An antioxidant peptide HYHE derived from dietary protein and its application

Abstract

The present invention relates to the technical field of antioxidant peptides, and specifically relates to an antioxidant peptide HYHE derived from dietary proteins and its application. The amino acid sequence of the antioxidant peptide is HYHE, as shown in SEQ ID NO: 1. This antioxidant peptide was predicted based on the antioxidant peptide prediction platform Pred5AOP; 27 dietary proteins were virtually hydrolyzed by trypsin and pepsin to screen out the antioxidant peptide HYHE; the antioxidant peptide HYHE as a biological peptide has advantages such as strong stability, low immunogenicity, and high purity compared with chemical drugs; at the same time, the present invention further demonstrates the rich nutritional value of dietary proteins, provides a new idea for resource recycling, and has good social and economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of antioxidant peptides, and specifically to an antioxidant peptide HYHE derived from dietary proteins and its applications. Background Art

[0002] Excessive free radicals and reactive oxygen species in cells can lead to oxidative stress, which has a negative impact on human health and is associated with various diseases, including chronic kidney disease, hypertension, and cancer; the Kelch-like ECH-associated protein 1 (Keap1)-Nuclear factor erythroid 2-related factor 2 (Nrf2) / antioxidant response element (ARE) signaling pathway is a key pathway for cellular oxidative stress and has become a hot topic in antioxidant research in recent years; Keap1 mainly maintains the intracellular redox balance by regulating the antioxidant stress response.

[0003] In recent years, the research on antioxidant peptides obtained from dietary proteins has become a hot topic in the field of functional peptide research. Bioactive peptides usually consist of 2 - 20 amino acids, and these peptide segments have no activity in the parent protein. After being absorbed and digested in the gastrointestinal tract, bioactive peptides are released from the parent protein and exhibit biological functions. Compared with synthetic antioxidants, antioxidant peptides extracted from dietary proteins have the advantages of easy absorption and non-toxicity. Hydrolysates and peptides extracted from various inexpensive and low-utilization dietary proteins can be used to produce antioxidant peptides.

[0004] The present invention uses a strategy combining machine learning and experimental exploration to screen antioxidant peptides, so as to accelerate the screening process and reduce costs; screen potential antioxidant peptides in 27 dietary proteins, and evaluate the antioxidant activity of the polypeptides and their protective effects on cells in vitro; therefore, the method combining theoretical calculation and experimental evaluation can be used as an effective method for screening antioxidant peptides. Summary of the Invention

[0005] The purpose of the present invention is to provide an antioxidant peptide HYHE derived from dietary proteins and its applications, so as to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] An antioxidant peptide derived from dietary proteins, whose amino acid sequence is described as SEQ ID NO:1: HYHE, hereinafter simply referred to as antioxidant peptide HYHE.

[0008] The applications of the above antioxidant peptide HYHE in the preparation of health products, food additives, and cosmetics.

[0009] Compared with the prior art, the beneficial effects of this patent are as follows:

[0010] The multi-layer perceptron (MLP, Multilayer Perceptron) is a feed-forward neural network composed of an input layer, a hidden layer, and an output layer. The neurons in each layer are connected to the neurons in the adjacent layer by weights. Its core advantage is the ability to learn complex patterns through non-linear activation functions. The present invention applies the MLP algorithm to develop an antioxidant peptide prediction platform Pred5AOP; by virtual hydrolysis of 27 dietary proteins with trypsin and pepsin, the antioxidant peptide HYHE is screened out; polypeptide drugs have advantages such as strong stability, low immunogenicity, and high purity compared with chemical drugs; at the same time, the present invention further proves the rich nutritional value of dietary proteins, provides a new idea for resource recycling, and antioxidant peptides have good application prospects in the fields of health products, food additives, and cosmetics, with good social and economic benefits. Brief Description of the Drawings

[0011] Figure 1 It is a dataset diagram of the present invention;

[0012] Figure 2 It is a diagram of the scavenging activity of antioxidant peptide HYHE at different concentrations on DPPH free radicals (* p<0.05, **p<0.01, *** p<0.001, **** p<0.0001);

[0013] Figure 3 It is a diagram of the scavenging activity of antioxidant peptide HYHE at different concentrations on ABTS free radicals (* p<0.05, **p<0.01, *** p<0.001, **** p<0.0001);

[0014] Figure 4 It is a diagram of the relative cell viability after the action of antioxidant peptide HYHE at different concentrations on HepG2 (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001):

[0015] Figure 5 It is a diagram of the relative cell viability after the action of D-galactose at different concentrations on HepG2 (* p<0.05,** p<0.01, *** p<0.001, **** p<0.0001);

[0016] Figure 6Relative cell viability of HepG2 cells after treatment with different concentrations of antioxidant peptide HYHE in the present invention (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001);

[0017] Figure 7 ROS content of HepG2 cells after treatment with different concentrations of antioxidant peptide HYHE in the present invention (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001);

[0018] Figure 8 Fluorescence images of HepG2 cells after treatment with different concentrations of antioxidant peptide HYHE in the present invention;

[0019] Figure 9 Quantum chemical analysis and molecular docking results of different concentrations of antioxidant peptide HYHE in the present invention. Detailed implementation manners

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Please refer to Figures 1 to 9 , the present invention provides a technical solution:

[0022] Example 1: Rational design of antioxidant peptide HYHE

[0023] In the present invention, a dietary protein-derived database containing 76,343 peptide segments was constructed to systematically screen for peptide segments with antioxidant potential. The data was sourced from 27 dietary proteins (such as soybeans, dairy products, and fish). Pepsin and trypsin were used for enzymatic hydrolysis simulation to generate short peptide segments, and the physicochemical properties of each peptide segment, such as molecular weight, lipophilicity, and solubility, were fully recorded. The database further integrated functional data from public databases such as UniProt. Peptide segments with antioxidant properties were used as positive samples, and other functional peptide segments with antibacterial, anticancer, etc. functions were introduced as negative samples to construct a comprehensive training set and validation set, providing a high-quality data basis for subsequent screening. The specific data set is as Figure 1 shown.

[0024] During the screening process, the present invention adopted the deep learning-based Pred5AOP model to predict the antioxidant potential of peptides in the database. The model utilized the multi-layer perceptron (MLP) algorithm to extract information from the characteristics of peptide amino acid composition, amino acid sequence, and three-dimensional spatial structure, and used Morgan fingerprints to generate feature vectors with parameter settings of radius 2 and length 2048 bits. The training dataset included 1020 known antioxidant peptides. Through five-fold cross-validation, the prediction accuracy of the model reached 92%. By batch predicting a large database containing 76343 peptides, the model calculated the antioxidant scores of each peptide, and finally selected the top 5% peptides with the highest antioxidant scores (a total of 3817 peptides). These peptides were used as candidate peptides to enter the subsequent verification and experimental stages.

[0025] From the top 5% peptides with the highest antioxidant scores screened by Pred5AOP, 201 short peptides with scores greater than 0.999 were selected and subjected to cluster analysis using the distance method to obtain six types of representative antioxidant peptide molecules. Among them, the antioxidant peptide HYHE is characterized by having a glutamic acid residue at the end.

[0026] To further evaluate the potential of the screened antioxidant peptides, the present invention also conducted molecular property evaluations. The screened antioxidant peptides were subjected to ADMET (absorption, distribution, metabolism, excretion, and toxicity) prediction analysis using the SwissAdme tool, focusing on evaluating properties such as water solubility, lipophilicity, gastrointestinal absorption, blood-brain barrier (BBB) permeability, and bioavailability. The evaluation results showed that the screened peptides generally had good water solubility and hydrophilicity, which is crucial for their effective absorption in the body. In addition, the metabolic pathways of these peptides were predicted through the ADMETLAB 3.0 platform, and the results indicated that they did not participate in other metabolic pathways and did not show obvious side effects.

[0027] Example 2: Determination of the free radical scavenging activity of antioxidant peptide HYHE

[0028] The standard product of the antioxidant peptide HYHE involved in the present invention was provided by Liaoning New Base Biochemical Technology Co., Ltd. Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products and can be obtained through commercial channels.

[0029] The DPPH radical is dark purple. When the DPPH radical reacts with an antioxidant, the radical is scavenged, the color of the system becomes lighter, and the absorbance decreases. DPPH is dissolved in absolute ethanol to prepare a 0.1 mM DPPH radical. Five different concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) of the antioxidant peptide HYHE (dissolving the antioxidant peptide HYHE in deionized water) are prepared in the present invention. The experimental group (As) is 100 μL of the antioxidant peptide and 100 μL of the DPPH radical. The control group (Ac) is 100 μL of distilled water and 100 μL of the DPPH radical. The blank group (A) is 100 μL of the antioxidant peptide and 100 μL of absolute ethanol (0.1 mM). After reacting for 30 minutes in the dark at room temperature (25 °C), the absorbance is measured at a wavelength of 517 nm. The calculation formula for the DPPH radical scavenging rate (%) is as follows:

[0030] DPPH radical scavenging rate (%) = [(As - A) / (Ac - A)]×100;

[0031] The DPPH radical scavenging activity of the antioxidant peptide HYHE is as Figure 2 shown. As the concentration of the antioxidant peptide HYHE increases, the DPPH radical scavenging activity gradually increases. When the concentration of HYHE reaches 1.0 and 2.0 mg / mL, the DPPH radical scavenging rates reach 49.26% and 50.27%, respectively.

[0032] 15 mg of ABTS is dissolved in 4 mL of deionized water, and 10 mg of potassium persulfate is dissolved in 35 mL of deionized water. 500 μL of each of the ABTS solution and the potassium persulfate solution are mixed and left in the dark for 12 hours. The mixture is diluted 30 times with phosphate buffered saline (PBS) to prepare the ABTS radical. The ABTS radical is green and its color becomes lighter under the action of an antioxidant. Five concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) of the antioxidant peptide HYHE (dissolving the antioxidant peptide HYHE in deionized water) are prepared. The experimental group (As) is 50 μL of the antioxidant peptide and 150 μL of the ABTS radical. The control group is 50 μL of distilled water and 150 μL of the ABTS radical. It is incubated in the dark at room temperature (25 °C) for 10 minutes, and the absorbance at a wavelength of 405 nm is measured. The calculation formula for the ABTS radical scavenging rate (%) is as follows:

[0033] ABTS radical scavenging rate (%) = [(A - As) / A]×100;

[0034] The ABTS radical scavenging activity of the antioxidant peptide HYHE is shown in Figure 3, as the concentration of HYHE increased, the ABTS radical scavenging activity gradually enhanced; when the concentration of HYHE reached 1.0 and 2.0 mg / mL, the DPPH radical scavenging rates reached 85.18% and 86.54%, respectively.

[0035] Example 3: Effect of antioxidant peptide HYHE on cytotoxicity

[0036] In this invention, HepG2 cells (human hepatoma cells) were selected to explore the antioxidant effect of antioxidant peptide HYHE at the cellular level; cell viability is an important indicator for evaluating the cytotoxicity of exogenous additives; in this invention, HepG2 cells were cultured in DMEM medium (10% fetal bovine serum, 1% penicillin-streptomycin (10,000 units / mL of penicillin, 10 mg / mL of streptomycin)), and the DMEM medium was selected from Gibco. The following DMEM media had the same specifications. The cells were placed in a constant temperature CO 2 incubator (37 °C, 5% CO 2 ) for culture; HepG2 cells in the logarithmic growth phase were cultured in a 96-well plate at a density of 5×10 3 for 24 hours; after the cells adhered to the wall, the medium was discarded, and antioxidant peptide HYHE at different concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) (dissolved antioxidant peptide HYHE in DMEM medium according to the concentration) was added to the HepG2 cells, and then cultured for another 24 hours; the CCK-8 method was used to measure the cell viability of each group, and the absorbance at a wavelength of 450 nm was measured by an enzyme-linked immunosorbent assay (ELISA) reader; the calculation formula for cell viability (%) is as follows:

[0037] Relative cell viability (%) = [(As - A) / (Ac - A)] × 100

[0038] where As, Ac, and A are the absorbances of the sample group, the control group (without adding antioxidant peptide HYHE), and the blank group (without adding antioxidant peptide HYHE and HepG2 cells), respectively;

[0039] The cytotoxic effects of antioxidant peptide HYHE at five concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) on HepG2 cells are shown as Figure 4 follows; when the concentration of antioxidant peptide HYHE was 0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL, there was no obvious effect on HepG2 cells after 24 hours of treatment; the relative cell viability values of HepG2 cells were 108.61%, 100.74%, 94.14%, 102.63%, and 97.60%, respectively; the results showed that antioxidant peptide HYHE had no cytotoxic effect on HepG2 cells.

[0040] Example 4: Protective effect of antioxidant peptide HYHE on oxidatively damaged cells

[0041] D-galactose is metabolized in the body by galactose oxidase; when a large amount of D-galactose accumulates in cells, it will reduce the level of antioxidant enzymes, damage the body's antioxidant defense system, and induce cellular oxidative stress; in the present invention, D-galactose is used to construct a HepG2 cell oxidative damage model at different concentrations and different incubation times acting on HepG2 cells.

[0042] HepG2 cells in logarithmic growth phase were seeded at a density of 5×10 3 per well in a 96-well plate and divided into two groups: a sample group added with different concentrations of D-galactose (100, 200, 300, 400, and 500 mM) (dissolving D-galactose in the culture medium according to the concentration) and an untreated control group; the absorbance was measured at a wavelength of 450 nm using the CCK-8 method to determine the optimal induction concentration and time.

[0043] The relationship between the viability of HepG2 cells and the induction concentration and induction time of D-galactose is as Figure 5 shown; as the concentration of D-galactose and the incubation time increase, the cell viability gradually decreases; the cell survival rate for constructing the damage model should be around 50%; the results show that when the concentration of D-galactose is 300 mmol / L and the induction time is 24 hours, the viability of HepG2 cells reaches 52.12%, which is within the appropriate range for establishing a cellular oxidative stress model.

[0044] In order to explore the pre-protective effect of the antioxidant peptide on oxidatively damaged cells in the present invention, HepG2 cells in logarithmic growth phase were cultured in a 96-well plate at a density of 5×10 3 per well for 24 hours; before D-galactose induction, different concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) of antioxidant peptide HYHE were added to the protected group and cultured for 24 hours. The calculation formula for the relative cell viability (%) is as follows:

[0045] Relative cell viability (%) = [(As - A) / (Ac - A)]×100;

[0046] where As, Ac, and A are the absorbances of the sample group, the control group (without adding D-galactose and antioxidant peptide HYHE), and the blank group (without adding D-galactose, antioxidant peptide HYHE, and HepG2 cells), respectively.

[0047] The protective effect of antioxidant peptide HYHE on D-galactose-induced cellular oxidative damage is shown in Figure 6; The cell survival rate decreased to 52.08% after D-galactose induction, which was within the survival rate range of the injury model and could be regarded as successful modeling. After pre-protective culture with antioxidant peptide HYHE for 24 hours, the relative cell viabilities of antioxidant peptide HYHE at different concentrations (0.1, 0.3, 0.5, 1.0, and 2.0 mg / mL) were 43.67%, 55.63%, 67.19%, 86.68%, and 90.94% respectively. The relative cell viability was positively correlated with the concentration of antioxidant peptide HYHE. The experimental results showed that the pre-protective treatment with antioxidant peptide HYHE increased the relative viability of HepG2 cells under oxidative stress.

[0048] Example 5: Antioxidant peptide HYHE reduces the intracellular ROS content in HepG2 cells

[0049] Reactive oxygen species (ROS) are involved in various physiological processes in the human body. However, excessive ROS may produce toxic effects, be able to oxidize biomolecules, and even promote the development of various diseases.

[0050] In this study, the intracellular ROS level was detected by the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA), and the intensity of green fluorescence was proportional to the ROS level. Antioxidant peptide HYHE at five different concentrations (0.1, 0.3, 0.5, 1.0, 2.0 mg / mL) was prepared using DMEM medium. HepG2 cells in the logarithmic growth phase were inoculated into 6-well plates at a density of 2×10 4 and cultured for 24 hours. After discarding the medium, the cells were pretreated with antioxidant peptides at different concentrations for 24 hours. Subsequently, the cells were continuously cultured with 300 mM D-galactose (DMEM medium containing 1% penicillin-streptomycin) for 24 hours, and then the medium was discarded. 1 mL of ROS probe solution was added to each well and incubated for 30 minutes. Subsequently, the cells were washed three times with PBS, and the ROS level was observed using a green fluorescence microscope, and the intracellular ROS level was quantitatively analyzed using Image J software.

[0051] The intracellular ROS content in oxidative damaged HepG2 cells treated with antioxidant peptide is shown in Figure 7 and Figure 8 ; Compared with the injury group (D-galactose induction group), the ROS level in the oxidative damaged cells treated with antioxidant peptide decreased, and with the increase in the concentration of antioxidant peptide HYHE, the intracellular ROS fluorescence intensity gradually weakened.

[0052] Example 6: Taste characteristic analysis of antioxidant peptide HYHE

[0053] To evaluate the taste characteristics of antioxidant peptides, the antioxidant peptide HYHE sample was dissolved in deionized water and formulated into a concentration of 0.1 mg / mL. Each test included an initial taste measurement stage of 30 seconds, followed by a 3-second rinse with a reference solution and a 30-second aftertaste evaluation stage; the electronic tongue sensor was tested four rounds in total, and the data of the first round was discarded due to calibration adjustment. The final result was the average of the remaining three rounds, which was used to determine the final taste evaluation result; the taste characteristic results of antioxidant peptide HYHE are shown in Table 1 below:

[0054] Table 1: Taste characteristic results table of antioxidant peptide HYHE

[0055]

[0056] Antioxidant peptide HYHE showed significant bitterness and astringency, and had a certain degree of sourness and saltiness, but lacked umami and richness; compared with the tasteless group, antioxidant peptide HYHE had significant differences in taste characteristics, mainly presenting a bitter and astringent taste characteristic.

[0057] Example 7: Molecular mechanism of antioxidant peptide HYHE

[0058] Kelch-like ECH-associated protein 1 (Keap1) is a key protein in the antioxidant stress signaling pathway Keap1-Nrf2-ARE; under excessive oxidative stress, Keap1 will be downregulated, thereby reducing the ubiquitination and degradation of Nrf2, and then increasing the expression of a series of antioxidant genes.

[0059] The crystal structure and sequence information of Keap1 protein were obtained from the PDB database (PDB ID: 2FLU); molecular docking of antioxidant peptide HYHE and Keap1 protein was performed by AlphaFold3, and the docking results were analyzed and visualized using Pymol 2.6; quantum chemical calculations were performed on antioxidant peptide HYHE using Gaussian 09, and the LUMO and HUMO orbits of antioxidant peptide were visualized using Multiwfn software.

[0060] The energies of HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) are key parameters; the energy difference between the HOMO and LUMO orbits reflects the stability and reactivity of antioxidant peptides; a smaller energy difference usually indicates a higher reactivity of the molecule.

[0061] The quantum chemical analysis and molecular docking results of antioxidant peptides are shown in Figure 9; The LUMO energy level is 0.21 eV. The lower LUMO energy level indicates that the molecule may be more likely to accept electrons and may have a stronger binding ability to Keap1 protein; the HOMO energy level is 3.74 eV; the distribution of the HOMO energy level reflects the ability of the molecule to release electrons; the energy gap is 3.53 eV, indicating that the antioxidant peptide has a certain stability and also has a certain chemical reactivity, and can effectively interact with the active site of Keap1 protein.

[0062] The antioxidant peptide interacts with the R91, R159, Y10, Y248, and Q206 residues of Keap1 protein. R159 and R91 act synergistically to stabilize the carboxyl groups of glutamic acid and aspartic acid in the antioxidant peptide. However, adjacent tyrosine residues (such as Y248) usually form conjugate interactions with the side chains of adjacent amino acids in the antioxidant peptide. This interaction enables R91 and R159 to anchor acidic amino acids, while Y248 interacts with adjacent non-acidic amino acids, thereby fixing the antioxidant peptide in a stable conformation.

[0063] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An application of an antioxidant peptide HYHE derived from dietary protein in the preparation of antioxidant health products or cosmetics, characterized in that: The amino acid sequence of the antioxidant peptide HYHE is shown in SEQ ID NO:1.

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