A decapeptide, its extraction and purification method, and its application

Abstract

This invention discloses a decapeptide, its extraction and purification method, and its applications, belonging to the field of bioactive peptides. Its amino acid sequence is RGFPGGDGAA. The invention also discloses its preparation method and its application in the cosmetics field. The disclosed decapeptide, its extraction and purification method, and its applications involve processing pufferfish skin to extract, purify, and screen the central peptide that provides photoprotection. This central peptide can be used in cosmetics.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptides, and in particular to a decapeptide, its extraction and purification method, and its application. Background Technology

[0002] The skin is the largest organ in the human body, a crucial component of the innate immune system, and the first line of defense protecting internal organs from environmental damage. The skin is primarily composed of the dermis, epidermis, and subcutaneous tissue, and also includes hair follicles and sweat glands. The appearance of the skin directly reflects the degree of aging. Skin aging is divided into intrinsic and extrinsic aging, also known as endogenous and exogenous aging. Endogenous aging refers to the physiological and hereditary skin aging that occurs with age; exogenous aging refers to skin aging caused by factors such as sunlight, temperature, dust, air pollution, chemical factors, and bacterial infection. Skin aging caused by UV radiation is defined as photoaging. UV radiation includes three types: UVA, UVB, and UVC. UVA has a wavelength range of 320-400 nm, UVB has a wavelength range of 280-320 nm, and UVC has a wavelength range of 100-280 nm. Because of its shorter wavelength, most UVC rays are absorbed by the ozone layer. UVB rays are partially absorbed by the ozone layer and partially by the epidermis, but they can also penetrate into the upper dermis, damaging collagen and elastin. Compared to UVC and UVB, UVA rays have the longest wavelength and are therefore absorbed by the inner dermis, causing damage to the structure and cells of both the dermis and epidermis. The mechanisms by which UV rays cause skin aging include: UV-induced oxidative stress, UV effects on the extracellular matrix (ECM), UV-damaged DNA, and UV-induced skin inflammation.

[0003] The unique habitat of marine organisms has fostered distinctive metabolic pathways and adaptive mechanisms, distinct from those of terrestrial organisms, resulting in a wealth of novel and functionally unique bioactive compounds. Marine bioactive peptides are an important class of natural products among these, characterized by their small molecular weight, ease of absorption, and high bioavailability, making them promising functional factors in marine cosmetics and functional foods. Marine bioactive peptides are peptides derived from marine organisms that regulate physiological functions, with molecular weights generally less than 6 kDa. Compared to proteins, marine bioactive peptides are characterized by their small molecular weight, easy digestibility, low toxicity, and high bioavailability, making them a research hotspot in the biopharmaceutical, cosmetic, and food fields. Numerous studies have shown that marine bioactive peptides possess excellent anti-photoaging effects, with mechanisms including antioxidant activity, promotion of collagen synthesis, inhibition of collagen degradation, anti-inflammation, promotion of wound healing, and mitigation of apoptosis.

[0004] MMPs are a class of proteins that can degrade collagen in the dermis and contain Zn. 2+ and Ca 2+ Endopeptidases, specifically micropeptides (MMPs), are mainly secreted by fibroblasts and play a balancing role in collagen synthesis and degradation in the dermis. UV radiation upregulates MMP expression in the skin, accelerating collagen degradation and leading to an imbalance between collagen synthesis and degradation, resulting in wrinkles and premature skin aging. Therefore, MMPs are key enzymes contributing to photoaging. Farmed pufferfish skin is non-toxic and rich in collagen, making it an ideal source of collagen and its collagen peptides. Pufferfish skin collagen peptides (TBSCH) of different molecular weights exhibit anti-photoaging effects, with peptides <1 kDa showing the best photoprotective effect. The central active peptide sequence in pufferfish skin that exerts photoprotective effects remains unknown.

[0005] Furthermore, current peptide separation and purification technologies are mature, including membrane separation, chromatographic separation, and electrophoresis. However, these technologies are characterized by long processing times, high costs, and high loss rates. Virtual screening can rapidly identify promising compounds from a large number of compounds, but it has long been plagued by a high false positive rate. Summary of the Invention

[0006] This invention provides a decapeptide, its extraction and purification method, and its application. The purpose is to obtain the central peptide segment of an active peptide that can exert a photoprotective effect, to obtain the method for separating, screening, and purifying the central peptide segment, and to describe the application of the central peptide segment in various industries.

[0007] To achieve the above objectives, the present invention adopts the following solution: The present invention provides a decapeptide with the following amino acid sequence: RGFPGGDGAA.

[0008] The present invention provides a method for polypeptide extraction, purification, and screening using pufferfish as raw material, comprising the following steps: S1. Cut the pufferfish skin into pieces, remove impurities, endogenous proteases and lipids from the skin, freeze-dry the treated skin and then vacuum preserve it. S2. Add the fish skin treated in step S1 to the acid solution and stir. After centrifugation, add the precipitate to the acid solution again and stir and centrifuge. Combine the supernatants after the two stirring and centrifugation and collect the precipitate. S3. Take the supernatant obtained in step S2, add buffer solution, centrifuge, add acid solution to the precipitate to dissolve, dialyze through dialysate, freeze dry to obtain acid-soluble collagen. S4. Take the precipitate obtained in step S2, add acid solution and pepsin, enzymatically digest and centrifuge to obtain supernatant, add buffer to supernatant, centrifuge and add acid solution to precipitate to dissolve, dialyze through dialysate, freeze dry to obtain enzyme-soluble collagen. S5. Combine the acid-soluble collagen and enzyme-soluble collagen obtained in steps S3 and S4 to obtain collagen. S6. The collagen obtained in step S5 is hydrolyzed by papain to obtain collagen peptides. S7. The collagen peptides obtained in step S6 are separated and purified to obtain the above-mentioned polypeptides.

[0009] Optionally, in step S7, the collagen peptide separation and purification steps include: S71. Ultrafiltration The collagen peptides obtained in step S6 were subjected to ultrafiltration to obtain several small molecule peptides with a molecular weight of <1kDa. S72. Gel filtration chromatography separation The polypeptide obtained in step S71 was subjected to gel filtration chromatography and eluted to obtain several purified small polypeptide molecules. S73. Molecular Virtual Screening The purified small molecule peptides obtained in step S72 were tested, and seed peptides were obtained through molecular virtual screening; the activity of the seed peptides was measured to obtain the active target peptides.

[0010] Optionally, in step S71, the collagen peptides are passed through a ceramic membrane and an ultrafiltration membrane to obtain small molecule peptides with a molecular weight of less than 1 kDa, which are then freeze-dried under vacuum. In step S72, the small molecule peptide is dissolved in deionized water to prepare a peptide solution with a concentration of 10-15 mg / mL. The solution is then filtered through a 0.2-0.3 μm filter membrane, and the eluent is filtered through a 0.4-0.5 μm filter membrane. Ultrasonic sonication is used to remove air bubbles from the eluent. The peptide solution is injected into a gel filtration chromatography column, and elution is performed using PBS buffer at a flow rate of 1-5 mL / min. The eluent is detected at a wavelength of 215 nm, and the elution peak is collected at room temperature. The elution peak is desalted using a dialysis bag and then freeze-dried under vacuum to obtain the purified small molecule peptide. In step S73, the purified small molecule peptides are subjected to high performance liquid chromatography and mass spectrometry analysis to screen out peptides with high reliability and perform virtual molecular docking with MMP-1 receptor protein to obtain seed peptides; the obtained seed peptides are then subjected to cell activity tests to obtain active target peptides.

[0011] Optionally, in step S73, lead peptides with confidence level -10lgP > 20 and number of amino acids < 10 are screened, solid-phase synthesized, and their effects on UVB-induced cell activity of L929 cells are measured to screen out peptides with anti-photoaging effects as target peptides.

[0012] Optionally, in step S1, the removal of impurities includes the following process: adding fish skin to a 0.6-1.0 mol / L NaCl solution at a solid-liquid ratio of 1:15-1:25, stirring, and then rinsing.

[0013] Optionally, in step S1, removing endogenous proteases includes the following process: after removing impurities, the fish skin is immersed in a 0.05-0.15 mol / L NaOH solution at a solid-liquid ratio of 1:35-1:45 and then stirred.

[0014] Optionally, in step S1, the removal of lipids includes the following process: after removing endogenous proteases, the fish skin is immersed in a solution of 8%-12% n-butanol at a solid-liquid ratio of 1:15-1:25, stirred, and then rinsed.

[0015] Optionally, in step S2, the acid solution is specifically an acetic acid solution, the solid-liquid ratio of fish skin to acetic acid solution is 1:10-1:20, the concentration of acetic acid solution is 0.3-0.7 mol / L, and the centrifugation time is 10-20 min.

[0016] Optionally, in step S3, the buffer solution is a NaCl Tris-HCl buffer solution, and the concentrations of the added NaCl solution and Tris-HCl buffer solution are 3-7 mol / L and 0.03-0.07 mol / L, respectively; the acid solution is an acetic acid solution, and the concentration of the acetic acid solution used to dissolve the precipitate is 0.3-0.7 mol / L; the dialysate is a colloid composed of acetic acid and water, and the concentration of acetic acid in the dialysate is 0.05-0.15 mol / L.

[0017] Optionally, in step S4, the acid solution for dissolving the precipitate is an acetic acid solution with a concentration of 0.3-0.7 mol / L; the buffer solution is a NaCl Tris-HCl buffer solution with concentrations of 3-7 mol / L and 0.03-0.07 mol / L for the added NaCl solution and Tris-HCl buffer solution, respectively; the concentration of the acetic acid solution for dissolving the precipitate is 0.3-0.7 mol / L; and the dialysate is a colloid of acetic acid and water with a concentration of acetic acid of 0.05-0.15 mol / L.

[0018] Optionally, in step S4, the enzymatic hydrolysis conditions for pepsin are as follows: under the conditions of 0.3-0.7 mol / L acetic acid solution, the enzyme activity of pepsin is 20 U / g, the temperature is 4℃, and the enzymatic hydrolysis time is 48 h.

[0019] Optionally, in step S5, the papain hydrolysis conditions are: a material-to-liquid ratio of 1:200-1:300, a temperature of 48°C, a pH of 5.35, a papain content of 51000 U / g, and a hydrolysis time of 4 h.

[0020] Optionally, the pufferfish is the two-spotted pufferfish.

[0021] The present invention provides the application of a decapeptide or a polypeptide prepared by a decapeptide extraction and purification method in the preparation of anti-photoaging cosmetics.

[0022] Optionally, the decapeptide is present in the cosmetic at a concentration range of 12.5-400 μM.

[0023] Optionally, the decapeptide is present in the cosmetic at a concentration range of 25-200 μM.

[0024] The present invention has the following beneficial effects: 1. The peptide of this invention has high activity, and the score for docking with MMP-1 is -9.6271, indicating high docking activity.

[0025] 2. The polypeptide of this invention is obtained by extraction, purification and screening of pufferfish skin. It belongs to marine bioactive peptides and has the characteristics of small molecular weight, easy absorption, low toxicity and high bioavailability.

[0026] 3. The polypeptide of this invention has good anti-photoaging effects, and its mechanism of action includes anti-oxidation, promoting collagen synthesis, inhibiting collagen degradation, anti-inflammation, promoting wound healing and alleviating cell apoptosis, etc.

[0027] 4. This invention employs an acid-enzyme complex method to extract collagen, followed by gel filtration chromatography to separate and purify TBAPP (molecular weight <1kDa) obtained through ultrafiltration, screening for components with photoprotective effects. Mass spectrometry is used to identify the amino acid sequence of this component. Using MMP-1 as a target, molecular docking virtual screening is employed to obtain lead peptide sequences interacting with MMP-1. These sequences are then synthesized in a solid phase, and their effects on the cell activity of UVB-irradiated mouse L929 cells are measured, screening for peptides with anti-photoaging effects. The screened peptides exhibit an inhibitory effect on MMP-1 activity, with an inhibition rate as high as 33.71%. Attached Figure Description

[0028] Figure 1 This is a flowchart of the extraction method for ASC and PSC from marine organisms.

[0029] Figure 2 This is an elution diagram of Superdex gel column separation and purification.

[0030] Figure 3 The effects of multiple lead peptides on the activity of UVB-induced photoaging L929 cells were investigated.

[0031] Figure 4 These are schematic diagrams of the two-dimensional structure of the anti-photoaging peptide and the three-dimensional structure of MMP-1, where (a) is a schematic diagram of the two-dimensional structure of RGFPGGDGAA; and (b) is a schematic diagram of the three-dimensional structure of MMP-1 (PDB: 966C).

[0032] Figure 5 This is a schematic diagram of the docking results between the anti-photoaging peptide and the MMP-1 molecule, showing the three-dimensional structure (a) and two-dimensional structure (b) of the docking between RGFPGGDGAA and the MMP-1 molecule.

[0033] Figure 6 This is the chromatogram of RGFPGGDGAA.

[0034] Figure 7 This is the mass spectrum of RGFPGGDGAA.

[0035] Figure 8 This study investigated the effect of RGFPGGDGAA on the survival rate of L929 cells.

[0036] Figure 9 This study investigated the effect of RGFPGGDGAA on UVB-induced L929 cell survival.

[0037] Figure 10 This study investigated the effect of RGFPGGDGAA on ROS levels in UVB-induced L929 cells. Detailed Implementation

[0038] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] Example The present application proposes a polypeptide with the following amino acid sequence: RGFPGGDGAA.

[0040] This polypeptide can be extracted from pufferfish skin, such as... Figure 1 As shown, the specific extraction process includes the following steps: The skin of the spotted pufferfish was cut into small pieces using a meat slicer. The skin was then freeze-dried to remove moisture and vacuum-packed for preservation. Next, the dried skin was added to a 0.8 mol / L NaCl solution at a solid-liquid ratio of 1:20 and stirred for 30 min, changing the solution every 10 min. The skin was then rinsed with cold distilled water to remove impurities. Then, the skin was immersed in a 0.1 mol / L NaOH solution at a solid-liquid ratio of 1:40 and stirred at low temperature for 48 h, changing the solution every 12 h and rinsing thoroughly with cold distilled water to remove non-collagenous proteins and prevent endogenous proteases from affecting collagen. Finally, the skin was immersed in a 10% n-butanol solution at a solid-liquid ratio of 1:20 and stirred at 4°C for 48 h, changing the solution every 12 h and rinsing thoroughly with cold distilled water to remove lipids. The treated skin was then freeze-dried and vacuum-packed for preservation.

[0041] Fish skin, from which non-collagenous proteins and lipids have been removed, was added to a 0.5 mol / L acetic acid solution at a solid-liquid ratio of 1:15. The mixture was stirred at 4°C for 48 h, centrifuged at 10000×g at 4°C for 15 min, and the precipitate was treated once more under the same conditions. The supernatants from both treatments were combined, and the precipitate was collected. The supernatant was used for the extraction of acid-soluble collagen, and the precipitate was used for the extraction of enzyme-soluble collagen.

[0042] Acid-soluble collagen and enzyme-soluble collagen were obtained by using both acid extraction and enzymatic extraction methods. The combined acid-soluble collagen and enzyme-soluble collagen were termed TBSC.

[0043] The TBSC obtained above was enzymatically hydrolyzed with papain under the following conditions: a solid-liquid ratio of 1:250, a temperature of 48 ℃, a pH of 5.35, a papain dosage of 51000 U / g, and a hydrolysis time of 4 h to obtain TBSCH. TBSCH was then passed through a ceramic membrane and an ultrafiltration membrane to obtain TSSCH-L with a concentration less than 1 kDa, which was then freeze-dried under vacuum and stored at -20℃.

[0044] The lyophilized peptides were dissolved in deionized water to prepare a 12 mg / mL peptide solution, which was then filtered through a 0.22 μm filter. Before the experiment, the eluent was filtered through a 0.45 μm filter and sonicated for 20 min to remove air bubbles. The gel column was equilibrated with deionized water before and after the experiment to maintain a stable baseline. 1.2 mL of the peptide solution was injected into a HiLoad 16 / 600 Superdex 30 pg column using a syringe. Elution was performed with PBS buffer at a flow rate of 1 mL / min. The eluent was detected at 215 nm using an AKTA purifier, and the elution peak was collected into centrifuge tubes at room temperature. The peak was automatically plotted using UNICORN software. The eluent was then desalted using a dialysis bag for 24 h. The desalted eluent solution was then lyophilized under vacuum and stored at -20°C or -80°C. The effects of each elution fraction at concentrations of 1 mg / mL and 0.5 mg / mL on UVB-induced L929 cell activity were investigated to screen for peptides with anti-photoaging properties.

[0045] The obtained peptide fraction with anti-photoaging properties was first desalted using a C18 column. The sample was then analyzed using a Q Exactive™ Plus mass spectrometer. An Acclaim PepMap C18 analytical column was used for sample separation. The column flow rate was controlled at 300 nL / min, the column temperature at 40 °C, and the electrospray voltage at 2 kV. The gradient started at 2% with a B phase, increased non-linearly to 35% within 47 min, then to 100% within 1 min, and maintained for 12 min. The mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition. The mass spectrometry parameters were set as follows: MS parameters: scan range (m / z): 200-1800; resolution: 70,000; AGC target: 3e6; maximum injection time: 50 ms; HCD-MS / MS parameters: resolution: 17,500; AGC target: 1e5; maximum injection time: 45 ms; collision energy: 28; dynamic exclusion time: 30 s.

[0046] like Figure 6 , 7As shown, based on the mass spectrometry analysis results, peptides were initially screened using the confidence level of the spectral identification (-10lgP) and the number of amino acids. MMP-1 (PDB: 966C) was selected as the receptor protein, and its 3D structure was downloaded from the RCSBPDB database. Before simulation screening, MOE 2015 software was used to remove useless water molecules from the receptor protein, add hydrogen, add charge, and optimize energy. The peptides were plotted as small molecules as ligands using Discovery Studio 2019 Client software to determine the active pockets of MMP-1. The S1′ active pocket contained Tyr240, Ala182, His218, Thr241, Ala234, Val215, and Arg214; the S3′ active pocket contained residues Tyr240 and Tyr210; and the S1 active pocket contained residues Phe185 and Gln186. Molecular simulation screening was performed using DOCK 6.9, and lead peptides were screened based on Grid Score and binding mode.

[0047] Using a Superdex pre-packed column, TBAPP was separated and purified according to its molecular weight. The separation results are as follows: Figure 2 As shown, three peaks appeared after elution with Superdex.

[0048] The peptide sequences of the components were identified using nano-HPLC-MS / MS. In the peptide results, the -10lgP index represents the confidence level of the corresponding spectral identification; a higher value indicates a better match. Based on the mass spectrometry analysis, peptides with a confidence level of -10lgP > 20 and an amino acid count < 10 were screened, ultimately resulting in 178 peptide sequences. These 178 peptides were then molecularly docked with the MMP-1 (PDB: 966C) receptor protein. Based on the grid score and binding mode, 12 lead peptide sequences were selected from these 178 peptides.

[0049] The effects of the 12 lead peptides screened using the above methods on UVB-induced L929 cell viability are as follows: Figure 3As shown, normal cells exhibited significantly reduced cell viability after UVB irradiation. Compared to the model control group, the addition of 200 μM of RGFPGGDGAA, GPAGPRGA, FPGGPGAK, and RGFPGGDGAA significantly improved cell survival, demonstrating a certain protective effect against UVB-induced L929 cells. Specifically, RGFPGGDGAA and GPAGPRGA increased the viability of UVB-induced L929 cells by 13.86% and 13.90%, respectively, while FPGGPGAK and RGFPGGDGAA increased the viability by 8.48% and 6.97%, respectively. Other peptides had no significant effect on cell viability. Based on these results, four anti-photoaging peptides—RGFPGGDGAA, GPAGPRGA, FPGGPGAK, and RGFPGGDGAA—were preliminarily identified.

[0050] Table 1-1 Relevant parameters of the docking results between anti-photoaging peptides and MMP-1

[0051] Table 1-1 shows the relevant parameters for docking results. A lower score indicates higher activity during receptor-ligand docking. As shown in Table 1-1, the score for MMP-1 docking with the anti-photoaging peptide is -9.6271, indicating high docking activity. The RMSD parameter describes the changes in protein-ligand interactions in the crystal structure before and after docking. A lower RMSD value indicates that the conformation of the docked ligand is closer to the target protein binding site. The RMSD values ​​for both MMP-1 and the anti-photoaging peptide docking are relatively low, indicating that the docked peptide conformation is close to the target protein binding site.

[0052] Depend on Figure 4 (a) Figure 4 (b) Figure 5 (a) Figure 5 (b) It is known that RGFPGGDGAA forms hydrogen bonds with residues Glu219 and Ala182 in the active pocket of the MMP-1 protein, and forms salt bridges with residues Gln247, Asp245, Thr241, Ile232, and Tyr240 through water molecules. From the above results, it can be seen that TBAPP and MMP-1 mainly bind through hydrogen bonds and salt bridges.

[0053] MMP-1 is an enzyme involved in the degradation of matrix metalloproteins. MMP-1 plays a very important role in skin photoaging. This experiment investigated the in vitro inhibition of MMP-1 activity by different concentrations of TBAPP. The results are shown in Table 1-2: RGFPGGDGAA has an inhibitory effect on MMP-1 activity.

[0054] Table 1-2 Inhibition rate of TBAPP against MMP-1 in vitro

[0055] (1) The toxic effect of RGFPGGDGAA on L929 cells L929 cells are mouse fibroblasts, and many studies have used the L929 cell line as an in vitro model of UVB-induced photoaging and photodamage. This study used the MTS assay to determine L929 cell viability and assessed the photoprotective effect of RGFPGGDGAA based on cell viability. The effect of RGFPGGDGAA on L929 cell cytotoxicity is as follows: Figure 8 As shown.

[0056] (2) Effect of RGFPGGDGAA on UVB-induced L929 cell viability The effect of RGFPGGDGAA on UVB-induced L929 cell viability is as follows: Figure 9 As shown, compared with the group without UVB irradiation, L929 cells were treated with 40 mJ / cm². 2 After UVB irradiation, cell survival rate decreased by about 40% (p < 0.0001), indicating that UVB irradiation caused a certain degree of photodamage to cells. When culture medium containing 25-200 μM RGFPGGDGAA was added, cell survival rate increased significantly compared with the model control group, indicating that RGFPGGDGAA has a protective effect against UVB-induced photodamage to L929 cells.

[0057] (3) Effect of RGFPGGDGAA on ROS content in UVB-induced L929 cells This study used the fluorescent probe DCFH-DA to measure intracellular ROS. DCFH-DA can freely enter the cell membrane. Once inside the cell, the non-fluorescent DCFH-DA is hydrolyzed to generate non-fluorescent DCFH, which is further oxidized by intracellular ROS to generate fluorescent DCF. The fluorescence intensity is detected using a photomicroplate reader, and the fluorescence intensity can measure the intracellular ROS content.

[0058] The effect of RGFPGGDGAA on ROS content in UVB-induced L929 cells is as follows: Figure 10 As shown, compared with the normal control group, L929 cells showed significantly enhanced fluorescence intensity after UVB irradiation (p < 0.0001).

Claims

1. A decapeptide, characterized in that, Its amino acid sequence is as follows: RGFPGGDGAA.

2. The application of the decapeptide according to claim 1 in the preparation of anti-photoaging cosmetics.

3. The application according to claim 2, characterized in that: The decapeptide is present in the cosmetic at a concentration range of 12.5-400 μM.

4. The application according to claim 2, characterized in that: The decapeptide is present in the cosmetic at a concentration range of 25-200 μM.

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