Active peptide with effects of resisting oxidation and protecting alcoholic liver injury and application of active peptide

Abstract

The invention discloses an active peptide with effects of resisting oxidation and protecting alcoholic liver injury and application of the active peptide. According to the invention, walnut protein hydrolysate is taken as a raw material, and two novel peptide fragments HGEPGQQQR and VAPFPEVFGK with high antioxidant activity are obtained through screening. The two active peptides show good antioxidant activity on the cellular level, and the EC50 values of the two active peptides are 0.0120 mg / mL and 0.0068 mg / mL respectively, which are both superior to 0.0122 mg / mL of positive control glutathione. In an ethanol injury model, the survival rates of cells subjected to ethanol injury treatment can be respectively increased from 47.35% to 62.69% and 57.05% by the two antioxidant peptides. The results show that the walnut protein source antioxidant peptides HGEPGQQQR and VAPFPEVFGK can be developed and applied as a potential antioxidant and a liver protection active component.

Description

Technical Field

[0001] The invention belongs to the technical field of anti-oxidation, and in particular relates to active peptides with anti-oxidation and alcoholic liver damage protection effects and applications thereof. Background Art

[0002] During the metabolic process of organisms, reactive oxygen species (ROS) or free radicals are naturally generated through the oxidation reaction of respiration. 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 accumulation of free radicals and reactive oxygen species in cells will cause oxidative stress, leading to damage to the organism. Oxidative stress states can lead to many non-communicable chronic diseases, including, for example, cardiovascular disease, diabetes, inflammatory diseases and aging. The development of natural antioxidants has great practical significance in preventing 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 regulating physiological functions such as antioxidant, anti-hypertensive, anti-thrombotic, anti-bacterial, anti-cancer, anti-inflammatory, anti-diabetic, anti-obesity, cholesterol-lowering, immunomodulatory and mineral binding. At the same time, food-derived bioactive peptides also have the function of preventing and treating diseases in a healthy diet. Not only that, 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. Peptides with antioxidant properties can scavenge free radicals, inhibit peroxides and chelate metal ions (Fe 2+ / Cu 2+ ) to exert antioxidant effects. At present, antioxidant peptides from different sources have been reported, such as pearl oyster meat, oyster, cottonseed, buckwheat, etc.

[0004] Walnut is one of the four most consumed dried fruits in the world and is considered an important oil-bearing tree species, economic tree species and ecological tree species. However, most of the walnut meal is currently used as feed or discarded after oil extraction, resulting in a waste of resources. Therefore, how to efficiently utilize walnut meal protein is of great practical significance. Studies have shown that peptides isolated from walnut protein have activities such as lowering blood pressure, anti-inflammatory, neuroprotection, and lowering uric acid.

[0005] Alcoholic liver disease (ALD) is one of the most common liver diseases in the world, mainly caused by long-term excessive drinking. Alcohol can cause the accumulation of ROS in cells, affect the activity of liver lipase, and lead to cell damage and death. Many studies have shown that natural antioxidants with antioxidant activity can prevent alcoholic liver damage through antioxidant mechanisms such as scavenging free radicals, restoring antioxidant enzyme activity, and maintaining the homeostasis of the liver's antioxidant defense system.

[0006] To solve the related problems, the primary purpose of the present invention is to provide an antioxidant active peptide.

[0007] Another object of the present invention is to provide the application of the above-mentioned antioxidant active peptide.

[0008] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0009] An antioxidant peptide having the following amino acid sequence (1) or (2):

[0010] (1)His-Gly-Glu-Pro-Gly-Gln-Gln-Gln-Arg(HGEPGQQQR);

[0011] (2) Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys(VAPFPEVFGK).

[0012] Furthermore, the antioxidant active peptides are prepared by enzymatic hydrolysis of walnut protein or solid phase synthesis.

[0013] A walnut protein hydrolysate containing the above-mentioned antioxidant active peptides.

[0014] A solid phase synthesis product comprising the above antioxidant active peptide.

[0015] Application of the above antioxidant active peptides, walnut protein hydrolysis products or solid phase synthesis products in the preparation of antioxidants.

[0016] Furthermore, the antioxidant is an oral preparation or an external preparation.

[0017] Furthermore, the antioxidant is used to remove free radicals in cells.

[0018] Furthermore, the antioxidant is used to protect cells from oxidative damage caused by alcohol.

[0019] Application of the above antioxidant active peptides, walnut protein hydrolysis products or solid phase synthesis products in the preparation of drugs for preventing and / or treating alcoholic hepatocyte damage.

[0020] Application of the above antioxidant active peptides, walnut protein hydrolysis products or solid phase synthesis products in the preparation of antioxidant additives.

[0021] Furthermore, the additive is used in food or medicine with antioxidant and / or liver protection effects.

[0022] Compared with the prior art, the present invention has the following advantages and effects:

[0023] The present invention uses walnut protein hydrolysate as raw material and screens out two new high antioxidant activity peptide segments HGEPGQQQR (1189.584Da) and VAPFPEVFGK (1089.586Da). The two active peptides show good antioxidant activity (CAA) at the cellular level, and their EC 50 The values ​​were 0.0120 mg / mL and 0.0068 mg / mL, respectively, both better than the positive control glutathione (EC 50 :0.0122mg / mL). In the ethanol injury model, the two antioxidant peptides can increase the survival rate of cells treated with ethanol from 47.35% to 62.69% and 57.05%, respectively. These results indicate that the walnut protein-derived antioxidant peptides HGEPGQQQR and VAPFPEVFGK can be developed and used as a potential antioxidant and liver-protecting active ingredient. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The figure shows the effect of different concentrations of antioxidant peptides on the viability of HepG2 cells; different letters indicate significant differences (P < 0.05).

[0025] Figure 2 This is a study result of the effect of antioxidant peptides on the antioxidant activity of cells; different letters indicate significant differences (P < 0.05).

[0026] Figure 3 The figure shows the research results of the protective effect of antioxidant peptides on ethanol-induced cell damage; A is the result of the construction of the ethanol-damaged HepG2 cell model, and different letters indicate significant differences (P < 0.05); B is the protective effect of antioxidant peptides on cells, and * indicates a significant difference from the damage group (P < 0.05).

[0027] Figure 4 This is the docking site map of antioxidant peptides and Keap1 molecules; among them, A is HGEPGQQQR and B is VAPFPEVFGK. DETAILED DESCRIPTION

[0028] The present invention is further described in detail below in conjunction with examples and drawings, but the embodiments of the present invention are not limited thereto.

[0029] Example 1: Synthesis of Antioxidant Peptides

[0030] Two antioxidant peptides were screened from walnut meal protein hydrolysate, and their amino acid sequences were: HGEPGQQQR and VAPFPEVFGK. They were commissioned to be synthesized by Nanjing Jiepeptide Biotechnology Co., Ltd. The purity of the synthesized peptide was 98% as determined by HPLC. The synthesized antioxidant peptides were stored at -20°C for subsequent experiments.

[0031] Example 2: Analysis of the toxicity of antioxidant peptides to HepG2 cells

[0032] HepG2 cells were cultured in complete medium at 37°C and 5% carbon dioxide. Cytotoxicity was determined by MTT assay, as follows: 100 μL of HepG-2 cell suspension was inoculated into each well of a white 96-well plate to obtain a seeding density of 1×10 4 cells, and the outer wells of the 96-well plate were not inoculated with cells, but filled with PBS. After inoculation, the 96-well plate was placed in a 37°C, 5% CO2 incubator for culture. After 24 hours of culture, the medium was changed. The experimental group was added with 100 μL of culture 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), and the blank control group was added with 100 μL of fresh culture medium. After the cells were cultured for another 24 hours, the culture medium was aspirated, 100 μL of MTT (0.5 mg / mL) solution was added, and the cells were incubated in the dark for 4 hours. The MTT solution was discarded, 100 μL of DMSO was added, and the blue-purple crystals were completely dissolved after shaking for 10 minutes. The OD value was measured with an enzyme reader at a wavelength of 490 nm. The cell survival rate was calculated according to formula (1):

[0033] Survival rate (%) = OD value of experimental group / OD value of blank control group × 100 (1)

[0034] The results are as follows Figure 1 As described above, after treating HepG2 cells with the two antioxidant peptides HGEPGQQQR and VAPFPEVFGK in the concentration range of 0.025-2 mg / mL, the cell survival rate was above 90%, indicating that they had no toxic effect on cells. Subsequent studies can be conducted within this concentration range.

[0035] Example 3: Cellular Antioxidant Activity (CAA) Assay

[0036] The cells were divided into a control group, a blank group, and an experimental group. The cell suspension was inoculated in a black 96-well plate to obtain an inoculation density of 6 × 10 per well. 4cells. After 24 hours of cell culture, the cell medium was changed, and DCFH-DA (50 μL, 50 μM) working solution and sterile water (50 μL) were added to the control group and blank group; DCFH-DA (50 μL, 50 μM) working solution and 50 μL culture medium containing different concentrations of antioxidant peptides (0.005 mg / mL, 0.01 mg / mL, 0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL) were added to the experimental group, and incubated at 37°C for 1 hour. The culture medium was discarded, and the PBS was discarded after washing with 100 μL PBS. 100 μL HBSS was added to the blank group, and AAPH (100 μL, 600 μM) working solution was added to the control group and experimental group. The cells were placed in an ELISA instrument, and the fluorescence value was measured at a measurement wavelength of 528 nm and an excitation wavelength of 485 nm, and the measurement was performed every 5 minutes within 1 hour. Glutathione (GSH) was used as a positive control. AUC represents the integrated area under the fluorescence intensity-time curve. The CAA value is calculated according to formula (2), and the half effective concentration EC of the sample is also calculated. 50 and standard antioxidant Trolox equivalent (mM TE / g Petide) to represent CAA value.

[0037]

[0038] Among them, AUC T : Fluorescence curve area of ​​the experimental group; AUC P : Fluorescence curve area of ​​the control group; AUC N : Fluorescence curve area of ​​the blank group.

[0039] The CAA value represents the intracellular antioxidant activity. Its working principle is that in the presence of reactive oxygen species (ROS), DCFH is oxidized into highly fluorescent dichlorofluorescein (DCF). The intracellular fluorescence intensity is positively correlated with the ROS level. The higher the detected fluorescence intensity, the higher the ROS level. The larger the CAA value, the lower the fluorescence intensity, which means that the intracellular ROS clearance rate is higher and the antioxidant capacity of the sample is stronger. Cellular antioxidant capacity (CAA value) of HGEPGQQQR, VAPFPEVFGK and positive control (GSH) Figure 2 EC of HGEPGQQQR and VAPFPEVFGK 50 The concentrations of the two peptides were 0.0120 mg / mL and 0.0068 mg / mL, respectively, which were lower than the positive control (GSH) of 0.0122 mg / mL (Table). It is worth noting that the two peptides can exert antioxidant capacity superior to that of the positive control (GSH) even at very low concentrations (0.005 mg / mL). [1] , Mulberry source [2] , soy protein source [3] EC of antioxidant peptides50 The EC values ​​of the monomeric peptides in this study were 2.85 mg / mL, 0.24 mg / mL, and 0.715 mg / mL, respectively. 50 The above results show that this antioxidant peptide can effectively scavenge intracellular free radicals, reduce ROS levels, and exert antioxidant effects.

[0040] Table 1 Cellular antioxidant activity EC of antioxidant peptides 50 Value and Trolox equivalent value.

[0041]

[0042] Example 4: Protective effect of antioxidant peptides on ethanol-damaged HepG2 cells

[0043] Construction of ethanol-damaged HepG2 cell model: The cell suspension was inoculated in a white 96-well plate to obtain a seeding density of 2×10 per well. 4 cells, and the medium was changed after 24 hours of cell culture. After adding 100 μL of fresh culture medium and culturing for 24 hours, the medium was changed again. The experimental group added culture medium containing different concentrations of ethanol (5mg / mL, 10mg / mL, 15mg / mL, 20mg / mL, 25mg / mL, 30mg / mL, 35mg / mL, 40mg / mL), and the blank group added fresh culture medium and cultured for 24 hours. The cell survival rate was determined by the MTT method to explore the ethanol concentration at which the cell survival rate was 50%.

[0044] The results are as follows Figure 3 As shown in A in FIG, when the ethanol concentration is 40 mg / mL, the cell viability is closest to 50%, so the cell injury model was constructed with an ethanol concentration of 40 mg / mL.

[0045] Pre-protection of HepG2 cells damaged by ethanol by antioxidant peptides: The cells were divided into a blank group, a damaged control group, and an experimental group. 100 μL of cell suspension was inoculated in a white 96-well plate to obtain an inoculation density of 2×10 4 cells, and after culturing for 24 hours, the culture medium was replaced. 100 μL of culture medium containing different concentrations of antioxidant peptides (0.025 mg / mL, 0.05 mg / mL, 0.1 mg / mL) was added to the experimental group, and the blank group and the injury control group were replaced with fresh culture medium and cultured for 24 hours. After that, the culture medium was replaced again, and 100 μL of culture medium containing 40 mg / mL ethanol was added to the experimental group and the injury control group, and 100 μL of fresh culture medium was added to the blank group and cultured for 24 hours. The cell survival rate was determined by the MTT method. Metadoxine was used as a positive control.

[0046] Ethanol can induce oxidative stress, increase ROS in the body and impair antioxidant defense, leading to alcohol poisoning, such as liver cell damage. Although lipid accumulation was first observed in ethanol-induced steatosis, oxidative stress plays a key role in ethanol-induced cell death. Therefore, antioxidant peptides with excellent antioxidant activity may have the potential to protect against alcoholic liver damage caused by ethanol.

[0047] The results are as follows Figure 3 As shown in Figure B, when the ethanol concentration was 40 mg / mL, the cell viability of the ethanol injury control group was 47.36%, which was significantly lower than that of the blank group (P < 0.05), indicating that the cell injury model was successfully established. In this model, Metadoxine, a commonly used drug for the treatment of alcoholic liver disease, was selected as a positive control.

[0048] The cell viability of HGEPGQQQR at a concentration of 0.025 mg / mL was 62.69%, significantly higher than that of the injury group (P < 0.05), and also better than the positive control (Metadoxine). The cell activity of VAPFPEVFGK at a concentration of 0.1 mg / mL was 57.06%, significantly higher than that of the injury group (P < 0.05). This indicates that under the pre-protection of different concentrations of HGEPGQQQR and VAPFPEVFGK, the damage of ethanol to cells can be reduced, so the two antioxidant peptides in this study have the potential to pre-protect alcoholic liver injury.

[0049] Example 5: Molecular docking and visualization analysis

[0050] AutoDockvina was used for molecular docking analysis. First, the 3D structure of the antioxidant peptide was drawn using ChemDraw, and then the energy was minimized and exported to mol2 format for subsequent docking. RCSB PDB: Homepage ) was downloaded from the 3D structure of the receptor molecule Keap1 protein (4L7B). The receptor molecule and the peptide were pre-processed by hydrogenation and exported to pdbqt format using AutoDockTools 1.5.7 software and then docked using AutoDockvina. The center of the docking box of Keap1 was set to x=-2.636, y=3.267 and z=-27.443, and the size of the docking box was set to x=15, y=15 and z=15. Visual analysis was performed using Discovery Studio Visualizer based on the results of the lowest docking binding energy.

[0051] Molecular docking is a biomolecular simulation method based on integrated bioinformatics analysis. It simulates the interaction between molecules and can depict the structure-activity relationship of peptides. It has been widely used in the research of screening bioactive peptides. Keap1 / Nrf2 is the main system for regulating cellular antioxidant response. Under normal circumstances, Nrf2 binds to Keap1 and is degraded, inhibiting the antioxidant response. If the antioxidant peptide can bind to Keap1 and inhibit the Keap1-Nrf2 interaction, it can activate and release the potential of Nrf2, and Nrf2 can migrate to the cell nucleus, activate the transcription of antioxidant genes, and reduce oxidative stress.

[0052] Molecular docking analysis revealed that the docking binding energy values ​​of HGEPGQQQR and VAPFPEVFGK were both -8.9 (Table 2), indicating that they have strong binding ability with Keap1. Studies have shown that the key binding sites of Nrf2 in the Kelch region of Keap1 protein are Tyr334, Arg 380, Arg 415, Arg 483, Ser363, Asn382, Ser555, Asn 387, His 436, and Tyr525. If the antioxidant peptide can form an interaction force with the above sites, it can effectively interfere with the binding of Nrf2 to Keap1 protein and reduce oxidative stress. Figure 4 The interaction between the two monomeric peptides and Keap1 was demonstrated from two-dimensional and three-dimensional perspectives. Both monomeric peptides can be embedded in the active cavity of Keap1 to form a stable docking conformation. Among them, HGEPGQQQR forms 20 hydrogen bonds with Asn382, Asp385, Tyr572, Gln530, Tyr334, Asn387, Ala556, Gly509, Arg415, Gly433, Arg380, Pro384, Gly574, Gly386, and Gly433 of Keap1. Asn382, Tyr334, Asn387, Arg415, and Arg380 are key sites, and also form a hydrophobic interaction force with the key site Tyr334 ( Figure 4 VAPFPEVFGK forms 13 hydrogen bonds with Asn387, Arg415, Ser555, Gly433, Arg483, Ser508 and Tyr334 of Keap1, among which Asn387, Arg415, Ser555, Arg483 and Tyr334 are key sites, and forms 3 hydrophobic interactions with Phe478, Arg415 and Ala556, and 2 electrostatic interactions with the key site Arg380 ( Figure 4 B). Similar binding sites are also found in cottonseed protein (Arg380, Arg415, Ser508) [4], fermented milk source (Arg380, Asn382, Asn387, Arg415, Gly433, Ser508, Gln530) [5] In general, HGEPGQQQR and VAPFPEVFGK can bind tightly to the key amino acids and surrounding residues of Keap1 through different non-covalent bonds, and have the potential to compete with Keap1 / Nrf2 to exert antioxidant effects.

[0053] Table 2 Docking binding energy values

[0054]

[0055] In general, the two antioxidant peptides HHGEPGQQQR and VAPFPEVFGK isolated and screened in the present invention showed excellent cellular antioxidant activity in cell experiments, effectively reduced the level of intracellular ROS, and were also found to have the ability to protect the vitality of ethanol-damaged cells. In addition, they can act on key sites in the Keap1-Nrf2 pathway, reduce oxidative stress, and provide new options for natural antioxidants.

[0056] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

[0057] References:

[0058] [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.

[0059] [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.

[0060] [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.

[0061] [4]Wang L,Ma M,Yu Z,et al.Preparation and identification ofantioxidant peptides from cottonseed proteins[J].Food Chemistry,2021,352:129399.

[0062] [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. An antioxidant active peptide, Features: It has the following amino acid sequence (1) or (2): (1)His-Gly-Glu-Pro-Gly-Gln-Gln-Gln-Arg; (2) Val-Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys.

2. The antioxidant peptide according to claim 1, Features: The antioxidant active peptide is prepared by enzymatic hydrolysis of walnut protein or solid phase synthesis.

3. A walnut protein hydrolysate comprising the antioxidant active peptides according to claim 1.

4. A solid phase synthesis product comprising the antioxidant active peptide according to claim 1.

5. Use of the antioxidant active peptide described in claim 1 or 2, the walnut protein hydrolysate described in claim 3 or the solid phase synthesis product described in claim 4 in the preparation of antioxidants.

6. The use according to claim 5, Features: The antioxidant is used to remove free radicals in cells.

7. The use according to claim 5, Features: The antioxidants described serve to protect cells from oxidative damage caused by alcohol.

8. The use according to claim 5, Features: The antioxidant is a kind of oral preparation and external preparation.

9. Use of the antioxidant active peptides described in claim 1 or 2, the walnut protein hydrolysate described in claim 3 or the solid phase synthesis product described in claim 4 in the preparation of drugs for preventing and / or treating alcoholic hepatocyte damage.

10. Use of the antioxidant active peptide described in claim 1 or 2, the walnut protein hydrolysate described in claim 3 or the solid phase synthesis product described in claim 4 in the preparation of antioxidant additives, Features: The additive is used for food or medicine with anti-oxidation and / or liver protection effects.

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