Tortoise shell bioactive peptide as well as preparation method and application thereof

Abstract

The invention discloses a tortoise shell bioactive peptide and a preparation method and application thereof.The tortoise shell bioactive peptide comprises one or more of EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS and DPKMNGLK. The preparation method comprises the steps of preparation of tortoise shell protein hydrolysate, ultrafiltration separation, anion exchange resin separation, gel column separation, de novo sequencing analysis, molecular docking screening and solid-phase synthesis. The tortoise shell bioactive peptide can be applied to the fields of oxidation resistance and immunoregulation. The method disclosed by the invention is a separation, purification and identification technical method of the tortoise shell specific antioxidant polypeptide based on activity guidance, and has the advantages of clear components, clear activity, clear mechanism and the like; the obtained specific polypeptide has important significance for functional product development of polypeptide compounds with clear tortoise shell components.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug development, and particularly relates to a turtle shell bioactive peptide, a preparation method thereof, and an application thereof. Background Art

[0002] As an important substance involved in various cellular functions in the human body, polypeptide is an important physiological regulator in the human body. Based on the antioxidant activity of peptides that can scavenge free radicals in vitro and regulate antioxidant pathways in vivo, it has always been the focus of research on natural antioxidants. Bioactive peptides are a class of peptides with biological activities that can have certain effects on the body, playing an important role in maintaining the stability and health of the body, and having the advantages of wide application range, high efficiency, no toxic and side effects, etc. Currently, the development and utilization of animal drugs from marine organisms such as crustaceans, fish, mollusks, and fishery wastes are rich sources of bioactive peptides, and the proteins isolated from them have strong antioxidant activities, as well as anti-cancer, antibacterial, antihypertensive, anti-diabetic, anti-obesity, immunomodulatory, neuroprotective, and analgesic effects.

[0003] Turtle shell, also known as Shenwu, turtle shell, tortoise plastron, and turtle ventral shell, is the dried ventral shell and dorsal shell of the turtle (Chinemys reevesii) of the tortoise family. Recently, studies have shown that turtle shell can inhibit apoptosis, improve immunity, and has a preventive and therapeutic effect on osteoporosis. The new Biejiajian Pills inhibit the proliferation of hepatic stellate cells and induce their apoptosis through the TGF-β1 / Smad axis, thereby exerting an anti-heart failure effect. At the same time, Biejiajian Pills can improve the clinical efficacy of levonorgestrel intrauterine for endometriosis, thereby reducing the protein levels of programmed cell death and its ligands and improving the immune function of cells. Turtle shell glue, a solid glue made by decocting and concentrating turtle shell, contains 15 kinds of amino acids, fatty acids, lipids, sterols, and various essential elements for the human body. Studies have shown that turtle shell glue can reduce the content of MDA in the serum of rats, increase the level of SOD, and enhance the activity of antioxidant enzymes, thereby regulating the cytokine disorder in rats in the state of kidney yin deficiency and enhancing their antioxidant capacity. Therefore, turtle shell has a broad market prospect due to its rich bioactive substances, high nutritional value, and antioxidant activity.

[0004] Modern nutrition has confirmed that the meat of Chinemys reevesii is rich in protein, vitamins, and fatty acids, and the content of bone collagen, protein, fat, and peptides in turtle shell is sufficient. Studies have shown that the enzymatic hydrolysate of turtle muscle protein contains rich polypeptides and has excellent anti-tumor activity. However, currently, the research on turtle shell protein and polypeptides is lacking, mainly focusing on the formation of turtle shell, the identification of genuine and fake turtle shell glue and other traditional Chinese medicine glue products, etc. There are few studies on the extraction, separation, identification, and functional activities of its polypeptides, which also limits the further development of turtle shell-related functional foods or drugs.

[0005] At present, the research on turtle shell mainly includes chemical analysis and pharmacological activities. In terms of chemical analysis, studies have shown that turtle shell contains a large amount of proteins and polypeptides, which can be used as important markers for the identification of its origin. For example, ultra-high performance liquid chromatography - electrospray quadrupole time-of-flight mass spectrometry was used, and the characteristic peptide ions of cowhide were used as the detection object to determine the turtle shell glue from two sources. The results showed that this method could distinguish the turtle shell glue of Trachemys scripta elegans and the turtle shell glue doped with cowhide components, and could also be used for the quality control of turtle shell glue. At the same time, the medicinal parts of different origin animals (donkey skin, turtle shell and deer antler disc) are rich in proteins and are distributed in the range of 10 kDa - 250 kDa. The main differences in the SDS-PAGE and 2-DE protein profiles of the above three kinds of traditional Chinese medicine glue are as follows: the molecular weight range of proteins in donkey-hide gelatin is between 15 kDa - 250 kDa, or even higher, while the antler glue and turtle shell glue are basically <50 kDa, and the regions are different, which can provide certain reference for the identification of traditional Chinese medicine glue. After digestion with trypsin, 1 characteristic protein was identified from the turtle shell by MALDI-TOF / TOF-MS analysis and database matching retrieval; taking the protein spots separated by 2-DE as the source, 6 other characteristic proteins were identified from the turtle shell by MALDI-TOF / TOF-MS analysis and database matching retrieval. The total proteins of donkey-hide gelatin, antler glue and turtle shell glue were digested with collagenase respectively to obtain peptide mixtures, and were analyzed by Nano LC-Orbitrap MSD. Then, the amino acid sequences of the peptides were analyzed by Mascot retrieval and de novo sequencing with PEAKS software, and a total of 14 potential characteristic peptides were found, 5 of which were from turtle shell glue. In addition, in terms of pharmacological activities, studies have shown that different extracts of turtle shell have various functional activities, including anti-tumor, anti-osteoporosis, immunomodulatory and other activities.

[0006] However, both the above-mentioned research on chemical analysis and pharmacological activities focus on the research related to polypeptides and proteins of turtle shell glue itself or its extracts. There is no research on the separation, purification, identification of specific polypeptides of turtle shell and the biological activities of its specific active peptides, especially the identification and application of new peptides. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art. In particular, the current research on tortoise shell protein and polypeptide focuses on the study of tortoise shell glue itself or its extracts, and there is no specific polypeptide research on tortoise shell yet. The present invention provides a tortoise shell bioactive peptide, its preparation method and application. The method is a separation, purification and identification technical method of tortoise shell specific antioxidant polypeptide based on activity guidance, with advantages such as clear composition, clear activity and clear mechanism. At the same time, through molecular docking screening and verification of cell-level activity of the identified polypeptides, 5 specific polypeptides can be screened out, which is of great significance for the development of functional products of tortoise shell polypeptide compounds with clear and in-depth components.

[0008] To solve the above technical problems, the present invention adopts the following technical solutions.

[0009] A tortoise shell bioactive peptide, the tortoise shell bioactive peptide includes one or more of EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS and DPKMNGLK;

[0010] The molecular structural formula of the EHTVVY is as follows:

[0011]

[0012] The molecular structural formula of the GPKKDQK is as follows:

[0013]

[0014] The molecular structural formula of the SPLSDVRGH is as follows:

[0015]

[0016] The molecular structural formula of the SENADKGALS is as follows:

[0017]

[0018] The molecular structural formula of the DPKMNGLK is as follows:

[0019]

[0020] As a general technical concept, the present invention also provides a preparation method of the above-mentioned tortoise shell bioactive peptide, including the following steps:

[0021] (1) The turtle shell of Chinese pond turtle (Chinemys reevesii) was broken into powder, and the turtle shell powder was air-dried and placed in five centrifuge tubes. Trypsin, alkaline protease, neutral protease, papain, and pepsin were added respectively for hydrolysis. Then, the hydrolyzate was heated to inactivate enzymes and centrifuged. The obtained supernatant was concentrated to obtain an enzyme hydrolyzate. The antioxidant activities of the five enzyme hydrolyzates were analyzed respectively, and the best enzyme was found to be alkaline protease. The alkaline protease was treated by water bath, heated to inactivate enzymes, and centrifuged to obtain a hydrolyzed supernatant, which was freeze-dried and named GTPH;

[0022] (2) The obtained GTPH above was divided into three segments. First, it was treated with a 10 kDa ultrafiltration membrane and centrifuged. The upper part was collected and denoted as GTPHa-I. The lower part was taken and treated with a 3 kDa ultrafiltration membrane and centrifuged. The upper part obtained was denoted as GTPHa-II, and the lower part obtained was denoted as GTPHa-III. Then, the antioxidant activities of GTPHa-I, GTPHa-II, and GTPHa-III were analyzed to obtain the part with the best antioxidant activity;

[0023] (3) The part with the best antioxidant activity above was separated by anion exchange chromatography using DEAE-52 packing material with antioxidant activity guidance. According to the elution order, three components were separated. After the antioxidant activity analysis, the part with the highest antioxidant activity was obtained;

[0024] (4) The part with the highest antioxidant activity above was analyzed using a Sephadex gel column. The antioxidant activities of the three components obtained were analyzed, and finally a homogeneous polypeptide component with the best antioxidant effect was obtained;

[0025] (5) The obtained polypeptide component above was subjected to de novo sequencing analysis based on mass spectrometry. All identified polypeptide sequences were defined as ligands, and the docking target protein Keap1 was defined as the receptor. Through molecular docking technology, polypeptides that had binding sites with the 8 key residues TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, and ARG380 in Keap1 and the absolute value of the docking energy was greater than that of the positive drug were screened out. Then, the screened polypeptides were synthesized by solid phase synthesis to obtain the bioactive peptides from turtle shell EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS, and DPKMNGLK.

[0026] For the above-mentioned method for preparing turtle shell bioactive peptides, preferably, in step (1), during hydrolysis: the temperature of trypsin is controlled at 36.5°C to 38.5°C, and the pH value is controlled at 6.0 to 8.0; the temperature of alkaline protease is controlled at 40°C to 60°C, and the pH value is controlled at 7.5 to 9.5; the temperature of neutral protease is controlled at 40°C to 60°C, and the pH value is controlled at 6.0 to 8.0; the temperature of papain is controlled at 40°C to 60°C, and the pH value is controlled at 7.5 to 9.5; the temperature of pepsin is controlled at 36.5°C to 38.5°C, and the pH value is controlled at 1.0 to 3.0;

[0027] The temperature for heat inactivation is 90°C to 100°C, the rotation speed for centrifugation is 10000 rpm to 12000 rpm, and the temperature for centrifugation is 15°C to 20°C.

[0028] For the above-mentioned method for preparing turtle shell bioactive peptides, preferably, in step (2), the centrifugal force for centrifugation after 10 kDa ultrafiltration membrane treatment is 5000×g to 6000×g, the temperature for centrifugation is 15°C to 25°C, and the time for centrifugation is 20 min to 30 min; the centrifugal force for centrifugation after 3 kDa ultrafiltration membrane treatment is 5000×g to 6000×g, the temperature for centrifugation is 15°C to 25°C, and the time for centrifugation is 50 min to 90 min.

[0029] For the above-mentioned method for preparing turtle shell bioactive peptides, preferably, in step (3), during anion exchange chromatography separation, elution is carried out successively with ultrapure water, 0.1 mol / L sodium chloride, 0.3 mol / L sodium chloride, 0.5 mol / L sodium chloride, and 1 mol / L sodium chloride, fractions are collected, and the detection wavelength is set at 280 nm.

[0030] For the above-mentioned method for preparing turtle shell bioactive peptides, preferably, in step (4), separation and purification are carried out through a Sephadex G-15 dextran gel column, elution is carried out with ultrapure water, each fraction is 3 mL, and detection is carried out with an enzyme-labeling instrument at 280 nm.

[0031] For the above-mentioned method for preparing turtle shell bioactive peptides, preferably, in step (5), the denovo sequencing analysis based on mass spectrometry is carried out as follows: dithiothreitol solution is added to the sample to make its final concentration 10 mmol / L, and it is incubated in a water bath at 56°C for 60 min; indoleacetic acid solution is added to make its final concentration 50 mmol / L, and the reaction is carried out in the dark for 40 min; desalting is carried out using a self-packed desalting column, vacuum centrifugal concentration and drying are carried out at 45°C, then liquid chromatography-mass spectrometry analysis is carried out, and then the software PEAKS Studio (8.5) is used to carry out polypeptide sequence analysis by the de novo method.

[0032] As a general technical concept, the present invention also provides an application of the above-mentioned turtle shell bioactive peptide or the turtle shell bioactive peptide obtained by the preparation method of the above-mentioned turtle shell bioactive peptide in the fields of antioxidant and immunomodulation.

[0033] Compared with the prior art, the advantages of the present invention are as follows:

[0034] (1) The turtle shell bioactive peptide provided by the present invention has significant antioxidant effects. The antioxidant polypeptide has a relatively simple structure, is convenient for artificial synthesis, and has strong antioxidant activity, which is convenient for subsequent development and utilization, and can be widely applied in the fields of antioxidant and immunomodulation.

[0035] (2) The preparation method of the turtle shell bioactive peptide of the present invention separates and purifies the turtle shell by means of enzymatic hydrolysis, ultrafiltration separation, anion exchange chromatography, gel separation, etc., and simultaneously examines the antioxidant activity indexes of DPPH·, HO·, ABTS· + scavenging activities. Through this activity-guided separation means, a homogeneous bioactive peptide with strong antioxidant activity is obtained, and the monomer polypeptides therein are further identified by de novo sequencing means. Using molecular docking as a means, polypeptide monomers with specific Keap1 binding ability are screened, and the activities of these specific polypeptides against H2O2-induced cell oxidative damage are further verified, including cytotoxicity, intracellular ROS levels, antioxidant enzyme activities, and the expression of antioxidant-related proteins. Compared with the current research on turtle shell proteins and polypeptides, which mainly focuses on the research of turtle shell glue itself or its extracts, the present invention is based on the separation, purification, identification, and screening technical methods of turtle shell-specific antioxidant polypeptides guided by activity, and has the advantages of clear composition, clear activity, and clear mechanism. At the same time, through molecular docking screening and cell-level activity verification of the identified polypeptides, 5 specific polypeptides are screened out, which is of great significance for the development of functional products of turtle shell with clear and well-defined polypeptide compounds. Description of the Drawings

[0036] Figure 1 It is an analysis diagram of the antioxidant activity of the peptide segments under alkaline protease measured after the enzymatic hydrolysis (five enzymes) of the turtle shell extract and ultrafiltration separation in Example 1 of the present invention. Among them, A, B, and C are the results obtained by the enzymatic hydrolysis method. A is the DPPH· scavenging activity, B is the HO· scavenging activity, and C is the ABTS· + scavenging activity, and D, E, and F are the results obtained after ultrafiltration separation. D is the DPPH· scavenging activity, E is the HO· scavenging activity, and F is the ABTS· + scavenging activity.

[0037] Figure 2This is the analysis chart of the components obtained from each peak segment after GTPHa-III passed through the cellulose column in Example 1 of the present invention, and the antioxidant activities of each component. Among them, A is the component obtained from each peak segment after GTPHa-III passed through the cellulose column, B is the HO scavenging activity of each component, C is the DPPH scavenging activity of each component, and D is the ABTS·+ scavenging activity of each component.

[0038] Figure 3 This is the analysis chart of the components obtained from each peak segment after gel filtration chromatography in Example 1 of the present invention, and the antioxidant activities of each component. Among them, A and E are the components obtained from each peak segment after gel filtration, B is the DPPH scavenging activity of each component, C is the HO scavenging activity of each component, and D is the ABTS·+ scavenging activity of each component.

[0039] Figure 4 This is the molecular docking screening result (Figure A) in Example 1 of the present invention, and the two-dimensional (Figure B-F) and three-dimensional (Figure G-K) molecular docking diagrams of 5 polypeptides.

[0040] Figure 5 This is the mass spectrometry chart of the polypeptides in Example 1 of the present invention. Among them, A is EHTVVY (EHT), B is GPKKDQK (GPK), C is SPLSDVRGH (SPL), D is SENADKGALS (SEN), and E is DPKMNGLK (DPK).

[0041] Figure 6 This is the molecular structure diagram of 5 polypeptides in Example 1 of the present invention. Among them, A is EHTVVY (EHT), B is GPKKDQK (GPK), C is SPLSDVRGH (SPL), D is SENADKGALS (SEN), and E is DPKMNGLK (DPK).

[0042] Figure 7 This is the diagram of the protective effect of the polypeptides on HepG2 cells under H2O2-induced oxidative stress in Example 1 of the present invention. Among them, A-E are the polypeptides EHT (A), GPK (B), SPL (C), SEN (D), and DPK (E) pre-treating HepG2 cells for 24 h, treating the cells with 4 mM H2O2 for 24 h, and detecting the viability of HepG2 cells by CCK8. Mean±SD, n = 5, ** / ***P<0.01, *P<0.05.

[0043] Figure 8Graph showing the effect of the polypeptide in Example 1 of the present invention on ROS generation; wherein, A: HepG2 cells were pretreated with polypeptides EHT, GPK, SPL, SEN, and DPK for 24 h and then treated with 4 mM H2O2 for 24 h, and ROS in HepG2 was detected by DCFH-DA probe, and the scale bar represents 50 μM; B: Histogram showing the gray values of the results, Mean±SD, n = 3, ** / ***P<0.01, *P<0.05.

[0044] Figure 9 Graph showing the effect of EHT, GPK, SPL, SEN, and DPK in Example 1 of the present invention on antioxidant enzymes; wherein, A represents the effect of EHT, GPK, SPL, SEN, and DPK on the activity of glutathione reductase (GR), B represents the effect of EHT, GPK, SPL, SEN, and DPK on the activity of glutathione peroxidase (GPX), C represents the effect of EHT, GPK, SPL, SEN, and DPK on the activity of catalase (CAT), D represents the effect of EHT, GPK, SPL, SEN, and DPK on the activity of MDA (malondialdehyde), and in the histogram, A represents the effect of EHT on antioxidant enzymes, B represents the effect of GPK on antioxidant enzymes, C represents the effect of SPL on antioxidant enzymes, D represents the effect of SEN on antioxidant enzymes, and E represents the effect of DPK on antioxidant enzymes.

[0045] Figure 10 Graph showing the effect of DPK in Example 1 of the present invention on alleviating H2O2-induced Keap1-Nrf2 signal transduction; wherein, A: HepG2 cells were pretreated with 125, 250, and 500 μM DPK for 24 h and then 4 mM H2O2 was added for 24 h, and β-actin was used as a control, and the protein levels of Nrf2, Keap1, HO-1, NQO1, and the internal reference β-actin were detected by Western blot; B-E: Histogram showing the gray values of the Western blot results, Mean±SD, n = 3, ** / ***P<0.01, *P<0.05. Detailed implementation manners

[0046] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific preferred embodiments, but the protection scope of the present invention is not limited thereby. The materials and instruments used in the following examples are all commercially available.

[0047] In the following examples, when performing statistical analysis, all experimental data were analyzed using SPSS 21.0 and GraphPad Prism 9.0.0 software. Measurement data were expressed as mean±standard deviation. One-way analysis of variance was used for comparison of differences between groups, and independent samples t-test was used for comparison between two groups. A P<0.05 was considered statistically significant.

[0048] Example 1

[0049] A tortoise shell bioactive peptide of the present invention, the tortoise shell bioactive peptide being EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS or DPKMNGLK;

[0050] The molecular structural formula of EHTVVY is as follows:

[0051]

[0052] The molecular structural formula of GPKKDQK is as follows:

[0053]

[0054] The molecular structural formula of SPLSDVRGH is as follows:

[0055]

[0056] The molecular structural formula of SENADKGALS is as follows:

[0057]

[0058] The molecular structural formula of DPKMNGLK is as follows:

[0059]

[0060] A preparation method of the tortoise shell bioactive peptide of this example, comprising the following steps:

[0061] (1) Preparation of tortoise shell protein hydrolysate:

[0062] The tortoise shell of Chinemys reevesii is crushed with a hard object and ground into powder with a pulverizer. The powder is air-dried into a solid precipitate. Then, 1.6 g of each portion is weighed into a centrifuge tube and dissolved in an appropriate amount of ultrapure water. Trypsin (pH 7.8, 37.5 °C), alkaline protease (pH 8.5, 50 °C), neutral protease (pH 7.0, 50 °C), papain (pH 8.5, 50 °C) and pepsin (pH 2.0, 37.5 °C) are used for hydrolysis in a water bath at different temperatures. Mix well every half hour and adjust the pH. After 4 h, the hydrolysate is heated to 95 °C, cooled to room temperature and then centrifuged at 10,000 rpm and 15 °C for 15 min to separate the supernatant and the precipitate. The above enzyme-hydrolyzed supernatants are respectively stored in a 4 °C refrigerator and concentrated to 2 mL by using a water bath nitrogen blower. Subsequently, the antioxidant activities of the five enzyme hydrolysates are evaluated respectively to obtain the best enzyme, named GTPH.

[0063] The experiment used mg protein / mL enzyme hydrolysate and its components to represent the concentration. Using BSA as the standard protein, the protein concentration of the sample was determined by the dye-binding method of Bradford (1976).

[0064] The antioxidant activities of five enzyme hydrolysates were evaluated separately to obtain the best enzyme. Then, the preparation was carried out as follows: 100 g of solid precipitate was dispersed in distilled water (DW) at a ratio of 1:5 (w / v). The best protease was treated at its appropriate temperature and pH, with a total enzyme dosage of 2% (w / w, 2 g enzyme / 100 g solid precipitate). It was treated in a constant-temperature water bath stirring box for 5 h. The pH was adjusted every half hour, and then heated to inactivate the enzyme. The hydrolysate supernatant was obtained by centrifugation and freeze-dried.

[0065] The following were the DPPH· scavenging experiment, HO· scavenging experiment, and ABTS· + scavenging experiment. The DPPH· scavenging experiment, HO· scavenging experiment, and ABTS· + scavenging experiment in each step were all carried out according to the following operations.

[0066] DPPH Scavenging Experiment

[0067] Take 50 μL of samples diluted to different concentration gradients (0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.025 mg / mL, a total of 7 gradients) into a six-well plate, add 150 μL of DPPH· methanol solution (0.1 mg / mL), mix well, and react in the dark at room temperature for 30 min. At the same time, a blank control sample without the sample was prepared by replacing the DPPH· solution with methanol and measured at 517 nm.

[0068] HO· Scavenging Experiment

[0069] Take 40 μL of 1.87 mM 1,10-phenanthroline solution and 80 μL of samples (0.5, 0.25, 0.1, 0.05, 0.025, 0.01, 0.001 mg / mL, a total of 7 gradients) and add them to an enzyme plate for mixing. Subsequently, add 40 μL of FeSO4·7H2O (1.87 mM) solution and 40 μL of H2O (0.03%, v / v) to start the reaction. Incubate at 37 °C with shaking for 60 min, and measure the absorbance of the reaction mixture against the reagent blank at 536 nm. The reaction mixture without any antioxidant was used as the negative control, and the mixture without H2O2 was used as the blank test.

[0070] ABTS· + Scavenging Experiment

[0071] Add 20 μL of peroxidase working solution to each test well of a 96-well plate. Start the reaction after adding 10 μL of the sample solution (0.5, 0.25, 0.125, 0.1, 0.05 mg / mL) to 170 μL of ABTS working solution. The peroxidase working solution and ABTS working solution are prepared according to the instructions. Add 10 μL of distilled water to the blank control well, incubate for 6 min, and measure the absorbance at 414 nm.

[0072] In this example, alkaline protease, trypsin, neutral protease, pepsin, and papain were used to hydrolyze the tortoise shell protein extract respectively, and the antioxidant capacity of GTPH was detected by adjusting the concentrations of the five proteins to 0.0 - 0.5 mg / mL; as Figure 1 shown in the DPPH scavenging activity results in A, the EC50 values of neutral protease, trypsin, alkaline protease, papain, and pepsin were 0.153 mg / mL, 0.196 mg / mL, 0.090 mg / mL, 0.146 mg / mL, and 0.293 mg / mL respectively; as Figure 1 shown in B, the EC 50 values of neutral protease, trypsin, alkaline protease, papain, and pepsin for HO· scavenging activity were 0.013 mg / mL, 0.023 mg / mL, 0.0039 mg / mL, 0.051 mg / mL, and 0.077 mg / mL respectively; as Figure 1 shown in C, the EC + values of neutral protease, trypsin, alkaline protease, papain, and pepsin for ABTS· 50 scavenging activity were 0.287 mg / mL, 0.331 mg / mL, 0.208 mg / mL, 0.280 mg / mL, and 0.323 mg / mL respectively. The above results show that the EC50 values of alkaline protease are the lowest among the antioxidant indexes of DPPH, ABTS· + , and HO·, so alkaline protease was selected for hydrolysis to obtain the best hydrolyzate GTPHa.

[0073] (2) Ultrafiltration separation

[0074] The hydrolysis supernatant was divided into three segments by ultrafiltration membranes (3 kDa, 10 kDa). After rinsing the ultrafiltration tube with pure water 3 - 5 times, first use a 10 kDa ultrafiltration membrane, centrifuge at 5000×g and 20 °C for 30 min, suck the upper part with a 200 μL pipette gun and collect it in one tube, transfer the lower part of the tube as fully as possible to another tube, then change to a 3 kDa ultrafiltration membrane, centrifuge at 5000×g and 20 °C for 60 min, continue to process the lower part to obtain three components, collect and freeze-dry them separately, and perform activity determination.

[0075] In this step, GTPHa was separated into three segments by ultrafiltration tubes with molecular weights of 3 kDa and 10 kDa, named GTPHa-I (>10 kDa), GTPHa-II (3 kDa - 10 kDa), and GTPHa-III (<3 kDa), respectively. Further, the antioxidant activities of the three components were analyzed within the protein concentration range of 0.0 - 0.8 mg / mL. As Figure 1 shown in the D-1F result, GTPHa-III with the smallest molecular weight had the best antioxidant activity. The EC50 values of its DPPH scavenging activity, HO scavenging activity, and ABTS· + scavenging activity were 21.93 μg / mL, 0.95 μg / mL, and 0.1134 mg / mL, respectively. Based on this, GTPHa-III was selected for subsequent separation operations.

[0076] (3) Anion exchange resin separation

[0077] Take the sample with the best activity obtained from the above experiment and separate it using DEAE-52 (2.4 cm × 100 cm). Elute successively with water, 0.1 M, 0.3 M, 0.5 M, and 1 M sodium chloride, and collect the fractions. The packing material was first soaked and swollen with water for 1 h, and then the alcohol was washed off. After packing the column, it was pre-equilibrated with water for 3 - 5 column volumes, and then the sample was rinsed with each gradient for 250 mL at a flow rate controlled at 1 mL / min. Each bottle collected 5 mL. The detection wavelength was 280 nm. After collecting according to the peaks, it was dried with a water bath nitrogen blower, and each peak segment was formulated into a 20 mL sample to measure the activity.

[0078] In this step, anion exchange chromatography with DEAE-52 packing material was used to further separate GTPHa-III by antioxidant activity guidance, as Figure 2 shown in the A result. According to the elution order of the peaks, three components were separated and named GTPHa-III-A, GTPHa-III-B, and GTPHa-III-C. Further, through antioxidant activity analysis, as Figure 2 shown in the B-2D result, the EC + values of HO, DPPH, and ABTS· 50 scavenging activity tests of GTPHa-III-C were 0.9222 μg / mL, 2.264 μg / mL, and 2.457 μg / mL, respectively, all of which were the lowest, indicating its highest antioxidant activity, suggesting that GTPHa-III-C may contain more acidic and hydrophobic amino acid residues. Therefore, GTPHa-III-C was selected for subsequent further separation experiments.

[0079] (4) Gel column separation

[0080] Take the sample with the best activity obtained above, concentrate it, and use Sephadex G-15 (2.4 cm × 120 cm) as the packing material for analysis. The gel was stirred with pure water for 24 h in advance to remove floating substances. The column was packed in the wet state, loaded in one go, and equilibrated with pure water for 3 - 5 h before loading the sample. The flow rate was controlled at 0.7 mL / min, and water was used as the eluent. A total of 120 bottles of components were collected, 3 mL per bottle, and detected at a wavelength of 280 nm. After collecting according to the peak segments, it was dried with a water bath nitrogen blower, and each peak segment was formulated into a 5 mL sample to measure the activity. Finally, the component with the best measured activity was dried and reserved for use.

[0081] In this step, the GTPHa-III-C component with the highest antioxidant activity separated by anion chromatography was further purified by gel filtration chromatography. As Figure 3 shown in Figure A, GTPHa-III-C was divided into three components, but the second and third components were clearly not separated. Therefore, the middle two components were separated by secondary gel filtration to obtain Figure 3 E. After each component was collected, freeze-dried, and named GTPHa-III-C1, GTPHa-III-C2, and GTPHa-III-C3 in the order of elution peaks. As Figure 3 shown in Figure B, DPPH· scavenging activity: GTPHa-III-C2 (EC 50 = 4.838 μg / mL) > GTPHa-III-C1 (EC 50 = 17.75 μg / mL) > GTPHa-III-C3 (EC 50 = 45.97 μg / mL). As Figure 3 shown in Figure C, HO· scavenging activity: GTPHa-III-C2 (EC 50 = 7.842 μg / mL) > GTPHa-III-C1 (EC 50 = 93.61 μg / mL) > GTPHa-III-C3 (EC 50 = 447.2 mg / mL). As Figure 3 shown in Figure D, ABTS· + scavenging activity: GTPHa-III-C2 (EC 50 = 8.331 μg / mL) > GTPHa-III-C1 (EC 50 = 3.362 mg / mL) > GTPHa-III-C3 (EC 50 = 76429 mg / mL). The above results show that GTPHa-III-C2 has the highest scavenging activity. Therefore, the GTPHa-III-C2 component was selected, freeze-dried, and subjected to subsequent mass spectrometry analysis.

[0082] (5) Screening of antioxidant candidate polypeptides and synthesis

[0083] LC-MS is a commonly used method for identifying polypeptide sequences. Add an appropriate amount of sample to a dithiothreitol (DTT) solution to make its final concentration 10 mmol / L, and incubate in a water bath at 56 °C for 60 min; add an indoleacetic acid (IAA) solution to make its final concentration 50 mmol / L, and react in the dark for 40 min; desalt using a self-packed desalting column, centrifuge and concentrate to dryness under vacuum at 45 °C, and then perform liquid chromatography-mass spectrometry (LC-MS / MS) analysis. The parameter settings are as follows: analytical column: 150 μm i.d.×150 mm, packed with Acclaim PepMap RP LC C18, 3 μm, Mobile phase A: 0.1% formic acid (by volume); mobile phase B: 0.1% formic acid, 80% ACN; flow rate: 600 nL / min; gradient analysis is adopted, where mobile phase B is 4% at 0 min, 8% at 2 min, 40% at 45 min, 60% at 55 min, and 95% at 56 min and later, and the total analysis time is 66 min; the mass spectrometry parameters are as follows: primary mass spectrometry parameters: Resolution: 70000 AGC target: 3e6 Maximum IT: 100 ms Scan range: 300 to 1800 m / z; secondary mass spectrometry parameters: Resolution: 17500 AGC target: 1e5 Maximum IT: 50 ms TopN: 20 NCE / stepped NCE: 28. After detection, use the software PEAKS Studio (8.5) to perform polypeptide sequence analysis using the de novo method. The search parameters are as follows: (1) Fixed modifications: Carbamidomethyl (C) (2) Variable modifications: Oxidation (M), Acetylation (N-term) (3) Enzyme: Nonspecific (4) Maximum missed cleavage sites: (5) Peptide mass tolerance: 20 ppm (6) Fragment mass tolerance: 0.02 Da. This part of the experiment was carried out by Beijing Bio-Tech Pack Technology Co., Ltd.

[0084] The polypeptide components were identified by de novo sequencing based on LC-MS technology. All the identified polypeptide sequences were defined as ligands, and the docking target protein Keap1 (PDB ID: 7K2S) was defined as the receptor. Using Discovery Studio software, further through molecular docking technology and the semi-flexible docking method, polypeptides that could interact with 8 key residues (TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, ARG380) in Keap1 (with binding sites) and whose absolute value of the docking energy (-cdocker energy) was greater than that of the positive drug were screened. The results are as Figure 4 (shown in A-K). Five polypeptides, EHTVVY (EHT), GPKKDQK (GPK), SPLSDVRGH (SPL), SENADKGALS (SEN), and DPKMNGLK (DPK), had good overlap. The absolute values of their docking energies (-cdocker energy) were 130.555 kcal / mol, 129.979 kcal / mol, 123.555 kcal / mol, 159.504 kcal / mol, and 123.865 kcal / mol respectively. The absolute value of the docking energy of the positive drug (Nrf2 polypeptide fragment) used for comparison was 122.313 kcal / mol. The mass spectra of these 5 polypeptides are as Figure 5 shown in A-5E, and the molecular structure diagrams are as Figure 6 shown in A-6E. The above 5 polypeptides were synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. using the solid-phase synthesis method, namely, the bioactive peptides from tortoise shell EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS, and DPKMNGLK were obtained.

[0085] Using the above 5 polypeptides as the research objects, cell verification experiments were carried out.

[0086] a) Cell culture:

[0087] Human hepatocellular carcinoma HepG2 cells were cultured in 5a medium (Gibco, USA) containing 10% fetal bovine serum and 1% double antibiotics (penicillin 100 U / mL and streptomycin 100 μg / mL). The culture flask was placed in an incubator at 37 °C and 5% CO2. When the cell confluence rate reached about 80%, cell passage was carried out.

[0088] b) Optimal concentration of H2O2-induced oxidative damage in HepG2 cells

[0089] HepG2 cells were seeded at a density of 2×10 4Cells were seeded in 96-well plates at a density of [cells / well]. After culturing in an incubator for 24 h, the cells were treated with H2O2 at gradient concentrations (0, 1.0, 2.0, 4.0, 8.0 mM) for 24 h. After 24 h, 10 μL of CCK8 was added to each well, and the plates were incubated in the incubator for another 2 h. Subsequently, the 96-well plates were taken out, and the OD values of each well were measured at 450 nm using a microplate reader. The cell survival rate was calculated, and the experiment was repeated three times. After calculation, a H2O2 concentration of 4.0 mM was finally selected for subsequent experiments.

[0090] c) Detection of the effect of tortoise shell polypeptide on cell viability by CCK8

[0091] HepG2 cells were seeded in 96-well plates at a density of 2×10 4 cells / well. After culturing in an incubator for 24 h, the cells were treated with tortoise shell polypeptide at concentrations of 0, 62.5, 125, 250, 500, 1000 μM for 24 h. After 24 h, 10 μL of CCK8 was added to each well, and the plates were incubated in the incubator for another 2 h. Subsequently, the 96-well plates were taken out, and the OD values of each well were measured at 450 nm using a microplate reader. The cell survival rate was calculated, and the experiment was repeated three times.

[0092] d) Detection of the protective effect of tortoise shell polypeptide on cells by CCK8

[0093] HepG2 cells were seeded in 96-well plates at a density of 2×10 4 cells / well. After culturing in an incubator for 24 h, the cells were pretreated with tortoise shell polypeptide at gradients of 250 μM and 500 μM for 24 h, and then 2 mM H2O2 was added for continuous treatment for 24 h. After 24 h, 10 μL of CCK8 was added to each well, and the plates were incubated in the incubator for another 2 h. Subsequently, the 96-well plates were taken out, and the OD values of each well were measured at 450 nm using a microplate reader. The cell survival rate was calculated, and the experiment was repeated three times.

[0094] e) Detection of reactive oxygen species in cells by ROS experiment

[0095] HepG2 cells in the logarithmic phase were seeded on coverslips in 12-well culture plates at a density of 2×10 5 . They were continuously cultured in a 37 °C, 5% CO2 incubator for 24 h. Then they were grouped into a blank control group, an H2O2 group, and a tortoise shell polypeptide group (250 μM, 500 μM). The cell culture medium was removed. In the negative control wells (untreated), positive control wells (positive reagent added), and sample groups, 1 mL of diluted DCFH-DA was added, and the plates were incubated in a 37 °C cell culture incubator for 30 min. After 30 min, the culture plates were taken out, and the cells were washed three times with serum-free cell culture medium. The cell coverslips were taken out and placed on glass slides, and directly detected under a fluorescence microscope at an excitation wavelength of 488 nm. All samples were measured under the same parameters, and the experiment was repeated three times.

[0096] f) Detection of antioxidant enzymes

[0097] To investigate the activity of antioxidant enzymes in HepG2 cells induced by H2O2 by the polypeptide and detect its antioxidant activity. Logarithmic-phase HepG2 cells were seeded in 6-well culture plates at a density of 5×10 5 . After continued culture in an incubator at 37°C and 5% CO2 for 24 h, after grouping and treating the cells according to e), cells in the blank control group, H2O2 model group, and tortoise shell polypeptide groups at different concentrations were collected. The activities of glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT), and malondialdehyde (MDA) were measured according to the instructions of each kit.

[0098] g) Detection of protein expression by Western blot

[0099] Logarithmic-phase HepG2 cells were seeded in 6-well culture plates at a density of 5×10 5 . After continued culture in an incubator at 37°C and 5% CO2 for 24 h, they were grouped into a blank control group, an H2O2 group, and tortoise shell polypeptide groups (125 μM, 250 μM, 500 μM). Total proteins of cells in the blank control group, H2O2 model group, and tortoise shell polypeptide protection groups at different concentrations were extracted. The protein concentration was measured by the BCA method. Equal amounts of proteins were added to the wells of the SDS-PAGE gel. After the protein bands were separated, protein transfer was performed using a PVDF membrane with a pore size of 0.45 μm. It was blocked with skim milk for 1 h and rinsed 6 times with TBST, once every ten minutes. Primary antibodies against rabbit (mouse) anti-human Nrf2, p-Nrf2, Keap1, HO-1, NQO1, and β-actin were added at a concentration of 1:1000 and incubated overnight at 4°C. The membrane was washed 3 times with TBST at room temperature. After incubation with a horseradish peroxidase-labeled secondary antibody for 1 min, detection was performed by ECL chemiluminescence method. Analyzed with Image J software.

[0100] Analysis of cell experiment results:

[0101] The above 5 polypeptides were synthesized by the solid-phase synthesis method, and their antioxidant activities and effects on Keap1 and Nrf2 were verified through an H2O2-induced HepG2 cell oxidative stress model. First, the results of the protective effect on HepG2 cells are as Figure 7 shown in A-7E. Compared with the blank control group, the cell survival rate of cells treated with 4 mM H2O2 decreased significantly (p<0.05). After pretreatment of HepG2 cells with polypeptides at concentrations of 250 μM and 500 μM for 24 h, the cell survival rate increased significantly, indicating that the polypeptides EHT, GPK, SPL, SEN, and DPK can effectively protect the survival of HepG2 under oxidative stress conditions.

[0102] The influence on ROS generation is as follows Figure 8 As shown in Figures 8A and 8B, compared with the control group, the intracellular ROS level in HepG2 cells increased significantly after induction with H2O2, while the ROS accumulation was eliminated after pretreatment with the polypeptide. In particular, the polypeptide DPK dose-dependently inhibited the intracellular ROS level (p<0.05), indicating that the polypeptides EHT, GPK, SPL, SEN, and DPK can play a protective role by reducing the production of intracellular ROS.

[0103] The activities of antioxidant enzymes GR, GPx, CAT, and the level of MDA in HepG2 cells were further measured. As Figure 9 shown in Figures 9A-9C, compared with the control group, the treatment of HepG2 cells with H2O2 significantly reduced the activities of GR, CAT, and GPx. After pretreatment with each polypeptide, compared with the model group, the polypeptides EHT, GPK, SPL, and SEN only increased the antioxidant activities of GR, GPx, and CAT in cells at a high concentration of 500 μM, and had no effect at a concentration of 250 μM, while the polypeptide DPK dose-dependently increased the antioxidant activities of GR, GPx, and CAT in cells. As Figure 9 shown in Figure 9D, the treatment of cells with H2O2 significantly increased the activity level of MDA. After pretreatment with the polypeptide, the polypeptides SPL, SEN, and DPK down-regulated the MDA level in HepG2 cells at concentrations of 250 μM and 500 μM, and the polypeptide DPK had the best effect.

[0104] The above cell experiment results show that all 5 polypeptides have a protective effect on H2O2-induced oxidative stress in HepG2 cells, but the polypeptide DPK has the best antioxidant activity. Therefore, in the present invention, DPK is selected as the representative polypeptide to study its effect on the expression of proteins related to Keap1 and Nrf2. As Figure 10 shown in Figures 10A-10E, after treating the cells in the model group, the expression of Keap1 was up-regulated and the expression of Nrf2 was down-regulated. After pretreatment with different concentrations of DPK, it could restore the expression of Keap1 and simultaneously up-regulate the expression of Nrf2. At the same time, DPK could significantly increase the expression of HO-1 and NQO1 in HepG2 cells treated with H2O2. This indicates that DPK can achieve significant antioxidant capacity through the Keap1 / Nrf2 pathway.

[0105] From the above content, it can be seen that the tortoise shell bioactive peptide of the present invention can be applied to the field of antioxidant or immunomodulation.

[0106] The above are only the preferred embodiments of the present invention, and do not impose any form of limitation on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A turtle shell bioactive peptide, characterized in that, The tortoise shell bioactive peptides include one or more of EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS, and DPKMNGLK; The molecular structural formula of the above-mentioned EHTVVY is as follows: The molecular structural formula of the above-mentioned GPKKDQK is as follows: The molecular structural formula of the above-mentioned SPLSDVRGH is as follows: The molecular structural formula of the above-mentioned SENADKGALS is as follows: The molecular structural formula of the above-mentioned DPKMNGLK is as follows:

2. A preparation method of the tortoise shell bioactive peptide as described in claim 1, characterized in that, It includes the following steps: (1) Break the tortoise shell of Chinemys reevesii into powder, air-dry the tortoise shell powder and place it in five centrifuge tubes. Add trypsin, alkaline protease, neutral protease, papain, and pepsin for hydrolysis respectively. Then heat-inactivate and centrifuge the hydrolyzate. The obtained supernatant is concentrated to obtain an enzyme hydrolyzate. Analyze the antioxidant activities of the five enzyme hydrolyzates respectively to obtain the best enzyme as alkaline protease. Subject the alkaline protease to water bath treatment, heat inactivation, and centrifugation to obtain a hydrolyzed supernatant, which is freeze-dried and named GTPH; (2) Divide the above-mentioned obtained GTPH into three segments. First, treat it with a 10 kDa ultrafiltration membrane and centrifuge. Collect the upper part and label it as GTPHa-I. Take the lower part and treat it with a 3 kDa ultrafiltration membrane and centrifuge. The upper part obtained is labeled as GTPHa-II, and the lower part obtained is labeled as GTPHa-III. Then analyze the antioxidant activities of GTPHa-I, GTPHa-II, and GTPHa-III to obtain the part with the best antioxidant activity; (3) Subject the part with the best antioxidant activity obtained above to antioxidant activity-guided separation using an anion exchange chromatography with DEAE-52 packing material. According to the elution order, three components are separated. Analyze the antioxidant activities to obtain the part with the highest antioxidant activity; (4) Analyze the part with the highest antioxidant activity obtained above using a Sephadex gel column. Analyze the antioxidant activities of the three components obtained to finally obtain a homogeneous polypeptide component with the best antioxidant effect; (5) Perform de novo sequencing analysis based on mass spectrometry on the above-mentioned obtained polypeptide component. Define all identified polypeptide sequences as ligands and dock the target protein Keap1 as the receptor. Through molecular docking technology, screen out the polypeptides that have binding sites with the 8 key residues TYR334, TYR572, TYR525, ASN382, HIS436, ARG415, ARG483, and ARG380 in Keap1 and the absolute value of the docking energy is greater than that of the positive drug. Then solid-phase synthesize the screened polypeptides to obtain the tortoise shell bioactive peptides EHTVVY, GPKKDQK, SPLSDVRGH, SENADKGALS, and DPKMNGLK.

3. The preparation method of the turtle shell bioactive peptide according to claim 2, characterized in that, In step (1), during the hydrolysis: the temperature of the trypsin is controlled at 36.5°C to 38.5°C, and the pH value is controlled at 6.0 to 8.0; the temperature of the alkaline protease is controlled at 40°C to 60°C, and the pH value is controlled at 7.5 to 9.5; the temperature of the neutral protease is controlled at 40°C to 60°C, and the pH value is controlled at 6.0 to 8.0; the temperature of the papain is controlled at 40°C to 60°C, and the pH value is controlled at 7.5 to 9.5; the temperature of the pepsin is controlled at 36.5°C to 38.5°C, and the pH value is controlled at 1.0 to 3.0; The temperature for heat inactivation is 90°C to 100°C, the rotation speed for centrifugation is 10,000 rpm to 12,000 rpm, and the temperature for centrifugation is 15°C to 20°C.

4. The preparation method of the turtle shell bioactive peptide according to claim 2, wherein In step (2), the centrifugal force for centrifugation after treatment with the 10 kDa ultrafiltration membrane is 5,000×g to 6,000×g, the temperature for centrifugation is 15°C to 25°C, and the time for centrifugation is 20 min to 30 min; the centrifugal force for centrifugation after treatment with the 3 kDa ultrafiltration membrane is 5,000×g to 6,000×g, the temperature for centrifugation is 15°C to 25°C, and the time for centrifugation is 50 min to 90 min.

5. The preparation method of the turtle shell bioactive peptide according to claim 2, characterized in that In step (3), during the anion exchange chromatography separation process, elution is successively carried out with ultrapure water, 0.1 mol / L sodium chloride, 0.3 mol / L sodium chloride, 0.5 mol / L sodium chloride, and 1 mol / L sodium chloride, fractions are collected, and the detection wavelength is set at 280 nm.

6. The preparation method of the turtle shell bioactive peptide according to any one of claims 2 to 5, characterized in that, In step (4), separation and purification are carried out through a Sephadex G-15 dextran gel column, elution is carried out with ultrapure water, each fraction is 3 mL, and detection is carried out with an enzyme-linked immunosorbent assay instrument at 280 nm.

7. The preparation method of the turtle shell bioactive peptide according to any one of claims 2 to 5, characterized in that In step (5), the de novo sequencing analysis based on mass spectrometry is carried out as follows: a dithiothreitol solution is added to the sample to make its final concentration 10 mmol / L, and it is placed in a water bath at 56°C for 60 min; an indoleacetic acid solution is added to make its final concentration 50 mmol / L, and the reaction is carried out in the dark for 40 min; desalting is carried out using a self-packed desalting column, vacuum centrifugation concentration and drying are carried out at 45°C, then liquid chromatography-mass spectrometry analysis is carried out, and then the software PEAKS Studio (8.5) is used to carry out polypeptide sequence analysis by the de novo method.

8. Use of the tortoise shell bioactive peptide as described in claim 1 or the tortoise shell bioactive peptide obtained by the preparation method of the tortoise shell bioactive peptide as described in any one of claims 2 to 7 in the fields of antioxidant and immunomodulation.

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