Antioxidant peptide and application thereof in preparation of medicine for preventing and treating non-alcoholic fatty liver disease

Abstract

The invention discloses an antioxidant peptide and application thereof in preparation of drugs for preventing and treating non-alcoholic fatty liver diseases. The invention discloses an antioxidant peptide. The antioxidant peptide comprises one or more of polypeptides A1, A3, A5 and A8, wherein A1: Glu-Ala-Glu-Lys; a3: As < n >-Val-Gl < n >-Thr; a5: Lu-Glu-Ala-Pro-Arg-Asn, and A6: Lu-Glu- A < 8 >: Asp-Glu-Tyr-Val-Gly. The polypeptides A1, A3, A5 and A8 disclosed by the invention have better oxidation resistance. The invention also finds that A1, A3, A5 and A8 can reduce the content of active oxygen in cells and improve the activity of antioxidant protease; the oxidative stress condition caused by excessive fat of the NAFLD cell model can be improved, so that the non-alcoholic fatty liver disease is improved, and a new thought is provided for auxiliary treatment of the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to antioxidant peptides and applications thereof in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Harpadon nehereus is a fish of the family Harpadonidae. Fish meat is rich in nutrients. In addition to daily consumption, it is also a good sample for preparing antioxidant peptides. Many studies have shown that fish meat has immune regulation, anti-fatigue, antioxidant, and wound healing effects, but there are very few studies on the antioxidant properties of Harpadon fish meat.

[0003] Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver damage worldwide, and can develop into nonalcoholic hepatitis (NASH) to cirrhosis and even liver cancer. The main cause of NAFLD is excessive triglyceride synthesis in liver cells. Fatty acids are mainly stored in the form of triglycerides in fat droplets of white adipose tissue. However, in the state of insulin resistance, the anti-lipolysis effect of insulin is weakened, and white adipose tissue is broken down, resulting in a large release of free fatty acids. Then, excess fatty acids are stored in the liver in the form of triglycerides, forming ectopic lipid deposition and causing NAFLD. High-fat and high-sugar foods are also prone to lipid accumulation in NAFLD.

[0004] Under normal conditions, fatty acids provide energy to the body through β-oxidation. However, due to various reasons, the increase of fatty acids in the liver leads to damage to β-oxidation and mitochondrial dysfunction, resulting in inflammation, which in turn leads to oxidative stress. NAFLD patients show ultrastructural mitochondrial damage, reduced activity of respiratory chain complexes, and impaired adenosine triphosphate (ATP) synthesis. Mitochondria play a very important role in impaired fat oxidation and energy supply, but this process produces a large amount of ROS. Fatty acids pass through the inner membrane of mitochondria through CPT1. When the flow of fatty acids to mitochondria is reduced, mitochondrial β-oxidation is impaired. The components of the mitochondrial respiratory chain are over-reduced by electrons and react abnormally with oxygen, resulting in an increase in ROS. Excessive ROS oxidize lipids, releasing peroxides that damage liver cells and further damage the respiratory chain, leading to a vicious cycle. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention aims to provide an antioxidant peptide and its use in the preparation of a drug for preventing and treating non-alcoholic fatty liver disease.

[0006] The specific technical solutions of the present invention are as follows:

[0007] In one aspect, the present invention provides an antioxidant peptide, which comprises one or more of polypeptides A1, A3, A5, and A8, wherein the amino acid sequences of the polypeptides A1, A3, A5, and A8 are as follows:

[0008] A1: Glu-Ala-Glu-Lys, as shown in SEQ ID NO.1;

[0009] A3: Asn-Val-Gln-Thr, as shown in SEQ ID NO.3;

[0010] A5: Leu-Glu-Ala-Pro-Arg-Asn, as shown in SEQ ID NO.5;

[0011] A8: Asp-Glu-Tyr-Val-Gly, as shown in SEQ ID NO.8.

[0012] The present invention also provides the use of the antioxidant peptide in the preparation of health products, food additives, nutritional enhancers, animal additives or medicines.

[0013] The present invention also provides the use of the antioxidant peptide in preparing a drug for preventing and treating non-alcoholic fatty liver disease.

[0014] Furthermore, the antioxidant peptide improves lipid accumulation in non-alcoholic fatty liver disease.

[0015] The present invention also provides application of the antioxidant peptide in preparing antioxidant drugs.

[0016] The present invention also provides application of the antioxidant peptide in preparing a medicine for alleviating oxidative stress.

[0017] Furthermore, the antioxidant peptide reduces the oxidative stress caused by excessive liver fat in patients with non-alcoholic fatty liver disease.

[0018] The present invention also provides a drug for preventing and treating non-alcoholic fatty liver disease, which comprises one or more of polypeptides A1, A3, A5, and A8, and the amino acid sequences of the polypeptides A1, A3, A5, and A8 are as follows:

[0019] A1: Glu-Ala-Glu-Lys, as shown in SEQ ID NO.1;

[0020] A3: Asn-Val-Gln-Thr, as shown in SEQ ID NO.3;

[0021] A5: Leu-Glu-Ala-Pro-Arg-Asn, as shown in SEQ ID NO.5;

[0022] A8: Asp-Glu-Tyr-Val-Gly, as shown in SEQ ID NO.8.

[0023] Furthermore, the medicine also contains pharmaceutically acceptable excipients.

[0024] The beneficial effects of the present invention are:

[0025] The present invention uses dragon head fish meat as a research sample, and enzymatically hydrolyzes it to obtain polypeptides A1, A3, A5, and A8 with good antioxidant properties. The present invention further studies the protective mechanism of A1, A3, A5, and A8 on NAFLD cell models, and finds that A1, A3, A5, and A8 can reduce the content of intracellular reactive oxygen species and increase the activity of antioxidant proteases; they can improve the oxidative stress caused by excessive fat in NAFLD cell models, thereby improving non-alcoholic fatty liver disease. The present invention provides a new idea for the adjuvant treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The effect of FFA on the activity of HepG2 cells;

[0027] Figure 2 The effect of FFA on lipid accumulation in HepG2;

[0028] Figure 3 The effect of FFA on the TG content in HepG2 cells;

[0029] Figure 4 The effect of FFA on TC content in HepG2 cells;

[0030] Figure 5 The effect of polypeptides A1-A9 on the activity of HepG2 cells;

[0031] Figure 6 The effect of peptide A1-A9 on lipid accumulation in HepG2 cells, where A: Control group; B: 1.0 mM FFA; C: NAC positive drug group; DL experimental group: experimental group A1-A9 (200 μM) + FFA (1.0 mM); M: A1-A9 changes in the absorbance of lipid accumulation in HepG2 cells;

[0032] Figure 7 The effect of polypeptides A1-A9 on TG content in HepG2 cells;

[0033] Figure 8 The effect of polypeptides A1-A9 on TC content in HepG2 cells;

[0034] Fig. 9The effect of different concentrations of H2O2 on the survival rate of HepG2 cells;

[0035] Fig.10 The effect of A1-A9 on MDA content in HepG2 cells;

[0036] Figure 11 shows the effects of different concentrations of A1, A3, A5, and A8 on lipid accumulation in HepG2 cells, wherein A: blank group; B: FFA model group; C: NAC positive drug group; DF: sample group FFA+A1 (50 μM), FFA+A1 (100 μM), and FFA+A1 (200 μM); GI: sample group FFA+A3 (50 μM), FFA+A3 (100 μM), and FFA+A3 (200 μM); JL: sample group FFA+A5 (50 μM), FFA+A5 (100 μM), and FFA+A5 (200 μM); MO: sample group FFA+A8 (50 μM), FFA+A8 (100 μM), and FFA+A8 (200 μM); P: absorbance changes of different concentrations of A1, A3, A5, and A8 on lipid accumulation in HepG2 cells;

[0037] Fig.12 The effects of different concentrations of A1, A3, A5, and A8 on the TG content in HepG2 cells;

[0038] Fig.13 The effects of different concentrations of A1, A3, A5, and A8 on TC content in HepG2 cells;

[0039] Figure 14 shows the effects of different concentrations of A1, A3, A5, and A8 on the ROS content in HepG2 cells, where A: blank group; B: FFA model group; C: NAC positive drug group; DF: sample group FFA+A1 (50μM), FFA+A1 (100μM), FFA+A1 (200μM); GI: sample group FFA+A3 (50μM), FFA+A3 (100μM), FFA+A3 (200μM); JL: sample group FFA+A5 (50μM), FFA+A5 (100μM), FFA+A5 (200μM); MO: sample group FFA+A8 (50μM), FFA+A8 (100μM), FFA+A8 (200μM); P: the proportion of ROS content in cells of each group compared with the blank group;

[0040] Fig.15 The effects of A1, A3, A5, and A8 at different concentrations on CAT activity in HepG2 cells;

[0041] Fig.16The effects of A1, A3, A5, and A8 at different concentrations on SOD activity in HepG2 cells;

[0042] Fig.17 The effects of A1, A3, A5, and A8 at different concentrations on the activity of GSH-PX in HepG2 cells; DETAILED DESCRIPTION

[0043] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0044] Example 1: Preparation of antioxidant peptides

[0045] This embodiment provides a method for preparing an antioxidant peptide, comprising the following steps:

[0046] (1) Take some fresh dragon head fish and separate its internal organs and other parts from the fish meat. Use a high-speed grinder to grind the fish meat into minced meat. Put the dragon head fish meat and ethyl acetate mixture into an ultrasonic cleaner at a mass and volume ratio of 1:5 and perform ultrasonic degreasing for 5 hours. After the ultrasonic cleaning, vacuum filter and separate the fish meat. Then put the fish meat into an oven to dry. Use a traditional Chinese medicine powder grinder to grind the dried fish meat into fine powder to obtain fish meat powder, which is sealed and stored.

[0047] (2) Accurately weigh 1 gram of fish powder, and then add 20 mL of distilled water to it. Use 0.5 M hydrochloric acid and sodium hydroxide to adjust the solution to a pH of 7.0. Next, add 0.03 grams of papain to the mixture and perform enzymatic hydrolysis at 50°C for 5 hours. After the enzymatic hydrolysis is completed, the solution is heated at 100°C for 15 minutes to inactivate the enzymes, and the dragon head fish enzymatic hydrolysate is obtained, which is then ultrafiltered. Component LI (MW < 1 kDa) is subjected to DEAE-52 anion exchange chromatography to obtain LI-3 with higher ABTS free radical scavenging rate and DPPH free radical scavenging rate. Component LI-3 is then subjected to Sephadex G-15 gel chromatography to screen out component LI-3-1 with better activity. After separation and purification by HPLC technology, a short peptide chain small molecule compound is obtained. The sequence and molecular weight of the short peptide are determined by mass spectrometry and Edman degradation method. As shown in Table 1 below.

[0048] Table 1 Sequence and molecular weight of polypeptides A1-A9

[0049]

[0050] Furthermore, this example determined the half effective concentration EC of A1-A9 for four free radicals: ABTS, DPPH, hydroxyl anion, and superoxide anion. 50 , the results are shown in Table 2. EC 50 The lower the value, the stronger the antioxidant capacity of the polypeptide. As can be seen from Table 2, A1, A3, A5, and A8 have better antioxidant capacity, and A5 and A8 have higher antioxidant capacity due to their higher hydrophobic amino acids.

[0051] Table 2 Free radical scavenging activity of A1-A9 Note: Different letters indicate significant differences (p<0.05)

[0052]

[0053] Example 2: Screening for polypeptides that have an improving effect on NAFLD cell models

[0054] 1. The NAFLD cell model can be successfully established in human liver cancer cells (HepG2) using free fat (FFA)

[0055] The modeling solution was prepared according to the molar mass ratio of sodium oleate and sodium palmitate of 2:1. The HepG2 cells were divided into the Control group and the FFA group. Control group: HepG2 complete medium was added; FFA group: HepG2 complete medium and modeling solution were added. Specifically: HepG2 cells were inoculated in a 96-well culture plate, HepG2 complete medium was added to each well, and then it was placed in an incubator for 24 hours of culture. Next, the 96-well plate was divided into the Control group and the FFA group. 20 μL of culture medium was added to the Control group, and 20 μL of modeling solution with a concentration of 1.0 mM was added to the FFA group. The culture was continued for 24 hours.

[0056] After 24 hours of culture, the cell activity was measured by CCK8 method, the intracellular fat accumulation was observed by Oil Red O staining technique, the triglyceride (TG) content in HepG2 cells was detected by triglyceride kit, and the total cholesterol (TC) content in HepG2 cells was detected by total cholesterol kit.

[0057] The results of the effects of FFA on the activity of HepG2 cells are shown in Figure 1 As shown in Figure 2, when the concentration of the modeling solution was 1.0 mM, it had no effect on the growth activity of HepG2 cells. Figure 2 As shown in Figure 2, FFA can effectively induce HepG2 cells to accumulate fat. The results of the effect of FFA on the TG content in HepG2 cells are shown in Figure 2. Figure 3As shown in Figure 2, 1.0 mM FFA can effectively increase the TG content of HepG2 cells. The results of the effect of FFA on the TC content in HepG2 cells are shown in Figure 2. Figure 4 As shown, 1.0 mM FFA can effectively increase the TC content in HepG2 cells. In summary, the NAFLD cell model can be successfully established by using 1.0 mM FFA in the embodiment of the present invention, and the NAFLD cell model is established by using 1.0 mM FFA in subsequent experiments.

[0058] 2. Screening of peptides that improve NAFLD cell models

[0059] 1. Effects of peptides A1-A9 on HepG2 cell activity

[0060] HepG2 cells were divided into a Control group and a polypeptide group. Control group: HepG2 complete culture medium was added; polypeptide group: HepG2 complete culture medium and different polypeptides were added. Specifically: HepG2 cells were inoculated in a 96-well culture plate, HepG2 complete culture medium was added to each well, and then it was placed in an incubator for 24 hours of culture. Next, the 96-well plate was divided into a Control group and a polypeptide group. Complete culture medium was added to the Control group, and polypeptides at a concentration of 200 μM were added to the polypeptide group. The culture was continued for 24 hours, and then the cell activity was measured by the CCK8 method. The results of the effects of polypeptides A1-A9 on the activity of HepG2 cells are shown in the figure. Figure 5 , when the concentration was 200 μM, these peptides could not significantly inhibit the proliferation of HepG2 (p<0.05), indicating that these oligopeptides have good biocompatibility for HepG2 cells, and therefore they are suitable for subsequent experimental studies.

[0061] 2. Effects of peptides A1-A9 on lipid accumulation, triglycerides (TG) and total cholesterol (TC) in HepG2 cells

[0062] HepG2 cells were seeded in 6-well plates, and complete medium was added to each well. After culturing in an incubator for 24 hours, the cells were grouped and treated with drugs as follows: 1) Control group: complete medium was added and the cells were cultured in an incubator for 48 hours; 2) FFA group: complete medium was added and cultured for 24 hours, and then medium containing 1.0 mM modeling solution was added, and then cultured in an incubator for 24 hours; 3) Positive drug group: medium containing 1.0 mM modeling solution was added, and then cultured in an incubator for 24 hours, the liquid was discarded, complete medium was added again, and then 200 μM NAC was added to continue culturing for 24 hours; 4) Experimental group: medium containing 1.0 mM modeling solution was added, and then cultured in an incubator for 24 hours, the liquid was discarded, complete medium was added again, and then 200 μM A1-A9 was added, and then cultured in an incubator for 24 hours.

[0063] After the culture, the intracellular fat accumulation was observed by Oil Red O staining, the triglyceride (TG) content in HepG2 cells was detected by a triglyceride kit, and the total cholesterol (TC) content in HepG2 cells was detected by a total cholesterol kit.

[0064] The results of the effects of peptides A1-A9 on lipid accumulation in HepG2 cells are as follows Figure 6 The change trend of the red oil droplets observed by staining was the same as the change trend of the absorbance value measured at 570nm. Comparing the FFA group with the Control group, the red oil droplets and absorbance values ​​in the FFA group cells increased significantly (p<0.001), indicating that the NAFLD cell model was successfully established. After adding oligopeptides A1-A9, the number of red oil droplets in the experimental cells of the A3, A5, and A8 groups decreased, and their measured absorbance values ​​were significantly different from those of the model group, which were 1.410±0.003 (p<0.001), 1.456±0.010 (p<0.001), and 1.466±0.050 (p<0.001), respectively. The absorbance measured in the A9 group was significantly different from that in the model group, which was 1.584±0.012 (p<0.01).

[0065] The results of the effects of peptides A1-A9 on TG content in HepG2 cells are as follows Figure 7 , the TG content of the Control group was 2.55±0.64mmol / gprot, and the TG content of the FFA group was 4.85±0.50mmol / gprot, which was significantly higher than that of the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. After the addition of oligopeptides A1-A9, all oligopeptides could reduce the TG content in HepG2 cells. There was no significant difference in the TG content in the cells of the A1, A2, A6, and A7 groups compared with the FFA group (p>0.05). The TG content in the A4 and A9 groups was significantly different from that in the FFA group, which were 3.57±0.24mmol / gprot (p<0.05) and 3.54±0.30mmol / gprot (p<0.05), respectively. The TG content of A3 and A8 groups was significantly different from that of FFA group, which was 3.31±0.44mmol / gprot (p<0.01) and 3.43±0.43mmol / gprot (p<0.01) respectively. The TG content of A5 group was extremely significantly different from that of FFA group, which was 3.05±0.17mmol / gprot (p<0.001).

[0066] The results of the effects of peptides A1-A9 on TC content in HepG2 cells are shown in Figure 8The total cholesterol content in the Control group was 0.69±0.16mmol / gprot, and the TC content in the FFA group was 1.77±0.19mmol / gprot, which was significantly higher than that in the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. After the addition of oligopeptides A1-A9, all oligopeptides could reduce the TC content in HepG2 cells. There was no significant difference in TC content in the A1, A2, A6, and A7 groups compared with the FFA group (p>0.05), while there was a significant difference in TC content in the A4 and A9 groups compared with the FFA group, which were 1.38±0.09mmol / gprot (P<0.01) and 1.36±0.06mmol / gprot (p<0.01), respectively. The TC contents in A3, A5 and A8 groups were significantly different from those in FFA group, which were 0.80±0.04mmol / gprot (p<0.001), 0.84±0.04mmol / gprot (p<0.001) and 0.89±0.15mmol / gprot (p<0.001), respectively.

[0067] 3. Effects of peptides A1-A9 on malondialdehyde content in HepG2 cells modeled with hydrogen peroxide

[0068] Studies have shown that in addition to the positive lipid regulation of the AMPK pathway, it also causes oxidative stress, activates the Nrf2 pathway, and regulates the lipid content in the liver by controlling fat oxidation. Therefore, by establishing a hydrogen peroxide injury model for HepG2 and determining the most suitable hydrogen peroxide concentration for establishing the model, a new screening index is provided for the subsequent detection and screening of peptides in the malondialdehyde (MDA) kit.

[0069] 1. Establishment of hydrogen peroxide-damaged cell model

[0070] like Fig. 9 As shown in the figure, under the action of 0-500μM hydrogen peroxide, after 4 hours of injury, the survival rate of HepG2 cells decreased with the increase of hydrogen peroxide concentration. At 500μM, the cell survival rate reached 33.05±1.44% (p<0.001). The hydrogen peroxide concentration when the cell survival rate was about 50% was the most suitable modeling concentration. Therefore, when 400μM was selected, the survival rate was 50.11±0.91% (p<0.001) as the hydrogen peroxide modeling concentration.

[0071] 2. Effects of peptides A1-A9 on MDA in HepG2 cells

[0072] The effects of A1-A9 on the malondialdehyde content in HepG2 cells modeled with hydrogen peroxide were calculated by measuring the MDA kit. Fig.10As shown in the figure, the content of MDA in the Control group was low, and the MDA in the Model group modeled by 400μM hydrogen peroxide was significantly increased compared with the Control group (p<0.001), indicating that the hydrogen peroxide-damaged cell model was successfully established. After the addition of oligopeptides A1-A9, all oligopeptides could reduce the MDA content in HepG2 cells. There was no significant difference in the MDA content in the cells of the A2 and A4 groups compared with the Model group (p>0.05). The MDA content in the cells of the A6, A7, and A9 groups was significantly different from that of the Model group, which were 1.17±0.03nmol / mgprot (p<0.05), 1.13±0.10nmol / mgprot (p<0.05), and 1.25±0.09nmol / mgprot (p<0.05), respectively. The intracellular MDA content of A3 and A8 groups was significantly different from that of Model group, which was 1.06±0.10nmol / mgprot (p<0.01) and 1.10±0.25nmol / mgprot (p<0.01), respectively. The intracellular MDA content of A5 group was extremely significantly different from that of Model group, which was 0.95±0.13nmol / mgprot (p<0.001). Since A1 had the best antioxidant activity, it was also included in the subsequent experiments.

[0073] Based on the above indicators, it can be seen that the lipid-lowering effect of A3, A5, and A8 oligopeptides is better than that of other oligopeptides. Therefore, A3, A5, and A8 oligopeptides were selected for subsequent experiments. Since A1 has the strongest antioxidant activity, it was also selected for subsequent experiments.

[0074] Example 3: Effects of A1, A3, A5, and A8 on lipid accumulation and oxidative stress in NAFLD models

[0075] HepG2 cells were seeded in 6-well plates, and complete medium was added to each well. After culturing in an incubator for 24 hours, the cells were grouped and treated with drugs as follows: 1) Blank group: complete medium was added and the cells were cultured in an incubator for 48 hours; 2) FFA group: complete medium was added and cultured for 24 hours, and then medium containing 1.0 mM modeling solution was added, and cultured in an incubator for 24 hours; 3) Positive drug group: medium containing 1.0 mM modeling solution was added, and after culturing in an incubator for 24 hours, the liquid was discarded, complete medium was added again, and 200 μM NAC was added to continue culturing for 24 hours; 4-6) A1 (50-200 μM) group: medium containing 1.0 mM modeling solution was added, and after culturing in an incubator for 24 hours, the liquid was discarded, complete medium was added again, and (50-200 μM) )A1 was cultured for 24 hours; 7-9)A3 (50-200μM) group: culture medium containing 1.0mM modeling solution was added, and after culturing in the incubator for 24 hours, the liquid was discarded, complete culture medium was added again, and then (50-200μM) A3 was added and cultured for 24 hours; 10-12)A5 (50-200μM) group: culture medium containing 1.0mM modeling solution was added, and after culturing in the incubator for 24 hours, the liquid was discarded, complete culture medium was added again, and then (50-200μM) A5 was added and cultured for 24 hours; 13-15)A8 (50-200μM) group: culture medium containing 1.0mM modeling solution was added, and after culturing in the incubator for 24 hours, the liquid was discarded, complete culture medium was added again, and then (50-200μM) A8 was added and cultured for 24 hours.

[0076] 1. Effects of A1, A3, A5, and A8 on lipid accumulation in NAFLD models

[0077] After the culture, the intracellular fat accumulation was observed by Oil Red O staining, the triglyceride (TG) content in HepG2 cells was detected by a triglyceride kit, and the total cholesterol (TC) content in HepG2 cells was detected by a total cholesterol kit.

[0078] The results of the effects of different concentrations of A1, A3, A5, and A8 on lipid accumulation in HepG2 cells are shown in Figure 11. The absorbance of the Control group was 0.79±0.07, and the absorbance of the FFA group was 1.60±0.08. The absorbance of the FFA group was significantly different from that of the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. When the four oligopeptides A1, A3, A5, and A8 were at a concentration of 50 μM, the absorbance measured in the four groups was not significantly reduced compared with the FFA group (p>0.05). When the four oligopeptides were at a concentration of 100 μM, the absorbance measured in the four groups was significantly reduced compared with the FFA group (p<0.001, p<0.01, p<0.05). At a concentration of 200 μM, the absorbances of the four groups were significantly lower than those of the FFA group, which were 1.10±0.04 (p<0.001), 1.04±0.05 (p<0.001), 0.98±0.04 (p<0.001), and 1.03±0.06 (p<0.001). In general, the lipid accumulation content in HepG2 cells decreased with the increase of oligopeptide concentration.

[0079] The results of the effects of different concentrations of A1, A3, A5, and A8 on the TG content in HepG2 cells are shown in Fig.12 As shown in the figure, the TG content in the cells of the Control group was 0.29±0.86mmol / gprot, while the TG content in the cells of the FFA group was 4.72±0.44mmol / gprot. There was a significant difference between the FFA group and the Control group (p<0.001), indicating that the cell model of NAFLD was successfully established. After the addition of oligopeptides, at a concentration of 50μM, the TG content in the A1, A3, and A8 groups was not significantly reduced compared with the FFA group (p>0.05). At a concentration of 200 μM, the intracellular TG content of the four groups A1, A3, A5, and A8 was significantly reduced compared with the FFA group, which were 3.12±0.08mmol / gprot (p<0.001), 3.24±0.11mmol / gprot (p<0.001), 2.83±0.13mmol / gprot (p<0.001), and 3.23±0.09mmol / gprot (p<0.001), respectively. In general, the intracellular TG content of the sample groups after adding A1, A3, A5, and A8 decreased with the increase of oligopeptide concentration.

[0080] The results of the effects of different concentrations of A1, A3, A5, and A8 on the TC content in HepG2 cells are shown in Fig.13As shown in the figure, the TC content of the Control group was 0.75±0.13mmol / gprot, while the TC content of the FFA group was 1.57±0.02mmol / gprot. The TC content in the FFA group was significantly increased compared with that in the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. When the sample groups with A1, A3, A5, and A8 were added at 50μM, the TC content in the cells of the A1, A3, A5, and A8 groups was not significantly reduced compared with the FFA group (p>0.05). At a concentration of 100μM, the TC content in the cells of the A1, A3, A5, and A8 groups was significantly reduced compared with the FFA group (p<0.05, p<0.01, p<0.001). At a concentration of 200 μM, the TC content of cells in the four groups A1, A3, A5, and A8 was significantly lower than that in the FFA group, which were 1.25±0.01mmol / gprot (p<0.001), 0.97±0.01mmol / gprot (p<0.001), 0.88±0.01mmol / gprot (p<0.001), and 1.20±0.01mmol / gprot (p<0.001), respectively. In general, the TC content in cells of the sample groups after adding A1, A3, A5, and A8 decreased with the increase of oligopeptide concentration.

[0081] 2. Effects of A1, A3, A5, and A8 on oxidative stress in NAFLD models

[0082] After the culture, the antioxidant capacity of the oligopeptide was detected by measuring the activity level of intracellular antioxidant proteases (CAT, SOD, GSH-PX) after the addition of the oligopeptide, as well as the content of a large amount of ROS induced by fatty acids and oxygen free radicals that damage cells.

[0083] The ROS kit was used to detect and measure the induction effect of different concentrations of A1, A3, A5, and A8 on the content of reactive oxygen species in HepG2. The results of the effects of different concentrations of A1, A3, A5, and A8 on the content of ROS in HepG2 cells are shown in Figure 14. The level of ROS in the Control group was low, while the level of ROS in the FFA group was significantly increased (p<0.001), indicating that the NAFLD cell model has been successfully established. When A1, A3, A5, and A8 were added, at 50 μM, the content of ROS in the cells of the A3 and A8 groups was not significantly reduced compared with the FFA group (p>0.05), while the content of ROS in the cells of the A1 and A5 groups was significantly reduced compared with the FFA group (p<0.05, p<0.001). At 100 and 200 μM, the content of ROS in the cells of the A1, A3, A5, and A8 groups was extremely significantly reduced compared with the FFA group (p<0.001). In general, with the increase of oligopeptide concentration, the ROS content in HepG2 cells showed a downward trend.

[0084] The CAT detection kit was used to detect the effects of different concentrations of A1, A3, A5 and A8 on the catalase activity in HepG2 cells. The results of the effects of different concentrations of A1, A3, A5 and A8 on the CAT activity in HepG2 cells are shown in Figure 2. Fig.15 As shown in the figure, the catalase activity in the cells of the Control group was the highest, and the catalase activity in the FFA group was significantly lower than that in the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. When A1, A3, A5, and A8 were added, at 50 μM, the catalase activity in the cells of the four groups A1, A3, A5, and A8 was not significantly increased compared with the FFA group (p>0.05). At 100 μM, the catalase activity in the cells of the A1, A3, and A8 groups was significantly increased compared with the FFA group (p<0.01), but the catalase activity in the cells of the A5 group was not significantly increased compared with the FFA group (p>0.05). At 200 μM, the catalase activity in the cells of the A1, A3, A5, and A8 groups was extremely significantly increased compared with the FFA group (p<0.001). In general, the size of the catalase activity in HepG2 cells increased with the increase of oligopeptide concentration.

[0085] The SOD kit was used to measure the effects of different concentrations of A1, A3, A5, and A8 on superoxide dismutase activity in HepG2 cells. The results of the effects of different concentrations of A1, A3, A5, and A8 on SOD activity in HepG2 cells are shown in Figure 2. Fig.16 As shown in the figure, compared with the Control group, the SOD activity in the FFA group was significantly reduced (p<0.001), indicating that the NAFLD cell model was successfully established. When A1, A3, A5, and A8 were added, at 50 μM, the SOD activity in the A1 and A8 groups was not significantly increased compared with the FFA group (p>0.05), but the SOD activity in the A3 and A5 groups was significantly increased compared with the FFA group (p<0.01). At 100 and 200 μM, the SOD activity in the A1, A3, A5, and A8 groups was significantly increased compared with the FFA group (p<0.001). In general, the size of the SOD activity in HepG2 cells increases with the concentration of oligopeptides.

[0086] The GSH-PX kit was used to measure the effect of different concentrations of A1, A3, A5, and A8 on the activity of glutathione peroxidase in HepG2 cells. The results of the effects of different concentrations of A1, A3, A5, and A8 on the activity of GSH-PX in HepG2 cells are shown in the figure. Fig.17As shown in the figure, the GSH-PX activity in the cells of the Control group was higher, while the GSH-PX activity in the cells of the FFA group was significantly reduced compared with the Control group (p<0.001), indicating that the NAFLD cell model was successfully established. When A1, A3, A5, and A8 were added, at 100 μM, the GSH-PX activity in the cells of the A1, A3, A5, and A8 groups was significantly increased compared with the FFA group (p<0.01, p<0.001). At 200 μM, the GSH-PX activity in the cells of the A1, A3, A5, and A8 groups was extremely significantly increased compared with the FFA group (p<0.001). In general, the GSH-PX activity in HepG2 cells increased with the increase of oligopeptide concentration.

[0087] From the results of the effects of A1, A3, A5, and A8 on oxidative stress in the NAFLD model, it can be seen that A1, A3, A5, and A8 have a protective effect on HepG2 cells against oxidative stress, thereby reducing liver fat accumulation and peroxidation.

Claims

1. Antioxidant peptide, characterized in that The antioxidant peptide comprises one or more of polypeptides A1, A3, A5, and A8, and the amino acid sequences of the polypeptides A1, A3, A5, and A8 are as follows: A1: Glu-Ala-Glu-Lys, as shown in SEQ ID NO.1; A3: Asn-Val-Gln-Thr, as shown in SEQ ID NO.3; A5: Leu-Glu-Ala-Pro-Arg-Asn, as shown in SEQ ID NO.5; A8: Asp-Glu-Tyr-Val-Gly, as shown in SEQ ID NO.

8.

2. Use of the antioxidant peptide according to claim 1 in the preparation of health products, food additives, nutritional enhancers, animal additives or medicines.

3. Use of the antioxidant peptide according to claim 1 in the preparation of drugs for preventing and treating non-alcoholic fatty liver disease.

4. The use according to claim 3, characterized in that: The antioxidant peptide improves lipid accumulation in non-alcoholic fatty liver disease.

5. Use of the antioxidant peptide according to claim 1 in the preparation of antioxidant drugs.

6. Use of the antioxidant peptide according to claim 1 in the preparation of a drug for alleviating oxidative stress.

7. The use according to claim 6, characterized in that: The antioxidant peptide reduces the oxidative stress in patients with non-alcoholic fatty liver disease with excessive liver fat.

8. A drug for preventing and treating non-alcoholic fatty liver disease, characterized in that: The drug comprises one or more of polypeptides A1, A3, A5, and A8, and the amino acid sequences of the polypeptides A1, A3, A5, and A8 are as follows: A1: Glu-Ala-Glu-Lys, as shown in SEQ ID NO.1; A3: Asn-Val-Gln-Thr, as shown in SEQ ID NO.3; A5: Leu-Glu-Ala-Pro-Arg-Asn, as shown in SEQ ID NO.5; A8: Asp-Glu-Tyr-Val-Gly, as shown in SEQ ID NO.

8.

9. The drug according to claim 8, characterized in that The drug further comprises pharmaceutically acceptable excipients.

Images