Liver-protecting active peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of protein, and relates to a peptide with liver protection activity and a preparation method and application thereof, and the amino acid sequence of the peptide is PFPRPQP. The active peptide can be used for activating a Keap1 / Nrf2 signal channel by reducing the liver enzyme level and inhibiting CYP2E1 expression so as to play a role in protecting HepG2 damaged cells. The invention also provides a preparation method and application of the liver-protecting active peptide.

Description

Technical Field

[0001] This invention belongs to the field of protein technology, and in particular relates to a hepatoprotective active peptide, its preparation method, and its application. Background Technology

[0002] The liver is a vital organ involved in metabolism and detoxification. Liver disease can be caused by a variety of factors, such as chemical drugs, obesity, alcoholism, and genetic factors. Currently, liver disease is a common disease worldwide, including acute or chronic hepatitis, drug-induced liver injury, alcoholic or non-alcoholic fatty liver disease, and cirrhosis. Alcohol-related liver disease (ALD) caused by excessive alcohol consumption has become a global public health problem.

[0003] Clinical intervention for ALD still faces significant challenges, with existing alternative therapies failing to meet acceptable standards in terms of efficacy and safety. Alcohol abstinence is the preferred treatment strategy for alcoholic liver disease, but it suffers from poor patient adherence and high relapse rates in clinical practice. Among drug therapies, corticosteroids can reduce short- to medium-term mortality but increase the risk of infection in patients with alcoholic hepatitis (AH), and their effects are limited to the short term. Pentoxothecobalamin (PTX), as a TNF-α inhibitor, does not fully improve survival. The use of statins in patients with alcoholic cirrhosis (AC) remains controversial, with potential safety concerns, and they are not suitable for patients in the later stages of AC.

[0004] Soybean peptides possess high nutritional value, a balanced amino acid composition, and are rich in functional amino acids, giving them potential advantages in the early prevention of ALD. However, currently, there is a lack of efficient methods for preparing hepatoprotective active peptides, and an effective screening method is also lacking to identify peptide compounds with hepatoprotective potential. This limits the efficient production and application of hepatoprotective peptide products. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an active peptide with liver-protective function, or simply a hepatoprotective active peptide, as well as its preparation method and application. By screening and preparing an active peptide with hepatoprotective function, the liver's protective ability is enhanced, the occurrence of related liver diseases is prevented, and the side effects of taking drugs such as corticosteroids and pentoxifylline are avoided, thus providing a preventive measure to avoid the occurrence of alcohol-related liver diseases.

[0006] The objectives of this invention and the solutions to its technical problems can be achieved through the following technical solutions.

[0007] On one hand, this invention provides an active peptide with liver-protective function. The amino acid sequence of this peptide is Pro-Phe-Pro-Arg-Pro-Gln-Pro, or PFPRPQP, and its molecular weight is 838.46 Da. This peptide is rich in hydrophobic amino acids, including proline, phenylalanine, arginine, and glutamine.

[0008] On the other hand, the present invention provides a method for preparing the above-mentioned active peptides, comprising the following steps:

[0009] 1) Soy protein isolate was hydrolyzed by automatic pH gradient reduction using alkaline protease, papain, and flavor protease to obtain soybean peptides.

[0010] 2) Soybean peptides were subjected to in vitro simulated digestion using pepsin and pancreatic enzymes to obtain soybean peptide digestion products.

[0011] 3) The digestion products of soybean peptides were subjected to in vitro simulated absorption. The absorbed soybean peptides were isolated using a Caco-2 monolayer cell model; and...

[0012] 4) The absorbed soybean peptides were identified, and the active peptide PFPRPQP was obtained.

[0013] In a third aspect, the present invention provides a composition having liver-protective function, referred to as a hepatoprotective active composition, comprising the above-mentioned active peptides.

[0014] In a fourth aspect, the present invention provides the use of hepatoprotective active peptides in the manufacture of medicaments having hepatoprotective functions.

[0015] The bioactive peptides provided by this invention can protect HepG2-damaged cells by reducing AST and ALT levels, inhibiting CYP2E1 expression, and activating the Keap1 / Nrf2 signaling pathway. Furthermore, the bioactive peptides of this invention are prepared by enzymatic hydrolysis, separation, and identification of soybean protein isolate, thus utilizing widely available raw materials and employing a simple and easy-to-implement preparation process. Attached Figure Description

[0016] Figure 1 This is a diagram showing the binding energies of different peptide segments to CYP2E1 and Keap1 receptor molecules.

[0017] Figure 2 The effect of different concentrations of peptide PFPRPQP on the viability of HepG2 cells;

[0018] Figure 3 The effect of peptide PFPRPQP on AST and ALT levels in HepG2 cells;

[0019] Figure 4The effect of peptide PFPRPQP on the expression of CYP2E1 and Keap1 / Nrf2 proteins in HepG2 cells;

[0020] Figure 5 This is a visualization of the molecular docking of peptide PFPRPQP with CYP2E1 and Keap1. Detailed Implementation

[0021] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments described below are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] On the one hand, the present invention provides a hepatoprotective active peptide with the amino acid sequence Pro-Phe-Pro-Arg-Pro-Gln-Pro, or PFPRPQP, and a molecular weight of 838.46 Da.

[0023] In an embodiment of the present invention, the active peptide can exert a protective effect on HepG2-damaged cells by reducing liver enzyme levels, inhibiting CYP2E1 expression, and activating the Keap1 / Nrf2 signaling pathway.

[0024] On the other hand, the present invention provides a method for preparing the above-mentioned active peptides, comprising the following steps:

[0025] 1) Soy protein isolate was hydrolyzed by automatic pH gradient reduction using alkaline protease, papain, and flavor protease to obtain soybean peptides.

[0026] 2) Soybean peptides were subjected to in vitro simulated digestion using pepsin and pancreatic enzymes to obtain soybean peptide digestion products.

[0027] 3) The digestion products of soybean peptides were subjected to in vitro simulated absorption. The absorbed soybean peptides were isolated using a Caco-2 monolayer cell model; and...

[0028] 4) The absorbed soybean peptides were identified, and the active peptide PFPRPQP was obtained.

[0029] In an embodiment of the present invention, in step 1), the amount of alkaline protease added is 2.25% of the soy protein isolate by mass fraction, the hydrolysis time is 10 min, the hydrolysis temperature is 50°C, and the pH is 8.0.

[0030] In an embodiment of the present invention, in step 1), the amount of papain added can be 0.28% of the soy protein isolate by mass fraction, the hydrolysis time can be 35 min, the hydrolysis temperature can be 50 °C, and the pH can be 7.5.

[0031] In an embodiment of the present invention, in step 1), the amount of flavor protease added can be 11.25% of the soy protein isolate by mass fraction, the hydrolysis time can be 195 min, the hydrolysis temperature can be 50 °C, and the pH can be 6.5.

[0032] In an embodiment of the method of the present invention, in step 1), after the enzymatic hydrolysis is completed, the enzyme can be inactivated by bathing in a 95°C water bath for 10 minutes.

[0033] In an embodiment of the present invention, step 2) specifically includes the following steps:

[0034] 2.1) Soybean peptides were dissolved in simulated oral fluid and the mixture was shaken to obtain a soybean peptide oral digestive fluid.

[0035] 2.2) Add simulated gastric juice to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin and shake to react, thus obtaining the soybean peptide gastric digestive fluid.

[0036] 2.3) Add simulated intestinal fluid to the soybean peptide gastric digestive fluid, adjust the pH to 7.0, then add pancreatic enzyme, and shake to obtain the soybean peptide intestinal digestive fluid.

[0037] 2.4) The soybean peptide intestinal digestive fluid was freeze-dried to obtain digested soybean peptide powder, i.e., soybean peptide digestion product.

[0038] The simulated oral fluid contains 15.1 mM KCl, 3.7 mM KH2PO4, 6.8 mM NaHCO3, 0.15 mM MgCl2(H2O)6, 0.06 mM (NH4)2CO3, 0.1 mM HCl, and 1.5 mM CaCl2(H2O)2, which are added separately before use.

[0039] The simulated gastric juice contains 6.9 mM KCl, 0.9 mM KH2PO4, 25.0 mM NaHCO3, 47.2 mM NaCl, 0.12 mM MgCl2(H2O)6, 0.5 mM (NH4)2CO3, 15.6 mM HCl, and 0.15 mM CaCl2(H2O)2, which are added separately before use.

[0040] The simulated intestinal fluid contains 6.8 mM KCl, 0.8 mM KH2PO4, 85.0 mM NaHCO3, 38.4 mM NaCl, 0.33 mM MgCl2(H2O)6, 8.4 mM HCl, and 0.6 mM CaCl2(H2O)2, which are added separately before use.

[0041] The reason why CaCl2(H2O)2 needs to be added separately before use in the above-mentioned simulated solution is that: 1) it simulates the ionic strength of calcium ions in gastrointestinal fluid; 2) calcium ions in the solution can improve the stability and activity of enzymes; 3) calcium ions participate in biological processes such as muscle contraction, nerve conduction, and blood coagulation. The reason for adding CaCl2(H2O)2 separately before use is that: 1) calcium ions have high reactivity and easily react with other components or form precipitates; 2) calcium ions affect the solubility of other components.

[0042] In a specific implementation plan, for example, based on 5g of soybean peptides, 5mL of simulated oral fluid and 0.025mL of 0.3M CaCl2(H2O)2 solution are added to a final volume of 10mL to obtain soybean peptide oral digestive fluid. During gastric digestion, 10mL of simulated gastric fluid and 0.005mL of 0.3M CaCl2(H2O)2 solution are added to the soybean peptide oral digestive fluid to a final volume of 20mL to obtain soybean peptide gastric digestive fluid. During intestinal digestion, 20mL of simulated intestinal fluid and 0.04mL of 0.3M CaCl2(H2O)2 solution are added to the soybean peptide gastric digestive fluid to a final volume of 40mL to obtain soybean peptide intestinal digestive fluid.

[0043] In an embodiment of the method of the present invention, in step 2), the pepsin hydrolysis time is 2 hours, the hydrolysis temperature is 37°C, and the pH is 3.0.

[0044] In an embodiment of the method of the present invention, in step 2), the pancreatic enzyme hydrolysis time can be 2 hours, the hydrolysis temperature can be 37°C, and the pH can be 7.0.

[0045] In an embodiment of the method of the present invention, in step 2), after the enzymatic hydrolysis is completed, the enzyme is inactivated by bathing in a water bath at 95°C for 10 minutes.

[0046] In an embodiment of the method of the present invention, in step 3), the soybean peptide digestion product is dissolved in HBSS and inoculated into Caco-2 monolayer cells, wherein the concentration of the soybean peptide digestion product in HBSS is 2 mg / mL.

[0047] In an embodiment of the method of the present invention, in step 3), the soybean peptide digestion products are simulated for in vitro absorption using a Caco-2 monolayer cell model, and the absorbed soybean peptides are collected. In a specific embodiment, Caco-2 cells are cultured for 21 days to stabilize their resistivity and ensure it exceeds 300 Ω·cm.2 The Caco-2 monolayer cell model was constructed, and then after 2 hours of absorption, the digested soybean peptides were transported and absorbed to obtain absorbed soybean peptides.

[0048] In a third aspect, the present invention provides a composition having liver-protective function, referred to as a hepatoprotective active composition, comprising the above-mentioned active peptides.

[0049] In a fourth aspect, the present invention provides the use of the above-mentioned hepatoprotective active peptide in the manufacture of a medicament having hepatoprotective function.

[0050] This invention prepares soy protein isolate through enzymatic hydrolysis, separation, and identification. The raw materials used in this method are widely available, and the preparation process is easy to implement.

[0051] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0052] Example 1. Preparation and screening of hepatoprotective active peptides

[0053] This embodiment provides a hepatoprotective active peptide with the amino acid sequence Pro-Phe-Pro-Arg-Pro-Gln-Pro, or PFPRPQP, and a molecular weight of 838.46 Da. The preparation process of the above-mentioned hepatoprotective active peptide is as follows:

[0054] (1) Prepare an 8% soy protein isolate solution using alkaline protease (Shanghai Yuanye Biotechnology Co., Ltd., S10154, with an alkaline protease addition amount of 2.25% of the soy protein isolate by mass fraction), papain (Shanghai Yuanye Biotechnology Co., Ltd., S10011, with papain addition amount of 0.28% of the soy protein isolate by mass fraction), and flavor protease (Shanghai Yuanye Biotechnology Co., Ltd., S10153, with flavor protease addition amount of 11.25% of the soy protein isolate by mass fraction) for automatic pH gradient hydrolysis. The alkaline protease hydrolysis time was 10 min, and the hydrolysis conditions were: hydrolysis temperature 50℃, pH 8.0; the papain hydrolysis time was 35 min, and the hydrolysis conditions were: hydrolysis temperature 50℃, pH 7.5; the flavor protease hydrolysis time was 195 min, and the hydrolysis conditions were: hydrolysis temperature 50℃, pH 7.5. 6.5; After enzymatic hydrolysis, the enzyme was inactivated in a 95℃ water bath for 10 min to obtain a soybean peptide solution. The soybean peptide solution was stored at -40℃ and then freeze-dried for 48 h to obtain soybean peptide powder.

[0055] (2) Prepare simulated gastric and intestinal fluids, each in 400 mL, as shown in Table 1.

[0056] Table 1. Preparation of simulated gastric juice and simulated intestinal juice

[0057]

[0058] In the oral digestion stage, 5g of soybean peptides were weighed, and simulated oral fluid and 0.025mL of 0.3M CaCl2(H2O)2 solution were added to a final volume of 10mL. The mixture was then reacted at 30℃ for 2min to obtain soybean peptide oral digestion fluid. In the gastric digestion stage, simulated gastric fluid and 0.005mL of 0.3M CaCl2(H2O)2 solution were added to the soybean peptide oral digestion fluid to a final volume of 20mL. The pH was adjusted to 3.0, and pepsin was added to achieve an activity of 2000U / mL in the resulting mixture. Digestion was carried out at 37℃ for 2h to obtain soybean peptide gastric digestion fluid. In the intestinal digestion stage, simulated intestinal fluid and 0.04mL of 0.3M CaCl2(H2O)2 solution were added to the soybean peptide gastric digestion fluid to a final volume of 40mL. The pH was adjusted to 7.0, and trypsin was added to achieve an activity of 100U / mL in the resulting mixture. Digestion was carried out at 37℃ for 2h to obtain soybean peptide intestinal digestion fluid. After digestion, the soybean peptide enteric digestion solution was heated in boiling water for 10 minutes to terminate digestion, followed by centrifugation at 4500 rpm for 30 minutes at 4°C. After centrifugation, the supernatant was collected and stored at -40°C for 48 hours using a vacuum freeze dryer. The simulated digested soybean peptides were collected for subsequent analysis.

[0059] (3) Caco-2 cells (from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in MEM medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (PS), and 1% non-essential amino acids (NEAA). The culture temperature was 37℃, and the humidity was 5% CO2 / 95% air. Every three days, when the cell density reached 80%, the cells were passaged using 0.25% trypsin-EDTA (TE) at a 1:3 ratio. An in vitro small intestinal absorption and transport experiment was conducted using a Caco-2 monolayer cell model. Caco-2 cells were cultured at a density of 4 × 10⁶ cells / year. 4 Cells / mL were inoculated into Transwell chambers, with a membrane growth area of ​​1.12 cm². 20.5 mL of complete culture medium was added to the upper (Apical, AP) side and 1.5 mL to the basal (BL) side of the Transwell chamber. The medium was changed every other day for the first two weeks, and daily for the following week. Transepithelial electrical resistance (TEER) was measured on days 3, 6, 9, 11, 15, 18, and 21 of culture. Cells were considered healthy after 21 days of culture if their TEER was >300 Ω·cm. 2 The translocation experiment of digested soybean peptides was conducted. Twelve hours before the experiment, the culture medium was changed. 1.5 mL of Hank's Balanced Salt Solution (HBSS) was added to the BL side of a 12-well Transwell plate, and 0.5 mL of HBSS was added to the AP side. The plates were incubated for 30 minutes to equilibrate. Subsequently, the HBSS was removed, and 1.5 mL of HBSS and 0.5 mL of 2 mg / mL digested soybean peptide solution (dissolved in HBSS) were added to the BL and AP sides of the plates, respectively. The plates were then incubated for 2 hours for translocation.

[0060] 1) After 2 hours of absorption, soybean peptides absorbed from the BL side of the Transwell chamber were analyzed using liquid chromatography-mass spectrometry (LC-MS) for peptide identification. First, sample pretreatment was performed: the soybean peptides absorbed for 2 hours were dissolved in ultrapure water, and dithiothreitol solution was added to a final concentration of 10 mmol / L. Reduction was carried out in a 56°C water bath for 1 hour. Iodoacetamide solution was added to a final concentration of 50 mmol / L, and the reaction was carried out in the dark for 40 minutes. Desalting was performed using a desalting column, and the solvent was evaporated in a vacuum centrifuge at 45°C. Then, LC-MS was performed using QExactive. TM Hybrid Quadrupole Orbit TM The mass spectrometer was connected to an Easy-NLC 1200 system (Thermo Fisher Scientific, USA) for detection. The capillary liquid chromatography pre-column was an Acclaim PepMap RPLC C18 (300μm id×5mm, 5μm, The analytical column was AcclaimPepMap RPLC C18 (50μm id×150mm, 1.9μm). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 80% acetonitrile / 0.1% formic acid aqueous solution. Gradient elution was used, with the following elution program: 0-5 min, 4%-10% B; 5-85 min, 10%-22% B; 85-110 min, 22%-40% B; 110-115 min, 40%-95% B; 115-120 min, 95% B. Flow rate was 600 nL / min, and injection volume was 5 μL. Spray voltage was 2.2 kV, and capillary temperature was 270 °C. Sample ion scan range: starting at 50 m / z. MS / MS data: The top 20 most abundant peptide ions were obtained in the preview scan. The raw MS file was analyzed using Byonic (https: / / www.uniprot.org / ), and a search was performed in protein databases based on sample type. The parameters were set as follows: protein modification was carbamoyl methylation (C) (fixed) or oxidation (M) (variable); enzyme specificity was set to non-specific; maximum missed cleavage count was set to 3; precursor ion mass tolerance was set to 20 ppm; and MS / MS tolerance was 0.02 Da. Peptides with high confidence were selected for downstream protein identification analysis.

[0061] 2) The top 20 abundant soybean peptides identified by mass spectrometry were molecularly docked with CYP2E1 and Keap1 receptors. The protein structures of CYP2E1 (PDB: 3E6I) and Keap1 (PDB: 1ZGK) were obtained from the RSCB PDB database (https: / / www.rcsb.org / ), and the peptide structures were obtained from PEP-FOLD 4.0 (https: / / mobyle2.rpbs.univ-paris-diderot.fr / cgi-bin / portal.py#forms:PEP-FOLD4). The binding energies of CYP2E1 and Keap1 with different peptides were obtained using AutoDockVina software. The results are shown below. Figure 1 As shown, the binding energies of 20 peptides to the CYP2E1 and Keap1 receptors were all negative, indicating that both can bind spontaneously. Among them, PIPFPR and PFPRPQP had the lowest binding energies to CYP2E1, at -8.7 and -8.6 kcal / mol, respectively, while GFGFVTY and PFPRPQP had binding energies to the Keap1 receptor at -9.9 and -9.6 kcal / mol, respectively. Based on the characteristics of low molecular weight, high UV absorption (A280), and high content of hydrophobic amino acids, PFPRPQP was selected for subsequent experiments. Finally, PyMol was used to visualize the molecular interactions between the synthesized peptides and proteins. The results are as follows... Figure 5As shown, PFPRPQP forms hydrogen bonds with THR58, GLN62, ASP394, SER395, and ASN400 in the CYP2E1 protein. PFPRPQP forms a relatively large number of hydrogen bonds with the amino acids in Keap1, with 9 hydrogen bonds forming. The more hydrogen bonds there are, the more stable the peptide binding to the protein.

[0062] Example 2. Protective effect of peptide PFPRPQP on damaged hepatocytes

[0063] In this embodiment, the protective effect of peptide PFPRPQP on damaged HepG2 cells was tested.

[0064] HepG2 cells (derived from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured in DMEM medium containing 10% FBS, 1% PS, and 1% NEAA, and incubated at 37°C in a 5% CO2 incubator. Subsequent experiments were performed after the HepG2 cells reached 80% confluence. (6 × 10⁻⁶ cells) 5 HepG2 cells were seeded at 0.25 mg / mL and cultured for 24 h until adherence. The effect of different concentrations of peptide PFPRPQP (0, 0.25, 0.5, 1, 1.5, 2, 2.5 mg / mL) on HepG2 cell viability was determined using the CCK-8 assay. The results are shown below. Figure 2 As shown in the figure, different concentrations of peptide PFPRPQP showed no toxicity to HepG2 cells. At a peptide concentration of 0.5 mg / mL, cell viability showed no significant change. Finally, soybean peptides were digested at a concentration of 0.5 mg / mL for subsequent experiments.

[0065] HepG2 cells were pretreated with peptide PFPRPQP (0.5 mg / mL) for 2 h, followed by treatment with ethanol (500 mM) for 24 h. A blank control group (cells seeded in MEM medium), a model group (cells seeded in DMEM medium containing ethanol), and a sample group (cells seeded in MEM medium containing PFPRPQP with added ethanol) were set up. AST and ALT levels were measured according to the instructions of the Nanjing Jiancheng Biotechnology Research Institute's aspartate aminotransferase (AST / GOT) and alanine aminotransferase (ALT / GPT) assay kits. Results are as follows: Figure 3 As shown, ethanol increases AST and ALT levels, causing damage to HepG2 cells. Pretreatment with peptide PFPRPQP significantly reduces AST and ALT levels, indicating that peptide PFPRPQP has a protective effect on damaged HepG2 cells.

[0066] Example 3. Study on the protective mechanism of peptide PFPRPQP against ethanol-induced HepG2 cell damage

[0067] In this embodiment, the effect of peptide PFPRPQP on protein expression in HepG2 cells after ethanol-induced injury was investigated. A cell injury model was established. HepG2 cells were treated, collected, and added to standard RIPA lysis buffer (tissue / cell) containing one PMSF vial from Solarbio Science & Technology Co., Ltd. The samples were lysed on ice for 30 min. After complete lysis, the samples were centrifuged at 10000g for 5 min at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA protein concentration kit. After standardizing protein concentration, 5× loading buffer (Soluble Biotech Co., Ltd.) was added, and the samples were denatured at 100°C and vortexed. The samples were loaded onto 10% SDS-PAGE and then analyzed using Trans-Blot Turbo. TM Electrotransfer printing was used to transfer the gel onto a PVDF membrane. The membrane was blocked in 5% skim milk powder for 2 hours, and then primary antibodies Nrf2, Keap1, CYP2E1 (Beyotime Biotechnology Co., Ltd. NRF2 Rabbit Polyclonal Antibody, KEAP1 Rabbit Polyclonal Antibody, CYP2E1 Rabbit Polyclonal Antibody), and GAPDH (from Wuhan Sanying Biotechnology Co., Ltd.) were added. The membrane was incubated overnight at 4°C. The membrane was washed 6 times for 5 minutes each time with 1× buffer prepared using Beyotime Biotechnology Co., Ltd. Secondary antibody (IgG (H+L)) was added and the membrane was incubated at 25°C for 1.5 hours. The results were visualized using Beyotime Biotechnology Co., Ltd.'s Western luminescence detection reagent BeyoECLPlus (high-sensitivity ECL chemiluminescence kit), and the grayscale values ​​were unified using ImageJ software. Results are shown below. Figure 4 As shown, ethanol induction significantly increased the expression level of CYP2E1 protein in the model group, while pretreatment with peptide PFPRPQP decreased the expression level of CYP2E1 protein. Furthermore, after HepG2 cells were stimulated by ethanol oxidation, the expression levels of Keap1 and Nrf2 proteins in the model group were significantly increased, indicating that the Keap1 / Nrf2 signaling pathway was activated. Peptide pretreatment decreased Keap1 protein levels and increased Nrf2 protein expression, suggesting that PFPRPQP pretreatment can alleviate hepatocyte oxidative damage by regulating the antioxidant system.

[0068] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A hepatoprotective active peptide, characterized in that, The peptide has the amino acid sequence PFPRPQP and a molecular weight of 838.46 Da.

2. The method for preparing the active peptide of claim 1, characterized in that, Includes the following steps: 1) Soy protein isolate was hydrolyzed by automatic pH gradient reduction using alkaline protease, papain, and flavor protease to obtain soybean peptides. 2) The soybean peptides were subjected to in vitro simulated digestion using pepsin and pancreatic enzymes to obtain soybean peptide digestion products; 3) The digested soybean peptides were subjected to in vitro simulated absorption, and the absorbed soybean peptides were separated using a Caco-2 monolayer cell model; and 4) The absorbed soybean peptide was identified to obtain the active peptide PFPRPQP.

3. The method according to claim 2, characterized in that, In step 1), the amount of alkaline protease added is 2.25% of the soy protein isolate by mass fraction, the amount of papain added is 0.28% of the soy protein isolate by mass fraction, and the amount of flavor protease added is 11.25% of the soy protein isolate by mass fraction.

4. The method according to claim 2, characterized in that, In step 1), the alkaline protease hydrolysis time is 10 min, the hydrolysis temperature is 50℃, and the pH is 8.0; the papain hydrolysis time is 35 min, the hydrolysis temperature is 50℃, and the pH is 7.5; the flavor protease hydrolysis time is 195 min, the hydrolysis temperature is 50℃, and the pH is 6.

5. After the hydrolysis is completed, the enzymes are inactivated by bathing in a 95℃ water bath for 10 min.

5. The method according to claim 2, step 2) specifically includes the following steps: 2.1) The soybean peptides were dissolved in simulated oral fluid and subjected to shaking reaction to obtain a soybean peptide oral digestive fluid. 2.2) Add simulated gastric juice to the soybean peptide oral digestive fluid, adjust the pH to 3.0, then add pepsin and shake to react, thus obtaining soybean peptide gastric digestive fluid. 2.3) Add simulated intestinal fluid to the soybean peptide gastric digestive fluid, adjust the pH to 7.0, then add pancreatic enzyme, and shake to obtain soybean peptide intestinal digestive fluid. 2.4) The soybean peptide enteric digestion solution is freeze-dried to obtain digested soybean peptide powder, i.e., the soybean peptide digestion product. The simulated oral fluid contains 15.1 mM KCl, 3.7 mM KH2PO4, 6.8 mM NaHCO3, 0.15 mM MgCl2(H2O)6, 0.06 mM (NH4)2CO3, 0.1 mM HCl, and 1.5 mM CaCl2(H2O)2, which is added separately before use. The simulated gastric fluid contains 6.9 mM KCl, 0.9 mM KH2PO4, 25.0 mM NaHCO3, 47.2 mM NaCl, 0.12 mM MgCl2(H2O)6, 0.5 mM (NH4)2CO3, 15.6 mM HCl, and 0.15 mM CaCl2(H2O)2, which are added separately before use. The simulated intestinal fluid contains 6.8 mM KCl, 0.8 mM KH2PO4, 85.0 mM NaHCO3, 38.4 mM NaCl, 0.33 mM MgCl2(H2O)6, 8.4 mM HCl, and 0.6 mM CaCl2(H2O)2, which are added separately before use.

6. The method according to claim 2, characterized in that, In step 2), the pepsin hydrolysis time is 2 hours, the hydrolysis temperature is 37°C, and the pH is 3.0; the pancreatin hydrolysis time is 2 hours, the hydrolysis temperature is 37°C, and the pH is 7.

0. After the hydrolysis is completed, the enzyme is inactivated by bathing in a 95°C water bath for 10 minutes.

7. The method according to claim 2, characterized in that, In step 3), the soybean peptide digestion product is dissolved in HBSS and inoculated into Caco-2 monolayer cells, wherein the concentration of the soybean peptide digestion product in HBSS is 2 mg / mL.

8. A hepatoprotective composition comprising the hepatoprotective active peptide of claim 1.

9. Use of the hepatoprotective active peptide according to claim 1 in the manufacture of a drug with hepatoprotective function.

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