Anti-digestive peptides for alleviating alcoholic liver damage, and methods of making and using the same

Abstract

The application belongs to the technical field of proteins, and relates to an anti-digestion peptide capable of relieving alcoholic liver injury, a preparation method and application of the anti-digestion peptide, and the amino acid sequence of the anti-digestion peptide is PIPFPR. The anti-digestion peptide can resist the degradation of gastrointestinal digestive enzymes and be absorbed by intestinal epithelial cells, can resist the decrease of cell viability, eliminate free radicals, resist ethanol-induced oxidative stress, and relieve liver cell injury. The application further provides a preparation method and application of the anti-digestion peptide capable of relieving alcoholic liver injury.

Description

Technical Field

[0001] This invention belongs to the field of protein technology, and particularly relates to an active peptide that resists digestion and alleviates alcoholic liver damage, as well as its preparation method and application. Background Technology

[0002] Alcohol consumption is a widespread global socio-cultural phenomenon. With the increasing prevalence of alcohol consumption, the public health problems it causes are becoming increasingly prominent, especially alcoholic liver damage caused by excessive drinking, which has become a significant threat to human health. Long-term heavy drinking disrupts alcohol metabolism pathways. During oxidative metabolism, the excessive production of acetaldehyde and reactive oxygen species (ROS) promotes the formation of DNA and protein adducts, leading to mitochondrial damage in liver cells and potentially inducing liver cancer. Furthermore, excessive acetaldehyde can react with mitochondrial glutathione (GSH), resulting in GSH depletion and a decline in the capacity of antioxidant enzyme systems.

[0003] Compared to chemical drugs, bioactive peptides are safer and more nutritious. Soy peptides are highly nutritious, have a balanced amino acid composition, and possess excellent antioxidant properties. Studies have shown that soy peptides can alleviate alcoholic liver damage by regulating oxidative stress. However, bioactive peptides need to resist degradation by gastrointestinal digestive enzymes after ingestion. Furthermore, before peptides can exert their biological effects in the bloodstream, they must cross the intestinal epithelial barrier. The difficulty in fully absorbing bioactive peptides after gastrointestinal digestion significantly hinders their effectiveness in alleviating alcoholic liver damage.

[0004] Therefore, enabling soybean peptides to overcome the degradation by gastrointestinal digestive enzymes after oral administration, and to be effectively absorbed by the intestines and protect the liver, would be an effective means of alleviating alcoholic liver damage. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an absorbable peptide that resists digestion and alleviates alcoholic liver damage, as well as its preparation method and application. By screening and preparing peptides that resist gastrointestinal digestive enzymes and have the effect of alleviating alcoholic liver damage, the invention ensures that the peptides enter the bloodstream in their intact form to exert their effects, thus solving the problem that peptides are difficult to absorb completely after digestion in the gastrointestinal tract, and providing an effective intervention for alleviating alcoholic liver damage.

[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 anti-digestive peptide that alleviates alcoholic liver injury. The amino acid sequence of this peptide is Pro-Ile-Pro-Phe-Pro-Arg, or represented as PIPFPR, and its molecular weight is 726.43 Da. This peptide is rich in hydrophobic amino acids, including proline, arginine, isoleucine, and phenylalanine.

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

[0009] 1) Soy protein isolate was hydrolyzed by an automatic pH gradient reduction process 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. Absorbable soybean peptides were obtained by isolating them using a Caco-2 monolayer cell model; and

[0012] 4) The absorbed soybean peptides were identified, and the anti-digestive peptide PIPFPR was obtained.

[0013] In a third aspect, the present invention provides a composition for alleviating alcoholic liver injury, comprising the above-mentioned active peptide.

[0014] In a fourth aspect, the present invention provides the use of the above-mentioned anti-digestive peptide in the manufacture of a medicament for alleviating alcoholic liver injury.

[0015] The anti-digestive peptides of this invention can resist degradation by gastrointestinal digestive enzymes, thereby being absorbed by intestinal epithelial cells. They also alleviate hepatocyte oxidative damage by inhibiting cell viability decline, scavenging free radicals, and resisting ethanol-induced oxidative stress. Furthermore, the bioactive peptides of this invention are prepared by enzymatic hydrolysis, separation, and identification of soy protein isolate, thus utilizing widely available raw materials and employing a simple and easy-to-implement preparation process. Attached Figure Description

[0016] Figure 1 The effect of different concentrations of digested soybean peptides on the viability of Caco-2 cells;

[0017] Figure 2 The electrical resistance value of Caco-2 monolayer cells after 21 days of continuous culture;

[0018] Figure 3 To investigate the effects of soybean peptides on AST and ALT levels in HepG2 cells;

[0019] Figure 4The effects of soybean peptides on intracellular fluorescence intensity, ROS, MDA, GSH, and SOD levels in HepG2 cells were investigated.

[0020] Figure 5 The effects of different concentrations of peptide PIPFPR and ethanol induction on the viability of HepG2 cells;

[0021] Figure 6 The effects of peptide PIPFPR on fluorescence intensity, ROS, MDA, and GSH oxidation levels in ethanol-stimulated HepG2 cells. Detailed Implementation

[0022] 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.

[0023] On the one hand, the present invention provides an absorbable peptide that is resistant to digestion and alleviates alcoholic liver injury. The amino acid sequence of the peptide is Pro-Ile-Pro-Phe-Pro-Arg, or represented as PIPFPR, and the molecular weight is 726.43 Da.

[0024] In an embodiment of the present invention, the active peptide is able to resist degradation by gastrointestinal digestive enzymes and be absorbed by intestinal epithelial cells, thereby alleviating ethanol-induced HepG2 cell damage by resisting decreased cell viability and oxidative stress.

[0025] On the other hand, the present invention provides a method for preparing the above-mentioned active peptides, comprising the following steps:

[0026] 1) Soy protein isolate was hydrolyzed by an automatic pH gradient reduction process using alkaline protease, papain, and flavor protease to obtain soybean peptides.

[0027] 2) Soybean peptides were subjected to in vitro simulated digestion using pepsin and pancreatic enzymes to obtain soybean peptide digestion products;

[0028] 3) The digestion products of soybean peptides were subjected to in vitro simulated absorption. Absorbable soybean peptides were obtained by isolating them using a Caco-2 monolayer cell model; and

[0029] 4) The absorbed soybean peptides were identified, and the anti-digestive peptide PIPFPR was obtained.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In an embodiment of the present invention, step 2) specifically includes the following steps:

[0035] 2.1) Soybean peptides were dissolved in simulated oral fluid and subjected to shaking reaction to obtain a soybean peptide oral digestive fluid.

[0036] 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.

[0037] 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.

[0038] 2.4) The soybean peptide intestinal digestive fluid was freeze-dried to obtain digested soybean peptide powder, i.e., soybean peptide digestion product.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The reason why CaCl2(H2O)2 needs to be added separately before use in the above-mentioned simulated solution is: 1) to simulate 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: 1) calcium ions have high reactivity and easily react with other components or form precipitates; 2) calcium ions affect the solubility of other components.

[0043] In a specific implementation plan, for example, based on 5 g of soybean peptides, 5 mL of simulated oral fluid and 0.025 mL of 0.3 M CaCl2(H2O)2 solution are added to a final volume of 10 mL to obtain soybean peptide oral digestive fluid. During gastric digestion, 10 mL of simulated gastric fluid and 0.005 mL of 0.3 M CaCl2(H2O)2 solution are added to the soybean peptide oral digestive fluid to a final volume of 20 mL to obtain soybean peptide gastric digestive fluid. During intestinal digestion, 20 mL of simulated intestinal fluid and 0.04 mL of 0.3 M CaCl2(H2O)2 solution are added to the soybean peptide gastric digestive fluid to a final volume of 40 mL to obtain soybean peptide intestinal digestive fluid.

[0044] In an embodiment of the method of the present invention, in step 2), the pepsin hydrolysis time is 2 h, the hydrolysis temperature is 37 °C, and the pH is 3.0.

[0045] In an embodiment of the method of the present invention, in step 2), the pancreatic enzyme hydrolysis time can be 2 h, the hydrolysis temperature can be 37 °C, and the pH can be 7.0.

[0046] 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 min.

[0047] 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.

[0048] In an embodiment of the method of the present invention, in step 3), the digestion products of soybean peptides are simulated for in vitro absorption using a Caco-2 monolayer cell model, and the absorbed soybean peptides are collected. In a specific embodiment, the electrical resistance tends to stabilize after culturing Caco-2 cells for 21 days, and finally stabilizes at 374 Ω·cm. 2 The Caco-2 monolayer cell model was constructed, and after 2 hours of absorption, the digested soybean peptides were separated to obtain absorbable soybean peptides.

[0049] 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.

[0050] In a third aspect, the present invention provides a composition for alleviating alcoholic liver injury, comprising the above-mentioned active peptide.

[0051] In a fourth aspect, the present invention provides the use of the above-mentioned anti-digestive peptide in the manufacture of a medicament for alleviating alcoholic liver injury.

[0052] In this invention, the anti-digestive peptide that alleviates alcoholic liver damage can also be simply referred to as anti-digestive peptide or active peptide.

[0053] 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.

[0054] Example 1. Preparation and Screening Identification of Anti-digestive Soybean Peptides

[0055] This embodiment provides an anti-digestive peptide for alleviating alcoholic liver injury, with the amino acid sequence Pro-Ile-Pro-Phe-Pro-Arg, or represented as PIPFPR, and a molecular weight of 726.43 Da. The preparation process of the above-mentioned active peptide is as follows:

[0056] (1) Prepare an 8% soy protein isolate solution and perform automatic pH gradient hydrolysis 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). The alkaline protease hydrolysis time is 10 min, and the hydrolysis conditions are: hydrolysis temperature 50℃, pH 8.0; the papain hydrolysis time is 35 min, and the hydrolysis conditions are: hydrolysis temperature 50℃, pH 7.5; the flavor protease hydrolysis time is 195 min, and the hydrolysis conditions are: hydrolysis temperature 50℃, pH 6.5. After hydrolysis, incubate in a 95℃ water bath for 10 minutes. Enzyme inactivation was performed to obtain a soybean peptide solution. The soybean peptide solution was stored at -40°C and then freeze-dried for 48 hours to obtain soybean peptide powder.

[0057] (2) Prepare simulated gastric and intestinal fluids, in 400 mL amounts, as shown in Table 1.

[0058] Table 1. Preparation of simulated gastric and intestinal fluids

[0059] reagents Simulated oral fluid volume (mL) Simulated oral fluid concentration (mM) Simulated gastric fluid volume (mL) Simulated gastric juice concentration (mM) Simulated intestinal fluid volume (mL) Simulated intestinal fluid concentration (mM) KCL 15.1 15.1 6.9 6.9 6.8 6.8 <![CDATA[KH2PO4]]> 3.7 3.7 0.9 0.9 0.8 0.8 <![CDATA[NaHCO3]]> 6.8 13.6 12.5 25 42.5 85 NaCL - - 11.8 47.2 9.6 38.4 <![CDATA[MgCl2(H2O)6]]> 0.5 0.15 0.4 0.12 1.1 0.33 <![CDATA[(NH4)2CO3]]> 0.06 0.06 0.5 0.5 - - 6M HCl 0.09 1.1 1.3 15.6 0.7 8.4 <![CDATA[CaCL2(H2O)2]]> 0.025 1.5 0.005 0.15 0.04 0.6

[0060] In the oral digestion stage, 5 g of soybean peptides were weighed, and simulated oral fluid and 0.025 mL of 0.3 M CaCl2(H2O)2 solution were added to a final volume of 10 mL. The mixture was then reacted at 30°C for 2 min to obtain the soybean peptide oral digestion solution. In the gastric digestion stage, simulated gastric fluid and 0.005 mL of 0.3 M CaCl2(H2O)2 solution were added to the soybean peptide oral digestion solution to a final volume of 20 mL. The pH was adjusted to 3.0, and pepsin was added to achieve an activity of 2000 U / mL in the resulting mixture. Digestion was carried out at 37°C for 2 h to obtain the soybean peptide gastric digestion solution. In the intestinal digestion stage, simulated intestinal fluid and 0.04 mL of 0.3 M CaCl2(H2O)2 solution were added to the soybean peptide gastric digestion solution to a final volume of 40 mL. The pH was adjusted to 7.0, and trypsin was added to achieve an activity of 100 U / mL in the resulting mixture. Digestion was carried out at 37°C for 2 h to obtain the soybean peptide intestinal digestion solution. 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.

[0061] (3) Caco-2 cells were cultured (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) 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. Cells were passaged every three days at a confluence of 80% using 0.25% trypsin-EDTA (TE) at a ratio of 1:3. The transport concentration of digested soybean peptides was determined by measuring their effect on Caco-2 cell viability. Results are as follows: Figure 1 As shown, different concentrations of digested soybean peptides had no toxic effect on Caco-2 cells, and the intermediate concentration of 2 mg / mL was ultimately selected as the transport concentration. An in vitro small intestinal absorption and transport experiment was conducted by constructing a Caco-2 monolayer cell model. Caco-2 cells were planted at a density of 4 × 10⁶ cells / mL. 4 Cells / mL were inoculated into Transwell chambers, with a membrane growth area of ​​1.12 cm². 2 0.5 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. Results are as follows... Figure 2 As shown, the TEER was 374 Ω·cm after 21 days of culture. 2 The transport of digested soybean peptides was performed. 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 min 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, respectively. The plates were then incubated for 2 hours for transport.

[0062] 1) Soybean peptides absorbed from the BL side of the Transwell chamber 2 h after absorption were analyzed by liquid chromatography-mass spectrometry (LC-MS). First, sample pretreatment was performed: soybean peptides absorbed for 2 h 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 h. Iodoacetamide solution was added to a final concentration of 50 mmol / L, and the reaction was carried out in the dark for 40 min. Desalting was performed using a desalting column, and the solvent was evaporated in a vacuum centrifuge at 45°C. Next, liquid chromatography-mass spectrometry (LC-MS) was used for detection. A QExactive™ hybrid quadrupole orbital mass spectrometer connected to an Easy-NLC 1200 system (Thermo Fisher Scientific, USA) was used for detection. The capillary LC pre-column was an Acclaim PepMap RPLC C18 (300 μm id × 5 mm, 5 μm, 100 Å), and the analytical column was an Acclaim PepMap RPLC C18 (50 μm id × 150 mm, 1.9 μm, 100 Å). 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 elution program as follows: 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. The flow rate was 600 nL / min, and the injection volume was 5 μL. The spray voltage was 2.2 kV, and the capillary temperature was 270 °C. The sample ion scan range started at 50 m / z. MS / MS data showed that 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 / ). A search was performed in the protein database based on the sample type. The parameters were set as follows: protein modification was carbamoyl methylation (C) (fixed) and oxidation (M) (variable); enzyme specificity was set to non-specific; the maximum number of missed cleavages was set to 3; the precursor ion mass tolerance was set to 20 ppm, and the MS / MS tolerance was 0.02 Da. Peptides with high confidence were selected for downstream protein identification analysis.

[0063] We selected peptides with high abundance for screening, and chose peptides with low molecular weight that were identified before and after transport and absorption. These peptides had the potential to alleviate alcoholic liver damage: peptides with a basic amino acid at the C-terminus.

[0064] Table 2. Peptide fragment analysis of soybean peptides after 2 hours of absorption

[0065] peptide sequence Score Molecular weight (Da) peptide length Pre-absorption peptide sequence C-terminal basic amino acid IGGEERKGME 334.4 1105.531 10 Not identified no SAGVAVTKVEQL 250.4 1243.689 12 Not identified no GSPIKRDF 249.4 961.510 8 Not identified no FCVDITDFCY 271.4 1339.533 10 Not identified no LMFVASLGIVLIMI 33.2 1519.899 14 Not identified no KRVTIMPKDIQL 49.7 1441.856 12 Not identified no VREIAQDFKTDL 155.7 1434.759 12 Not identified no PIPFPR 145.7 726.430 6 Identified yes PGTVALREI 276.6 955.557 9 Not identified no KTEWLDGKHVVF 311.2 1458.774 12 Not identified no

[0066] Example 2. Protective effect of anti-digestive soybean peptides against alcoholic liver injury.

[0067] In this embodiment, we tested whether the peptide PIPFPR could protect hepatocytes from ethanol-induced damage.

[0068] HepG2 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) and Caco-2 cells (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were cultured to establish a Caco-2 / HepG2 cell co-culture model (see Yao, M., Yao, Y., Qin, B., Pan, M., Ju, X., Xu, F., Wang, L. 2022, Screening and identiffcation of high bioavailable oligopeptides from rapeseed napin (Brassica napus) protein-derived hydrolysates via Caco-2 / HepG2 co-culture model. FoodResearch International, 155, 111101). After 21 days of monolayer culture of Caco-2 cells, HepG2 cells with a cell concentration of 80% were transferred from the 6-well plate to the Caco-2 cell monolayer. The culture medium was changed from complete medium (DMEM medium containing 10% FBS, 1% PS, and 1% NEAA) to serum-free DMEM medium. The HBSS from the AP side was decanted and 2 mg / mL of digested soybean peptide solution (dissolved in DMEM medium) was added. After transporting the cells in an incubator for 2 h, an ethanol-induced cell model was established. Three groups were established: a control group (cells seeded with basal medium), a model group (cells seeded with basal medium containing ethanol), and a sample group (cells seeded with basal medium containing transported absorbed soybean peptides and ethanol). The potential of absorbed soybean peptides to alleviate alcoholic liver injury was explored by detecting AST and ALT levels and oxidative damage indicators in HepG2 cells. Subsequent cell experiments will identify and validate the selected peptides.

[0069] Experimental Example 1

[0070] 1) AST and ALT activities in HepG2 cells were measured according to the instructions of the aspartate aminotransferase (AST / GOT) and alanine aminotransferase (ALT / GPT) assay kits from Nanjing Jiancheng Biotechnology Institute. The results are as follows: Figure 3 As shown, ethanol stimulation significantly increased AST and ALT levels in the model group, inducing HepG2 cell damage. Treatment with soybean peptides significantly reduced AST and ALT levels, alleviating ethanol-induced hepatocellular damage.

[0071] 2) Determine the release of ROS in HepG2 cells after treatment with soybean peptides. The fluorescent probe DCFH-DA (from Beyotime Biotechnology Co., Ltd. reactive oxygen species detection kit) was diluted 1:1000 with DMEM medium (Gibco, Thermo Fisher Scientific, USA). 1 mL of diluted DCFH-DA was added to each well of a six-well plate, and the cells were incubated at 37°C for 30 min. HepG2 cells were then washed three times with DMEM medium to remove any DCFH-DA that had not yet entered the Caco-2 cells. Observation and photography were performed using a fluorescence microscope. Finally, the fluorescence intensity was detected using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Results are as follows: Figure 4 As shown in Figures A and B, under ethanol stimulation, the fluorescence intensity and ROS level in the model group increased, indicating oxidative stress. However, treatment with soybean peptides resulted in decreased fluorescence intensity and a significant reduction in ROS levels, suggesting that soybean peptides can alleviate ethanol-induced oxidative damage by scavenging free radicals. Following the instructions of the Nanjing Jiancheng Biotechnology Institute's malondialdehyde (MDA) assay kit, reduced glutathione (GSH) assay kit, and total superoxide dismutase (T-SOD) assay kit, the MDA, GSH content, and SOD activity in HepG2 cells were measured. The results are as follows: Figure 4 As shown in C, D, and E, ethanol stimulation significantly increased MDA content and significantly decreased SOD activity, while GSH content also decreased. Treatment with soybean peptides, however, alleviated hepatocyte oxidative damage by reducing MDA content and increasing SOD activity. Overall, the absorption of soybean peptides can protect hepatocytes from ethanol-induced damage by lowering liver enzyme levels, scavenging free radicals, and regulating the antioxidant system.

[0072] Experiment Example 2

[0073] 1) Culture HepG2 cells until they reach 80% confluence density before proceeding with subsequent experiments. Use 6 × 10⁻⁶ HepG2 cells... 5 HepG2 cells were seeded at 0.5 mg / mL into 6-well plates and cultured for 24 h until cell adhesion. The effect of different concentrations of peptide PIPFPR (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 5As shown in Figure A. Different concentrations of peptide PIPFPR showed no toxicity to HepG2 cells. At a peptide concentration of 0.5 mg / mL, cell viability showed no significant change. To exclude the effect of the peptide on cell proliferation, a peptide concentration of 0.05 mg / mL was ultimately selected for subsequent experiments. HepG2 cells were pretreated with 0.5 mg / mL peptide PIPFPR for 2 h, followed by stimulation with 500 mM ethanol for 24 h to model cell growth. The effect of peptide PIPFPR on ethanol-stimulated HepG2 cell viability was then determined. The results are shown in Figure A. Figure 5 As shown in Figure B, the cell viability of the model group was significantly reduced, ethanol stimulation caused cell damage, and peptide PIPFPR pretreatment inhibited the decline in hepatocyte viability and had a protective effect on hepatocytes.

[0074] 2) The effect of PIPFPR pretreatment on ethanol-induced HepG2 cell damage was determined. ROS release in cells was measured using a reactive oxygen species (ROS) detection kit from Beyotime Biotechnology Co., Ltd. DCFH-DA was diluted 1:1000 with DMEM medium, and 1 mL of the diluted DCFH-DA was added to each well of a six-well plate. The cells were incubated at 37°C for 30 min, and then HepG2 cells were washed three times with DMEM medium to thoroughly remove any DCFH-DA that had not yet entered the Caco-2 cells. The cells were observed and photographed using a fluorescence microscope. Finally, the fluorescence intensity was detected using a fluorescence microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Results are as follows: Figure 6 As shown in Figures A and B, under ethanol stimulation, the fluorescence intensity of the model group increased, and the ROS level significantly increased. Treatment with the peptide PIPFPR significantly reduced the ROS level and scavenged free radicals. The MDA and GSH contents in HepG2 cells were measured according to the instructions of the MDA and GSH kits from Nanjing Jiancheng Bioengineering Institute. The results are as follows... Figure 6 As shown in C and D, ethanol stimulation significantly increased MDA levels and decreased GSH levels. Pretreatment with peptide PIPFPR significantly reduced MDA levels and increased GSH levels. Peptide PIPFPR can protect hepatocytes from ethanol-induced damage by scavenging free radicals, inhibiting lipid peroxidation, and protecting them.

[0075] 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. Use of an anti-digestive peptide in the manufacture of a medicament for alleviating alcoholic liver injury, wherein the amino acid sequence of the anti-digestive peptide is PIPFPR.

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