 as synthesis raw materials, weighing Fmoc-L-Leu-Wang Resin, soaking in a DMF solution, adding a DMF solution containing piperidine to deprotect, adding Fmoc-Cys(Trt)-OH, and adding a condensing agent DIC / HOBt to react; 2) according to step 1), sequentially condensing according to the sequence C-L-E-S-P-P-R-A-R-V to obtain Fmoc-condensed peptide-L-Leu-Wang Resin; finally, using TFA, water, EDT and TLS to prepare a mixed cleavage agent, and cutting the modified peptide LCLESPPRARV from Fmoc-L-Leu-Wang Resin; 3) chromatographically purifying the solution collected in step 2), using water and 0.01%-0.1% TFA as mobile phase A, using acetonitrile and 0.01%-0.1% TFA as mobile phase B, using a flow rate of 0.5-1 ml / min, using a detection wavelength of 220 nm, and collecting the modified peptide.

7. Use of the modified peptide of claim 5 in the preparation of a product having any one of the following functions: (a) a cosmetic product for promoting fibroblast proliferation; (b) an active cosmetic product for promoting fibroblast scratch repair; (c) a cosmetic product for promoting collagen synthesis.

8. A cosmetic composition characterized in that, The cosmetic composition comprises the Andrias davidianus hydrolyzed collagen active peptide of claim 1 or the modified peptide of claim 5, and a cosmetically acceptable carrier thereof.

9. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the Andrias davidianus hydrolyzed collagen active peptide of claim 1 or the modified peptide of claim 5, and a pharmaceutically acceptable carrier thereof; the weight ratio of the Andrias davidianus hydrolyzed collagen active peptide or the modified peptide to the pharmaceutically acceptable carrier is (1-1000):(1-1000).

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