A walnut protein octapeptide with the pre-protecting effect of alcoholic hepatocyte injury and a preparation method and application thereof

Abstract

This invention discloses a walnut protein octapeptide with pre-protective efficacy against alcoholic hepatocellular injury, its preparation method, and its applications. Using walnut protein hydrolysate as raw material, this invention screened and obtained a novel peptide with high antioxidant activity, HNVADPQR, with a molecular weight of 949.473 Da. This active peptide exhibits good antioxidant activity at the cellular level, with an EC50 of [missing information]. 50 The concentration was 0.0069 mg / mL, which was superior to the positive control glutathione (0.0122 mg / mL). In the ethanol injury model, the antioxidant peptide increased the survival rate of alcohol-damaged cells from 47.35% to 71.65%. These results indicate that the walnut protein-derived antioxidant peptide HNVADPQR can be developed and utilized as a potential antioxidant and hepatoprotective active ingredient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antioxidant, and particularly relates to a walnut protein octapeptide with the effect of pre-protecting alcoholic hepatocyte injury and a preparation method and application thereof. BACKGROUND

[0002] In the metabolic process of organisms, reactive oxygen species (ROS) or free radicals are naturally produced by respiratory oxidative reactions. Organisms have developed their own antioxidant defense system to combat excessive ROS. When the limited efficiency of the body cannot prevent all oxidative damage related to environmental conditions, excessive free radicals and reactive oxygen species accumulate in cells, causing oxidative stress, which leads to damage to the organism. The oxidative stress state can lead to many non-communicable chronic diseases, such as cardiovascular disease, diabetes, inflammatory disease and aging. It has great practical significance to develop natural antioxidants in the prevention of diseases.

[0003] In recent years, peptides have attracted widespread interest as the most important class of bioactive ingredients. A large number of studies have shown that bioactive peptides have important value in the regulation of physiological functions such as antioxidant, antihypertensive, antithrombotic, antibacterial, anticancer, anti-inflammatory, antidiabetic, anti-obesity, cholesterol-lowering, immune regulation and mineral binding. At the same time, food-derived bioactive peptides also have the function of preventing and treating diseases in healthy diet. Moreover, the most attractive feature of bioactive peptides is their ability to show very few side effects in the human body due to their natural origin. Peptide segments with antioxidant properties can exert antioxidant effects by scavenging free radicals, inhibiting peroxides and chelating metal ions (Fe 2+ / Cu 2+ ) ions. There have been reports of antioxidant peptides from different sources, such as pearl meat, oysters, cottonseed, bitter buckwheat, etc. Walnut is one of the four largest dry fruits in the world and is considered an important oil tree species, economic tree species and ecological tree species. However, most of the walnut meal is used as feed or discarded after oil extraction, resulting in waste of resources. Therefore, it is of great practical significance to efficiently utilize walnut meal protein. Previous studies have shown that peptides isolated from walnut protein have activities such as blood pressure reduction, anti-inflammatory, neuroprotection, uric acid reduction, etc.

[0004] Alcoholic liver disease (ALD) is one of the most common liver diseases worldwide, mainly caused by long-term excessive alcohol consumption. Alcohol can cause accumulation of intracellular ROS, affect the activity of liver lipase and cause cell damage and death. Many studies have shown that natural antioxidants with antioxidant activity can prevent alcoholic liver injury through antioxidant mechanisms such as scavenging free radicals, restoring antioxidant enzyme activity and maintaining the homeostasis of the liver antioxidant defense system. SUMMARY

[0005] To address these issues, the primary objective of this invention is to provide a walnut protein octapeptide with pre-protective effects against alcoholic hepatocellular damage.

[0006] Another object of the present invention is to provide the application of the above-mentioned walnut protein octapeptide with the pre-protective effect against alcoholic hepatocellular injury.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0008] A walnut protein octapeptide with pre-protective efficacy against alcoholic hepatocellular injury has the following amino acid sequence His-Asn-Val-Ala-Asp-Pro-Gln-Arg(HNVADPQR).

[0009] Furthermore, the walnut protein octapeptide is prepared by walnut protein hydrolysis or solid-phase synthesis.

[0010] Walnut protein hydrolysate containing the aforementioned walnut protein octapeptide.

[0011] Solid-phase synthesis products containing the aforementioned walnut protein octapeptide.

[0012] The application of the above-mentioned walnut protein octapeptide, walnut protein hydrolysis product or solid-phase synthesis product in the preparation of antioxidants.

[0013] Furthermore, the antioxidant is one of an oral preparation or a topical preparation.

[0014] Furthermore, the antioxidants are used to scavenge intracellular free radicals.

[0015] Furthermore, the antioxidants described herein are used to protect cells from oxidative damage caused by alcohol.

[0016] The application of the above-mentioned walnut protein octapeptide, walnut protein hydrolysis product or solid-phase synthetic product in the preparation of drugs for the prevention and / or treatment of alcoholic hepatocellular injury.

[0017] The above-mentioned walnut protein octapeptide, walnut protein hydrolysis product or solid-phase synthesis product are used in the preparation of antioxidant additives.

[0018] Furthermore, the antioxidant additives are used in foods or medicines that have antioxidant and / or liver-protective effects.

[0019] The present invention has the following advantages and effects compared with the prior art:

[0020] This invention utilizes walnut protein hydrolysate as a raw material to screen and obtain a novel high-antioxidant-activity peptide, HNVADPQR (949.473 Da). This active peptide exhibits good antioxidant activity (CAA) at the cellular level, with an EC50 of [missing value]. 50The value was 0.0069 mg / mL, which was superior to the positive control glutathione (EC). 50 (0.0122 mg / mL). In an ethanol injury model, the antioxidant peptide increased the survival rate of cells treated with ethanol from 47.35% to 71.65%. These results suggest that the walnut protein-derived antioxidant peptide HNVADPQR can be developed and utilized as a potential antioxidant and hepatoprotective active ingredient. Attached Figure Description

[0021] Figure 1 Figure showing the results of the study on the effect of different concentrations of antioxidant peptides on the viability of HepG2 cells; different letters indicate significant differences (P < 0.05).

[0022] Figure 2 Figure 1 shows the results of a study on the effects of antioxidant peptides on cellular antioxidant activity; different letters indicate significant differences (P < 0.05).

[0023] Figure 3 Figure 1 shows the results of a study on the protective effect of antioxidant peptides against ethanol-induced cell damage. In this figure, A represents the results of constructing the HepG2 cell model of ethanol damage, and different letters indicate significant differences (P < 0.05); B represents the protective effect of antioxidant peptides on cells, and * indicates a significant difference from the damaged group (P < 0.05).

[0024] Figure 4 This is a diagram showing the docking sites between the antioxidant peptide and the Keap1 molecule. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0026] Example 1: Synthesis of Antioxidant Peptides

[0027] An antioxidant peptide, with the amino acid sequence HNVADPQR, was screened from walnut meal protein hydrolysate. It was synthesized by Nanjing Jietai Biotechnology Co., Ltd. HPLC analysis showed the synthesized peptide to have a purity of 98%. The synthesized antioxidant peptide was stored at -20°C for subsequent experimental use.

[0028] Example 2: Toxicity analysis of antioxidant peptides on HepG2 cells

[0029] HepG2 cells were cultured in complete culture medium at 37°C and 5% CO2. Cytotoxicity was determined using the MTT assay, specifically as follows: 100 μL of HepG-2 cell suspension was seeded into each well of a white 96-well plate, resulting in a seeding density of 1 × 10⁶ cells per well. 4Cells were seeded in 96-well plates, but the outer wells were left unseeded. PBS was added to fill the wells. After seeding, the 96-well plates were placed in a 37°C, 5% CO2 incubator. After 24 hours of culture, the medium was changed. 100 μL of medium containing different concentrations of antioxidant peptides (0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL) was added to the experimental group, while 100 μL of fresh medium was added to the blank control group. After another 24 hours of cell culture, the medium was removed, and 100 μL of MTT (0.5 mg / mL) solution was added. The cells were incubated in the dark for 4 hours. The MTT solution was discarded, and 100 μL of LDMSO was added. The cells were shaken for 10 minutes to dissolve all the blue-purple crystals. The OD value was measured at 490 nm using a microplate reader. Cell viability was calculated using formula (1).

[0030] Survival rate (%) = OD value of experimental group / OD value of blank control group × 100 (1)

[0031] The results are as follows Figure 1 The antioxidant peptide HNVADPQR, when treated with HepG2 cells at concentrations ranging from 0.025 to 2 mg / mL, maintained a cell viability of over 90%, indicating that it has no toxic effect on cells. Further studies can be conducted within this concentration range.

[0032] Example 3: Assay of Cellular Antioxidant Activity (CAA)

[0033] Cells were divided into a control group, a blank group, and an experimental group. The cell suspension was seeded in black 96-well plates to obtain a seeding density of 6 × 10⁶ cells per well. 4 Cells were cultured for 24 hours, followed by medium replacement. The control and blank groups were treated with 50 μL of DCFH-DA (50 μM) working solution and 50 μL of sterile water. The experimental groups were treated with 50 μL of DCFH-DA (50 μM) working solution and 50 μL of medium containing different concentrations of antioxidant peptides (0.005 mg / mL, 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, and 0.1 mg / mL). Cells were incubated at 37°C for 1 hour. The medium was discarded, and the cells were washed with 100 μL of PBS. The PBS was then discarded. 100 μL of HBSS was added to the blank group, while 100 μL of AAPH (600 μM) working solution was added to the control and experimental groups. Cells were then placed in a microplate reader, and fluorescence values ​​were measured at a wavelength of 528 nm and an excitation wavelength of 485 nm, measured every 5 minutes for 1 hour. Glutathione (GSH) was used as a positive control. AUC represents the area under the fluorescence intensity-time curve. The CAA value is calculated according to formula (2), and the half-maximal effective concentration (EC) of the sample is also calculated. 50The CAA value is represented by the standard antioxidant Trolox equivalent (mM TE / g Petide).

[0034]

[0035] Among them, AUC T Area of ​​fluorescence curve in the experimental group; AUC P Area of ​​fluorescence curve in the control group; AUC N Area of ​​fluorescence curve in the blank group.

[0036] CAA value represents intracellular antioxidant activity. Its mechanism of action involves the oxidation of DCFH to highly fluorescent dichlorofluorescein (DCF) in the presence of reactive oxygen species (ROS). Intracellular fluorescence intensity is positively correlated with ROS levels; higher fluorescence intensity indicates higher ROS levels. A higher CAA value corresponds to lower fluorescence intensity, signifying a higher intracellular ROS scavenging rate and stronger antioxidant capacity of the sample. The cellular antioxidant capacity (CAA value) of HNVADPQR and the positive control (GSH) is shown in [link to relevant data]. Figure 2 HNVADPQR's EC 50 The concentration was 0.0069 mg / mL, lower than the positive control (GSH) of 0.0122 mg / mL (Table). Notably, the monomeric peptide exhibited superior antioxidant capacity compared to the positive control (GSH) even at very low concentrations (0.005 mg / mL). In related reports, corn gluten meal sources... [1] Mulberry Source [2] Soy protein source [3] EC of antioxidant peptides 50 The concentrations were 2.85 mg / mL, 0.24 mg / mL, and 0.715 mg / mL, respectively. The EC50 of the monomeric peptides in this study... 50 The values ​​are lower than those reported. These results indicate that this antioxidant peptide can effectively scavenge intracellular free radicals, reduce ROS levels, and exert its antioxidant effect.

[0037] Table 1. Cellular antioxidant activity of antioxidant peptides (EC) 50 Value and Trolox equivalent value.

[0038]

[0039] Example 4: Protective effect of antioxidant peptides on ethanol-damaged HepG2 cells

[0040] Construction of the ethanol-damaged HepG2 cell model: Cell suspension was seeded in white 96-well plates at a seeding density of 2 × 10⁶ cells per well. 4Cells were cultured for 24 hours, followed by a medium change. 100 μL of fresh culture medium was added, and the medium was changed again after 24 hours. The experimental groups were cultured with medium containing different concentrations of ethanol (5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, and 40 mg / mL), while the control group was cultured with fresh culture medium. Cell viability was determined using the MTT assay to investigate the ethanol concentration at which 50% cell viability was achieved.

[0041] The results are as follows Figure 3 As shown in A, the cell viability is closest to 50% when the ethanol concentration is 40 mg / mL, so a cell damage model was constructed using an ethanol concentration of 40 mg / mL.

[0042] Pre-protection of HepG2 cells from ethanol-damaged antioxidant peptides: Cells were divided into a blank control group, a damage control group, and an experimental group. 100 μL of cell suspension was seeded into white 96-well plates at a seeding density of 2 × 10⁶ cells per well. 4 Cells were cultured for 24 hours, and then the culture medium was changed. The experimental groups were given 100 μL of culture medium containing different concentrations of antioxidant peptides (0.025 mg / mL, 0.05 mg / mL, and 0.1 mg / mL), while the blank and damage control groups were given fresh culture medium. Cells were cultured for another 24 hours. Afterward, the culture medium was changed again. The experimental and damage control groups were given 100 μL of culture medium containing 40 mg / mL ethanol, while the blank group was given 100 μL of fresh culture medium. Cell viability was determined using the MTT assay. Metadoxine was used as a positive control.

[0043] The results are as follows Figure 3 As shown in Figure B, the cell viability of the ethanol-damaged control group was 47.36%, which was significantly lower than that of the blank group (P < 0.05), indicating that the cell damage model was successfully established. In this model, Metadoxine, a commonly used drug for treating alcoholic liver disease, was used as a positive control. The survival rates of HNVADPQR at concentrations of 0.05 mg / mL and 0.1 mg / mL were higher than the positive control, at 70.66% and 71.64%, respectively, and significantly higher than the damage group (P < 0.05). This indicates that pre-protection with different concentrations of HNVADPQR can reduce the damaging effects of ethanol on cells, thus the antioxidant peptides in this study have the potential for pre-protection against alcoholic liver injury.

[0044] Example 5: Molecular docking and visualization analysis

[0045] Molecular docking analysis was performed using AutoDockvina. First, the 3D structure of the antioxidant peptide was plotted using ChemDraw, and the energy was minimized before outputting it in mol² format for subsequent docking. The structure was then retrieved from the RCSB PDB database. RCSB PDB: Homepage The 3D structure of the receptor molecule Keap1 protein (4L7B) was downloaded from [website name - missing]. The receptor molecule and peptide were preprocessed using AutodockTools 1.5.7 software, including hydrogenation, and output as pdbqt format before docking using AutoDockvina. The center of the docking box for Keap1 was set to x = -2.636, y = 3.267, and z = -27.443, and the dimensions of the docking box were set to x = 15, y = 15, and z = 15. Visualization analysis was performed using Discovery Studio Visualizer based on the results of the minimum docking binding energy.

[0046] Molecular docking is a biomolecular simulation method based on integrated bioinformatics analysis. It simulates interactions between molecules and can depict the structure-activity relationship of peptides, and has been widely used in the screening of bioactive peptides. Keap1 / Nrf2 is a major system regulating cellular antioxidant responses. Under normal conditions, Nrf2 binds to Keap1 and is degraded, inhibiting antioxidant responses. If antioxidant peptides can bind to Keap1 and inhibit the Keap1-Nrf2 interaction, the potential of Nrf2 can be activated and released. Nrf2 can then migrate to the cell nucleus, activate the transcription of antioxidant genes, and reduce oxidative stress.

[0047] Molecular docking analysis revealed that HNVADPQR has a docking binding energy of -8.9 (Table 2), indicating a strong binding ability to Keap1. Previous studies have shown that the key binding sites of Nrf2 in the Kelch region of the Keap1 protein are Tyr334, Arg380, Arg415, Arg483, Ser363, Asn382, Ser555, Asn387, His436, and Tyr525. If antioxidant peptides can interact with these sites, they can effectively interfere with the binding of Nrf2 to the Keap1 protein, reducing oxidative stress. Figure 4The interaction between HNVADPQR and Keap1 was demonstrated from both two-dimensional and three-dimensional perspectives. HNVADPQR can embed into the active cavity of Keap1, forming a stable docking conformation. HNVADPQR forms 11 hydrogen bonds with Asn382, Asn387, Ser383, Ser602, Arg415, Ser508, Arg483, and Gly509 of Keap1, among which Asn382, Asn387, Arg415, and Arg483 are key sites, and a hydrophobic interaction is formed with the key site Tyr525. Similar binding sites are also found in cottonseed protein sources (Arg415, Ser508, Ser602). [4] Fermented milk source (Asn387, Arg415, Ser508) [5] Reported in the field of oxidized peptides. Overall, HNVADPQR can bind tightly to key amino acids and surrounding residues of Keap1 through different non-covalent bonds, and has the potential to exert antioxidant effects through competitive binding with Keap1 / Nrf2.

[0048] Table 2. Bonding Energy Values

[0049]

[0050] In summary, the antioxidant peptide HNVADPQR isolated and screened in this invention exhibits excellent cellular antioxidant activity in cell experiments, effectively reducing intracellular ROS levels. It was also found to protect cells from ethanol-damaged activity. Furthermore, it can act on key sites in the Keap1-Nrf2 pathway, reducing oxidative stress and providing a new option for natural antioxidants.

[0051] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0052] References:

[0053] [1]Wang L, Ding L, Yu Z, et al. Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells [J]. FOOD RESEARCH INTERNATIONAL, 2016, 90: 33-41.

[0054] [2]Sun C,Tang X,Ren Y,et al.Novel Antioxidant Peptides Purified fromMulberry(MorusatropurpureaRoxb.)Leaf Protein Hydrolysates with HemolysisInhibition Ability and Cellular Antioxidant Activity[J].JOURNAL OFAGRICULTURAL AND FOOD CHEMISTRY,2019,67(27):7650-7659.

[0055] [3]Yang R,Wang J,Lin S,et al.In vitro antioxidant activities of thenovel pentapeptides Ser-His-Glu-Cys-Asn and Leu-Pro-Phe-Ala-Met and therelationship between activity and peptide secondary structure[J].Journal ofthe Science of Food and Agriculture,2017,97(6):1945-1952.

[0056] [4]Wang L,Ma M,Yu Z,et al.Preparation and identification ofantioxidant peptides from cottonseed proteins[J].Food Chemistry,2021,352:129399.

[0057] [5]Tonolo F,Fiorese F,Moretto L,et al.Identification of New Peptidesfrom Fermented Milk Showing Antioxidant Properties:Mechanism of Action[J].ANTIOXIDANTS,2020,9(2)。

Claims

1. A walnut protein octapeptide with pre-protective efficacy against alcoholic hepatocellular injury, characterized in that: Its amino acid sequence is His-Asn-Val-Ala-Asp-Pro-Gln-Arg.

2. A solid-phase synthetic product comprising the walnut protein octapeptide described in claim 1.

3. The use of the walnut protein octapeptide as described in claim 1 or the solid-phase synthesis product as described in claim 2 in the preparation of antioxidants.

4. The application according to claim 3, characterized in that: The antioxidants described herein are used to protect cells from oxidative damage caused by alcohol.

5. The application according to claim 3, characterized in that: The antioxidant mentioned is one of the oral or topical preparations.

6. The use of the walnut protein octapeptide of claim 1 or the solid-phase synthetic product of claim 2 in the preparation of a medicament for the prevention and / or treatment of alcoholic hepatocellular injury.

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