Tilapia skin peptide, preparation method thereof and use thereof in preparing anti-aging related products

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to tilapia skin peptides, preparation methods thereof, and uses thereof in the preparation of anti-aging related products. The tilapia skin peptides described in the present invention are IL, FGP or LPGL. In the present invention, tilapia skin is subjected to enzymatic hydrolysis to prepare protein peptides, and peptide segments with high content and high credibility are selected by mass spectrometry identification, and a pure product is prepared using solid phase chemical synthesis. Taking GPH as the positive control group, it was found that IL, FGP and LPGL have a protective effect on UV-induced cell damage, have good antioxidant activity, and have the effect of repairing damaged cells, which is beneficial to protecting the skin barrier and delaying skin aging.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to tilapia skin peptide and a preparation method thereof and use thereof in preparing anti-aging related products. Background Art

[0002] Skin aging is a complex and diverse process, which can be divided into endogenous aging and exogenous aging. Endogenous aging includes inevitable physiological changes over time and is affected by gene expression, nutrition, endocrine and immunity. Exogenous aging is caused by external factors, such as air pollution, smoking, malnutrition, climate and ultraviolet rays.

[0003] In recent years, people have paid more attention to their skin health while paying attention to their physical health. As the concept of gentle, green and efficient skin care has been deeply rooted in people's minds, the skin care method has begun to change from external application to oral, avoiding irritation, inflammation and even allergies to the skin, improving the skin condition from the inside out and delaying skin aging.

[0004] Peptides are low molecular weight bioactive peptides with special functions obtained through deep hydrolysis. Compared with proteins, peptides have a higher human absorption rate, stronger biological activity, high safety and good biocompatibility. Based on the above excellent characteristics, people are increasingly favoring the use of peptides to delay skin aging. Compared with drugs, functional foods with anti-aging activity avoid the defects of long drug development cycle and clinical trial cycle, and have a broader development space.

[0005] The present invention aims to provide an efficient and reliable active peptide having a significant effect on delaying skin aging. Summary of the invention

[0006] In order to overcome the defects and shortcomings of the prior art, the purpose of the present invention is to screen the polypeptides in the enzymatic hydrolysate of tilapia skin, and then obtain peptide segments that have cell protection and repair functions, antioxidant activity, protect skin barrier function, inhibit matrix metalloproteinase activity and increase collagen content.

[0007] Specifically, the present invention is realized through the following technical solutions:

[0008] In a first aspect, the present invention provides a tilapia skin peptide having anti-aging activity, wherein the tilapia skin peptide is separated and identified from a tilapia skin enzyme product, and the tilapia skin peptide is IL, FGP or LPGL.

[0009] As an optional method, in the above-mentioned tilapia skin peptide, the preparation method of the tilapia skin enzymatic hydrolysate is as follows:

[0010] Take an appropriate amount of frozen tilapia skin, add water to thaw, then add 5-10% NaOH to soak and remove impurities, rinse it with clean water for several times until the pH of the washing water is neutral, add 0.05-2% alkaline protease, enzymolyze for 2-8 hours, then inactivate the enzyme in a boiling water bath, take the enzymolysis solution and pass it through a 200-mesh silk cloth to remove impurities, filter to remove suspended impurities, centrifuge at 4°C, take the supernatant, and freeze-dry to obtain the tilapia skin enzymolysis product.

[0011] In a second aspect, the present invention provides a method for preparing the tilapia skin peptide described in the first aspect, wherein the tilapia skin peptide is obtained by enzymatic separation from tilapia skin.

[0012] In a third aspect, the present invention provides a method for preparing the tilapia skin peptide described in the first aspect, wherein the tilapia skin peptide is prepared by solid phase synthesis.

[0013] In a fourth aspect, the present invention provides use of the tilapia skin peptide described in the first aspect in preparing anti-aging related products.

[0014] As an option, in the above use, the product is an oral product or an external product.

[0015] As an option, in the above use, the product is a medicine, a functional food, a health product, a beverage, a nutritional supplement or a cosmetic.

[0016] As an optional mode, in the above use, the product is an oral solution, tablet, pill, liquid suspension, injection, granule, powder, capsule, beverage, energy bar, chewing gum or candy.

[0017] As an option, in the above use, the product is toner, lotion, essence, cream or mask.

[0018] As an optional mode, in the above-mentioned use, the product has a protective effect on UV-induced cell damage, has antioxidant activity, repairs damaged cells, protects the skin barrier, and delays skin aging.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present inventors screened tilapia skin enzymatic hydrolysates and obtained for the first time three peptides that have the effects of protecting cell activity, inhibiting oxidative damage, protecting the skin barrier, increasing collagen synthesis, inhibiting collagen degradation, and repairing skin damage without obvious side effects.

[0021] (2) The innovation of the present invention is mainly reflected in the functions of IL, FGP and LGPL in anti-oxidation, repair, promotion of collagen synthesis and inhibition of collagen degradation. It comprehensively emphasizes the functions of the three polypeptides in protecting cells from oxidative damage, avoiding collagen loss and improving the ability to repair damage, and further emphasizes the role of the above three polypeptides in inhibiting skin aging.

[0022] (3) The composition of the present invention can be used to prepare various products related to anti-skin aging and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Total ion chromatogram of tilapia skin enzymatic hydrolysates.

[0024] Figure 2 : Molecular structure formula of GPH, IL, FGP and LPGL.

[0025] Figure 3 : Effects of peptides on the survival rate of UV-damaged cells, where A: MSF; B: HACAT.

[0026] Figure 4 :Effects of peptides on MDA content in UV-damaged cells. A: MSF; B: HACAT.

[0027] Figure 5 : Effects of four peptides on ROS content in cells.

[0028] Figure 6 :Effects of three peptides on barrier protein expression in cells.

[0029] Figure 7 :Effects of three peptides on the expression of type I collagen in cells.

[0030] Figure 8 : Inhibition rate of three peptides on collagenase activity.

[0031] Fig. 9 : The inhibition rate of three peptides on MMP protein expression.

[0032] Fig.10 : Effects of four peptides on the migration rate of HaCaT cells. DETAILED DESCRIPTION

[0033] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0034] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0035] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0036] Example 1: Preparation process of tilapia skin peptide

[0037] Take an appropriate amount of frozen tilapia skin, add water to thaw at a ratio of 1:3 (w / v), then add 7% NaOH to soak and remove impurities, rinse it with clean water for several times until the pH of the washing water is neutral, add 0.1% alkaline protease, hydrolyze for 5 hours, then inactivate the enzyme in a boiling water bath for 15 minutes, take the enzymatic hydrolyzate and pass it through a 200-mesh silk cloth to remove impurities, filter to remove suspended impurities, then centrifuge at 8000r / min and 4°C for 15 minutes, take the supernatant, and freeze-dry to obtain the tilapia skin enzymatic hydrolyzate.

[0038] Example 2: Peptide sequence identification

[0039] Sample pretreatment: The samples were desalted and then subjected to mass spectrometry detection.

[0040] Chromatographic separation: Mobile phase A is deionized water containing 0.1% formic acid, and mobile phase B is acetonitrile aqueous solution containing 0.1% formic acid (acetonitrile is 84%). First, 95% of liquid A is used to balance the chromatographic column (0.15 mm x 150 mm, RP-C18, Column Technology Inc.), and the sample is loaded into Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) by an automatic sampler, and then separated by liquid chromatography column. The relevant elution conditions are shown in Table 1:

[0041] Table 1: LC-MS / MS liquid phase elution conditions

[0042]

[0043] Mass spectrometry identification: The enzymatic hydrolysis products were separated by capillary high performance liquid chromatography and then analyzed by mass spectrometry using a Q Exactive mass spectrometer (ThermoFisher). The analysis time was 60 min. Detection method: positive ion. The mass-to-charge ratio of the peptide and its fragments was collected according to the following method: 10 fragment spectra (MS2 scan) were collected after each full scan (full scan).

[0044] Database search: The mass spectrometry test raw file (Raw File) was searched against the corresponding database using the software MaxQuant1.5.5.1, and finally the protein identification and quantitative analysis results were obtained.

[0045] The peptide sequences of tilapia skin enzymatic hydrolysates were identified by mass spectrometry, and peptide sequences with relatively high content were selected for chemical synthesis. Figure 1 The total ion chromatogram of tilapia skin enzymatic hydrolysate is shown, among which IL, FGP and LPGL have high credibility and large peak area. At the same time, the online screening tools https: / / www.innovagen.com / proteomics-tools and https: / / webs.iiitd.edu.in / raghava / toxinpred / show that these three peptides have high solubility, non-toxicity and good stability. Therefore, the three peptides of IL, FGP and LPGL were further studied.

[0046] Example 3: Synthesis of polypeptides GPH, IL, FGP, LPGL by solid phase synthesis

[0047] The resin was swollen, washed, and dried to remove the Fmoc protecting group. The amino acids that make up the peptide were weighed and reacted in a shaker at 30°C for 1 hour for condensation reaction. The removal-protection-condensation process was repeated until all amino acids were connected. The target peptide was separated from impurities by high performance liquid chromatography (HPLC) to obtain a pure peptide (>95%), and the target peptide was lyophilized into powder. Figure 2 It is the molecular structural formula of four peptide segments: GPH (control peptide), IL, FGP, and LPGL.

[0048] Example 4: Effects of peptides on the survival rate of UV-damaged MSF and HACAT cells

[0049] Adenosine triphosphate (ATP) is a constant substance in cells, and the amount of endogenous ATP in cells can reflect the activity of cells. ATP was used to quantitatively detect cell activity, where HACAT cells were purchased from Zhejiang Meisen Cell Technology Co., Ltd., and MSF cells were obtained by removing the head, tail and limbs of C57BL / 6J P0 suckling mice, leaving the trunk, cutting the mouse neck along the hips with surgical scissors, taking skin tissue, digesting the skin tissue with skin tissue digestion fluid, and isolating the obtained mouse skin fibroblasts.

[0050] MSF cells and HACAT cells were cultured at 7×10 3 / hole, 1×10 4 / well were inoculated in a 96-well plate, and DMEM medium was added. Six replicate wells were set up for each group. No cells were inoculated in the blank group, and the same volume of medium was added. After culturing for 24 hours, the model control group and the sample group were UV modeled for 70 minutes, and then the old medium was discarded, the blank control empty and model wells were replaced with fresh medium, and the sample group was replaced with medium containing samples. After continuing to culture for 18 hours, the old medium was discarded, 100 μL of ATP was added to each well, and the cells were treated at 37°C for 1 hour for lysis. The supernatant was taken to the ELISA plate, and the RLU value was determined by a chemiluminometer.

[0051] The results are as follows Figure 3 As shown in the figure, compared with the blank group, the survival rate of HACAT and MSF cells in the UV-induced group was significantly reduced, and after administration, the survival rate was significantly increased. Among them, the cell survival rates of the LGPL, FPG, and IL groups were all higher than those of the GPH group. This shows that the screened peptides are helpful in protecting cells, and their cell protection ability is higher than that of the GPH group.

[0052] Example 5: Effect of peptides on the antioxidant activity of UV-damaged cells

[0053] 1. Determination of MDA content

[0054] Add 100 μL RIPA lysis buffer to the cell sample, react at 4°C for 30 min, centrifuge at 10000 rpm for 10 min, collect the supernatant, adjust the density to 2 mg / mL after quantification. MDA standard curve: prepare the standard curve into 2mM MDA standard, standard curve: add 10 μL of 2mM MDA standard to 200 μL (ddH20); 600 μL TBA + standard or sample 200 μL, 95°C for 60 min, ice bath for 10 min; take 200 μL to 96 wells, detect OD value at 532nm with full wavelength microplate reader, and calculate MDA content.

[0055] The results are as follows Figure 4 As shown, the results showed that after UV irradiation, the MDA content in MSF and HACAT cells increased significantly. After intervention with LGPL, FPG, IL, and GPH, the MDA content in MSF cells decreased, and the MDA content in the LGPL, FPG, and IL groups was lower than that in the GPH group. In addition, LGPL, FPG, IL, and GPH also have an inhibitory effect on MDA in HACAT cells. At the same time, the effects of the four peptides on lipid peroxidation are LGPL, IL, FPG, and GPH from strong to weak. In summary, whether in HACAT cells or in MSF cells, the effects of LGPL, FPG, and IL on lipid peroxidation are obvious, and they are all higher than GPH.

[0056] 2. ROS content determination

[0057] MSF cells and HACAT cells were respectively 4 / hole and 15×10 4 / well were inoculated in a 6-well plate, and DMEM medium was added. Six replicate wells were set up for each group. No cells were inoculated in the blank group, and the same volume of medium was added. After culturing for 24 hours, the model control group and the sample group were UV modeled for 70 minutes, and then the old medium was discarded. The blank control empty and model wells were replaced with fresh medium, and the sample group was replaced with medium containing samples. After continuing to culture for 18 hours, the old medium was discarded. DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10μmol / L, the cell culture medium was removed, and PBS was used to rinse once. 1mL of diluted DCFH-DA was added to each well, and incubated at 37℃ cell box for 20 minutes. The cells were washed 3 times with serum-free medium to remove DCFH-DA that did not enter the cells and reduce the fluorescence background. Finally, the 6-well plate was photographed under a fluorescence microscope.

[0058] The results are as follows Figure 5 As shown in the figure, compared with the ROS in the blank group cells, the ROS content in MSF and HACAT cells after UV irradiation increased. After intervention with LGPL, IL, FGP and GPH, the ROS content in MSF cells decreased, but compared with GPH, the effect of LGPL, IL and FGP in inhibiting cell oxidative stress was more significant. In addition, after intervention with LGPL, IL, FGP and GPH, the ROS content in HACAT cells from low to high was LGPL, IL, FGP and GPH. The above results show that LGPL, IL and FGP can effectively inhibit UV-induced cell oxidative stress, reduce the release of intracellular ROS, and alleviate cell aging and decline caused by UVB.

[0059] Example 6: Protective effects of three peptides on skin barrier

[0060] In this example, the expression of Loricrin, Involucrin, Filaggrin and Aquaporin-3 in HACAT cells was detected by Western Blot. Cells were treated with GPH, IL, FGP and LGPL for 24 hours, digested, centrifuged and collected. The total protein concentration of each group of cells was extracted using RIPA protein high-efficiency lysis buffer. 20 mg of total protein was loaded on each group for SDS-PAGE gel electrophoresis, transferred to PVDF membrane after 2 hours, blocked with 5% BSA for 1 hour, and incubated with Loricrin, Involucrin, Filaggrin and Aquaporin-3 primary antibodies at 4°C overnight. After the primary antibody was recovered, it was washed 3 times with TBST and incubated with rabbit anti- / mouse anti-IgG secondary antibodies at room temperature for 1 hour. After the incubation, TBST was washed 3 times and detected by fluorescence development. The relative expression of each target protein was analyzed using ImageJ software.

[0061] The results are as follows Figure 6 As shown in the figure, compared with the blank group, the expression levels of Loricrin, Involucrin, Filaggrin and Aquaporin-3 increased after intervention with GPH, IL, FGP and LGPL. The protein expression levels of Loricrin, Filaggrin and Aquaporin-3 were higher than those of GPH after intervention with IL, FGP and LGPL. The above results show that GPH, IL, FGP and LGPL can increase the expression of water barrier moisturizing proteins, among which IL, FGP and LGPL have the best effects, indicating that IL, FGP and LGPL can effectively protect the skin barrier and keep the skin clean and moisturized.

[0062] Example 7: Effect of peptides on collagen synthesis

[0063] 1. Determination of type I collagen expression

[0064] Based on the established MSF cell oxidative damage model, Western Blot was used to explore the effects of GPH, IL, FGP and LGPL on CO-1 expression in UV-induced MSF cells. After 70 minutes of UV induction, each group of MSF cells were treated with GPH, IL, FGP and LGPL for 24 hours, digested, centrifuged, and collected. The cells were lysed with RIPA to extract proteins, and WB was performed and detected by fluorescence development. The relative expression of each target protein was analyzed using ImageJ software.

[0065] The results are as follows Figure 7As shown, compared with the blank group, the content of type I collagen in the model group cells after UV irradiation was significantly reduced. After the intervention of GPH, IL, FGP and LGPL, the expression of type I collagen in the cells increased, among which LGPL had the most significant effect, followed by IL, GPH and FGP. The results show that the three peptides IL, FGP and LGPL screened in this study have the same collagen synthesis effect as the positive control GPH.

[0066] 2. Determination of collagenase inhibition rate

[0067] Collagenase is an enzyme from the protease class in molecular biology that can break down collagen. Collagen determines the properties of connective tissue and maintains the toughness and firmness of the skin. When the collagen content in the skin is high, the skin becomes plump and firm, while skin lacking collagen becomes loose and inelastic.

[0068] Collagenase was dissolved in 0.1M Tris-HCl buffer (pH 7.5). To measure collagenase inhibitory activity, 150 μL collagenase and 250 μL of 0.1M Tris-HCl buffer (pH 7.5) containing substrate collagenase were added, followed by 100 μL of sample containing 4 mM CaCl2, mixed and incubated at 25°C for 20 min in the dark. Finally, 0.5 mL of 6% citric acid was added to terminate the reaction and then 1.5 mL of ethyl acetate was added to separate the reaction mixture. The absorbance of the supernatant was measured at 320 nm using a UV spectrophotometer. Collagenase inhibitory activity was determined as follows:

[0069]

[0070] Among them, the samples OD Values ​​and Sample Blanks OD The values ​​represent the optical density of the sample in the presence and absence of OD Values ​​represent optical density in the absence of collagenase.

[0071] The results are as follows Figure 8 As shown, the IC values ​​of GPH, IL, FGP and LGPL for inhibiting collagenase activity are 50 The values ​​are 3.42, 3.27, 2.70 and 3.11 respectively, indicating that these four peptides have a certain inhibitory effect on collagenase activity and can reduce the degradation of collagen in skin fibroblasts. At the same time, compared with GPH, IL, FGP and LGPL have higher collagenase activity inhibition rates. Therefore, IL, FGP and LGPL can more effectively maintain the toughness and firmness of the skin and delay skin aging.

[0072] Example 8: Effect of peptides on collagen degradation

[0073] Based on the established MSF cell oxidative damage model, the effects of GPH, IL, FGP and LGPL on the expression of MMP1, MMP2 and MMP9 in UV-induced MSF cells were investigated by protein immunoblotting. After 70 min of UV induction, each group of MSF cells were treated with GPH, IL, FGP and LGPL for 24 h, digested, centrifuged and collected. The cells were lysed with RIPA to extract proteins, and WB was performed and detected by fluorescence development. The relative expression of each target protein was analyzed using ImageJ software.

[0074] The results showed that compared with the blank group, the expression of MMP in the UV-induced group increased significantly, indicating that the model was successfully established. The four peptides in the experimental group had a significant inhibitory effect on the expression of MMP1, MMP2 and MMP9 proteins, and the inhibitory effects of IL, FGP and LGPL on MMP were higher than those of GPH, among which LGPL had the highest inhibitory effect on MMP ( Fig. 9 ).

[0075] Example 9: Effect of peptides on fibroblast repair activity

[0076] Experimental groups: blank group (HaCaT cells without irradiation treatment + serum-free culture medium), model group (HaCaT cells UV irradiation + serum-free culture medium), experimental group (HaCaT cells UV irradiation + 20 μM concentration sample solution prepared in serum-free culture medium).

[0077] First, use a marker to draw three horizontal lines evenly on the bottom of a 6-well plate. Inoculate HACAT in a 6-well plate (6×10 5 cells / well, 2mL culture medium / well), when the cell coverage rate reached 90%-100%, the serum-free culture medium was replaced according to the experimental groups for 3 hours, and a sterile pipette tip was used to scratch a straight scratch of similar thickness on the bottom of the 6-well plate. The suspended cells were washed 3 times with PBS, and then new serum-free culture medium was replaced according to the experimental groups; the cell migration status was photographed in the same field of view at 0, 6, 12, 24, 36, and 48 hours, and the scratch area was calculated using ImageJ software.

[0078]

[0079] In the cell scratch assay, Fig.10It can be seen that compared with the blank group, after intervention with IL, FGP, LGPL and GPH, the cells had a significant migration trend; the cell migration rates of the IL, FGP and LGPL groups were all higher than those of the GPH group, and the migration rates of the IL group and the LGPL group were significantly different. These results confirm that IL, FGP and LGPL have the effects of promoting cell proliferation, improving cell migration rate and repairing cell damage.

[0080] In summary, the following conclusions can be obtained through the experiments: (1) The present invention found that IL, FGP and LGPL can improve cell survival rate and have the effect of protecting cells; (2) The present invention found that IL, FGP and LGPL can inhibit lipid oxidation and oxidative stress in cells, thereby protecting cells from oxidative damage. (3) The present invention found that IL, FGP and LGPL can significantly increase the expression of proteins related to moisturizing and hydrating in the cell barrier, protect the skin barrier, and lock in skin moisture. (4) The present invention found that IL, FGP and LGPL have a significant inhibitory effect on the increased expression of MMP1, MMP2, and MMP9 proteins in MSF cells induced by UV, and are stronger than GPH; (5) The present invention found that IL, FGP and LGPL can significantly inhibit the decrease in collagen content caused by UV induction; (6) The present invention found that IL, FGP and LGPL can promote cell proliferation and increase cell migration rate, thereby playing a role in repairing skin damage.

[0081] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. The use of tilapia skin peptide in the preparation of anti-aging related products, characterized in that: The tilapia skin peptide is separated and identified from tilapia skin enzyme products, the tilapia skin peptide is IL, FGP or LPGL, and the product is a medicine or a cosmetic.

2. The use according to claim 1, characterized in that: The product is an oral product or an external product.

3. The use according to claim 2, characterized in that: The product is an oral solution, tablet, pill, liquid suspension, injection, granule, powder or capsule.

4. The use according to claim 2, characterized in that: The product is a toner, lotion, essence, cream or mask.

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