Antioxidant oligopeptide from tuna bone and preparation method thereof

Abstract

The present application belongs to the technical field of bioengineering, and particularly relates to a tuna fish bone antioxidant oligopeptide and a preparation method thereof.The tuna fish bone antioxidant oligopeptide has good scavenging activity, and has good thermal stability and digestion resistance; the mechanism of action is to promote the release of nitric oxide (NO), improve the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and total antioxidant capacity (T-AOC), and reduce the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH); JQYG-1 can protect HUVEC cells from oxidative damage induced by hydrogen peroxide; JQYG-1 can exert an antioxidant effect and protect vascular endothelial cells, and can provide a candidate drug for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis and heart failure.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to an antioxidant oligopeptide from tuna bone and its preparation method. Background Technology

[0002] The incidence and mortality rates of cardiovascular diseases are increasing year by year. According to the 2019 Global Burden of Cardiovascular Disease and Risk Factors study, there were approximately 523 million patients with cardiovascular disease in 2019, double the number in 1990, and approximately 18.6 million people died from cardiovascular disease. Most commonly used drugs for treating cardiovascular diseases are chemically synthesized, such as anticoagulants, antiplatelet drugs like aspirin, lipid-lowering drugs like atorvastatin, antiarrhythmic drugs like amiodarone, antihypertensive drugs like nifedipine, and beta-blockers like metoprolol. While these drugs can control the disease and alleviate symptoms, they have been found to cause adverse reactions such as hypotension, lower extremity edema, elevated blood potassium levels, taste disturbances, cough, and allergic reactions. Therefore, finding safe, effective drugs without strong toxic side effects has become an inevitable trend. In recent years, there has been a surge of research into finding antioxidant peptides from plant and animal-derived proteins as alternatives to cardiovascular disease drugs. Hypertension is a globally recognized major public health problem and a major risk factor for cardiovascular and cerebrovascular diseases, making its prevention and treatment particularly important.

[0003] Tuna accounts for more than 10% of the global seafood trade value. A large amount of by-products are generated during processing. These by-products contain not only a large amount of protein but also various bioactive substances, all of which have high utilization value. Studies have shown that one ton of tuna can produce 100 kilograms of tuna bones. Summary of the Invention

[0004] This invention uses tuna bones as raw material and employs enzymatic hydrolysis and chromatographic preparation techniques to prepare oligopeptides with antihypertensive and antioxidant functions. These oligopeptides are safe and have no toxic side effects. By exerting antioxidant effects and protecting vascular endothelial cells, these oligopeptides can provide candidate drugs for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, arteriosclerosis, and heart failure.

[0005] The first technical problem to be solved by the present invention is to provide an antioxidant oligopeptide from tuna bone, which has the amino acid sequence Gln-Glu-His-Glu (JQYG-1) and a molecular weight of 541.1 Da as determined by ESI-MS.

[0006] The second technical problem to be solved by the present invention is to provide a method for preparing antioxidant oligopeptides from tuna bones.

[0007] A method for preparing antioxidant oligopeptides from tuna bones: tuna bones are used as raw material, and then prepared by enzymatic hydrolysis, ultrafiltration, and chromatographic purification to obtain antioxidant oligopeptides from tuna bones.

[0008] A method for preparing antioxidant oligopeptides from tuna bones, specifically including the following steps:

[0009] 1) Tuna bone pretreatment process:

[0010] Tuna bones were dried in a constant temperature oven at 37°C for 48 hours, pulverized in a Chinese medicine pulverizer and passed through a 60-mesh sieve, defatted with ethyl acetate, and repeated three times. They were then dried in a constant temperature oven (37°C, 48h).

[0011] 2) Pretreatment of tuna bone samples:

[0012] Defatted tuna bone powder was dissolved in 50°C distilled water, salted out by adding saturated ammonium sulfate solution, and then filtered after standing in a 4°C refrigerator for 6 hours and dried at 37°C to obtain tuna bone protein.

[0013] 3) Proteolytic digestion of tuna bones:

[0014] Weigh out tuna bone protein, add deionized water and pepsin, adjust the pH to 2.0 with 0.5 mol / L NaOH and 0.1 mol / L HCl solution, and enzymatically hydrolyze at 37.5℃. Use the heating enzyme inactivation method to inactivate the enzyme hydrolysate in a 100℃ water bath. After enzyme inactivation, cool to room temperature, centrifuge at 8000 r / min for 10 min, collect the supernatant, and obtain tuna bone protein hydrolysate.

[0015] 4) Preparation of tuna bone antioxidant oligopeptides: The enzymatic hydrolysate of tuna bone protease was fractionated by ultrafiltration membranes with molecular weight cutoffs of 1kDa, 3kDa, 5kDa and 10kDa. The fractions were collected and subjected to antioxidant activity tests. The fraction with the best activity was selected and further purified by high performance liquid chromatography to obtain tuna bone antioxidant oligopeptide Gln-Glu-His-Glu (JQYG-1).

[0016] The high-performance liquid chromatography separation and purification are as follows:

[0017] 1) Sample pretreatment: Dissolve the obtained peptide in deionized water to prepare a solution with a final concentration of 10 mg / mL. Centrifuge at 8000 r / min for 1 min and collect the supernatant for later use.

[0018] 2) System: Agilent 1200 HPLC

[0019] 3) Column: Zorbax 300SB-C18 9.4X 250 5um

[0020] 4) Injection volume: 10 μL

[0021] 5) Buffer solution

[0022] Equilibration buffer: 0.06% TFA

[0023] Elution buffer: 0.05% TFA in methanol

[0024] 6) Gradient:

[0025] Solution A: 0.1% trifluoroacetic acid in 100% water;

[0026] Solution B: 0.1% trifluoroacetic acid in 100% acetonitrile;

[0027] 2%–2% elution for 2 min; 2%–30% B elution for 4 min; 30%–60% B elution for 27 min; 60%–100% B elution for 0.1 min; 100%–100% B elution for 5 min.

[0028] 7) Flow rate: 2.0 ml / min

[0029] 8) Detection: 280nm / 254nm / 214nm

[0030] In some embodiments of the present invention, the weight-to-volume ratio of tuna bone to ethyl acetate (0.05 mol / L) in step 1) is 1 g: 1 mL.

[0031] In some embodiments of the present invention, the weight-to-volume ratio of the pretreated tuna bone defatted powder to distilled water in step 2) is 1 g: 2 mL.

[0032] In some embodiments of the present invention, the weight-to-volume ratio of tuna bone protein to deionized water in step 3) is 1:40 (g / mL).

[0033] In some embodiments of the present invention, the amount of pepsin added in step 3) is 3% of the weight of tuna bone protein.

[0034] In some embodiments of the present invention, the enzymatic hydrolysis time of pepsin in step 3) is 4 hours.

[0035] Compared with existing technologies, the tuna bone antioxidant oligopeptide JQYG-1 provided by this invention has better scavenging activity and good thermal stability and digestibility. It can reduce the production of intracellular reactive oxygen species (ROS) and form a protective effect. Its mechanism of action is to promote the release of nitric oxide (NO), increase the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT), and total antioxidant capacity (T-AOC), and decrease the levels of malondialdehyde (MDA) and lactate dehydrogenase (LDH). JQYG-1 can protect against hydrogen peroxide-induced oxidative damage to HUVEC cells. By exerting antioxidant effects and protecting vascular endothelial cells, JQYG-1 can provide a candidate drug for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, arteriosclerosis, and heart failure. Attached Figure Description

[0036] Figure 1 This is the mass spectrum of Gln-Glu-His-Glu (JQYG-1) in Example 1.

[0037] Figure 2 The image shows the T-AOC test results for peptide JQYG-1.

[0038] Figure 3 The image shows the CAT test results for peptide JQYG-1.

[0039] Figure 4 This is a graph showing the quantitative analysis of Nrf2 by peptide JQYG-1. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments; however, these do not constitute a limitation or restriction on the scope of the present invention.

[0041] There are no particular restrictions on the solvents used in this invention; commercially available conventional solvents can be used.

[0042] NAC: N-acetylcysteine;

[0043] Total antioxidant capacity (T-AOC) test kit: purchased from Nanjing Jiancheng Biotechnology Research Institute;

[0044] CAT reagent kit: purchased from Nanjing Jiancheng Biotechnology Research Institute;

[0045] The steps for HPLC separation and purification are as follows:

[0046] Experimental steps

[0047] 1) Sample pretreatment: Dissolve the obtained peptide in deionized water to prepare a solution with a final concentration of 10 mg / mL. Centrifuge at 8000 r / min for 1 min and collect the supernatant for later use.

[0048] 2) System: Agilent 1200 HPLC

[0049] 3) Column: Zorbax 300SB-C18 9.4X 250 5um

[0050] 4) Injection volume: 10 μL

[0051] 5) Buffer solution

[0052] Equilibration buffer: 0.06% TFA

[0053] Elution buffer: 0.05% TFA in methanol

[0054] 6) Gradient:

[0055] Solution A: 0.1% trifluoroacetic acid in 100% water;

[0056] Solution B: 0.1% trifluoroacetic acid in 100% acetonitrile;

[0057] 2%–2% elution for 2 min; 2%–30% B elution for 4 min; 30%–60% B elution for 27 min; 60%–100% B elution for 0.1 min; 100%–100% B elution for 5 min.

[0058] 7) Flow rate: 2.0 ml / min

[0059] 8) Detection: 280nm / 254nm / 214nm

[0060] Example 1

[0061] 1) Tuna bone pretreatment process:

[0062] Tuna bones were dried in a constant temperature oven at 37°C for 48 hours, pulverized with a Chinese medicine pulverizer and passed through a 60-mesh sieve, defatted with ethyl acetate (material-to-liquid ratio 1:1 g / mL, repeated three times), and dried in a constant temperature oven (37°C, 48 h).

[0063] 2) Pretreatment of tuna bone samples:

[0064] 100g of defatted tuna bone powder was dissolved in 200mL of 50℃ hot water, and then salted out by adding saturated ammonium sulfate solution. After standing in a 4℃ refrigerator for 6 hours, the mixture was filtered and dried at 37℃ to obtain tuna bone protein.

[0065] 3) Proteolytic digestion of tuna bones:

[0066] Weigh 5g of tuna bone protein, add deionized water (solid-to-liquid ratio 1:40g / mL), add 3% pepsin, adjust the pH to 2.0 with 0.5mol / L NaOH and 0.1mol / L HCl solution, and enzymatically hydrolyze at 37.5℃ for 4h. Inactivate the enzyme solution by heating in a 100℃ water bath. After inactivation, cool to room temperature, centrifuge at 8000r / min for 10min, collect the supernatant, and freeze-dry for testing the ACE inhibitory activity of the enzyme solution.

[0067] 4) ACE inhibitory activity assay

[0068] ACE was dissolved in borate buffer to a final concentration of 0.1 U / mL for subsequent experimental determination. The ultrafiltration-lyophilized sample was dissolved in deionized water to prepare peptide solutions of different concentrations. Then, 180 μL of HCl solution was mixed with 30 μL of a peptide solution of a specific concentration, and the mixture was incubated at 37 °C for 5 min. Subsequently, 15 μL of 0.1 U / mL ACE was added to the mixture to initiate the reaction. The reaction was maintained at 37 °C for 60 min, and then 225 μL of 1M HCl was added to terminate the reaction. The reaction solution was filtered through a 0.22 μm filter membrane, and 20 μL of the filtered reaction solution was drawn using a syringe and loaded into RP-HPLC, with absorbance measured at 228 nm. All measurements were repeated three times. The ACE inhibitory activity was calculated as follows:

[0069]

[0070] Where AInhibitor is the relative area of ​​the hippuric acid (HA) peak obtained by the reaction of ACE and HHL with an inhibitor (ACEI). AControl is the relative area of ​​the hippuric acid (HA) peak obtained by the reaction of ACE and HHL without an inhibitor.

[0071] 5) Separation and purification of active peptides from enzymatically hydrolyzed peptides. Using DPPH and other free radical scavenging rates as evaluation indicators, ultrafiltration was first used to fractionate the peptides (starting the membrane ultrafiltration device, setting the frequency and pressure for ultrafiltration, cleaning the machine, and selecting ultrafiltration membranes of different molecular weights (1, 3, 5, 10 kDa) to fractionate the tuna bone enzymatic hydrolysate, obtaining five fractions. Each fraction was collected, rotary evaporated, and lyophilized. Antioxidant activity tests were performed, and the fractions with better activity were selected for further separation and purification. Subsequently, high-performance liquid chromatography (HPLC) was used for further separation, and mass spectrometry and amino acid analysis yielded the tuna bone antihypertensive oligopeptide Gln-Glu-His-Glu (JQYG-1), whose mass spectrum is shown below. Figure 1 As shown.

[0072] Example 2

[0073] Cell culture: HUVEC cells in logarithmic growth phase were seeded into 6-well plates at a density of 8.0 × 10⁶ cells / well. 5 Cells per well, 1.6 mL of culture medium per well, incubated at 37°C and 5% CO2 for 24 h. Sample loading was 10-fold. Sample loading was pre-grouped: blank and model groups were added with 200 μL PBS; the positive control group was added with 200 μL NAC (final concentration 1 mM); and the sample group was added with 200 μL peptide solution (final concentration 200 μM). After 24 h of incubation, the blank group was added with 200 μL PBS, and the remaining groups were added with 200 μL 500 μM hydrogen peroxide. Incubation was continued for 4 h.

[0074] Cell treatment: Remove culture medium with a pipette, wash twice with PBS, add lysis buffer, lyse on ice for 30-40 minutes, mix well by pipetting, and remove for later use.

[0075] Assay method: Total antioxidant capacity was determined using the T-AOC kit, following the instructions for the T-AOC kit. Results are as follows: Figure 2 As shown.

[0076] Example 3

[0077] Cell culture: HUVEC cells in logarithmic growth phase were seeded into 6-well plates at a density of 8.0 × 10⁶ cells / well. 5 Cells per well, 1.6 mL of culture medium per well, incubated at 37°C and 5% CO2 for 24 h. Sample loading was 10-fold. Sample loading was pre-grouped: blank and model groups were added with 200 μL PBS; the positive control group was added with 200 μL NAC (final concentration 1 mM); and the sample group was added with 200 μL peptide solution (final concentration 200 μM). After 24 h of incubation, the blank group was added with 200 μL PBS, and the remaining groups were added with 200 μL 500 μM hydrogen peroxide. Incubation was continued for 4 h.

[0078] Cell treatment: Remove culture medium with a pipette, wash twice with PBS, add lysis buffer, lyse on ice for 30-40 minutes, mix well by pipetting, and remove for later use.

[0079] Assay method: CAT concentration was determined using a CAT kit. Refer to the CAT kit instructions for testing. Results are as follows: Figure 3 As shown.

[0080] Example 4

[0081] (1) Polyacrylamide gel electrophoresis (SDS-PAGE): Prepare a 10% separating gel, mix well, pour into a container, seal with ultrapure water, and remove the ultrapure water after solidification. Prepare a 5% stacking gel, mix well, pour onto the separating gel, insert a comb, and ensure no air bubbles are generated during gel preparation. Load the sample and perform electrophoresis;

[0082] (2) Transfer: Soak the PVDF membrane in anhydrous methanol, clamp the required items according to the instructions, and continuously remove air bubbles. Transfer protein;

[0083] (3) Sealing: Wash the PVDF membrane after transfer with TBST 3 times, 15 min each time. Seal the PVDF membrane with 5% skim milk powder on a shaker for 2 h;

[0084] (4) Incubation with primary antibody: Wash the membrane 3 times with TBST, 15 min each time, add primary antibody, and incubate overnight at 4°C;

[0085] (5) Incubation with secondary antibody: Wash the membrane 3 times with TBST, 15 min each time, add secondary antibody, and incubate at room temperature for 2 h;

[0086] (6) ECL chemiluminescence development: Wash the membrane 3 times with TBST, 15 min each time, and develop and photograph.

[0087] To investigate the mechanism by which the peptide JQYG-1 protects HUVEC cells from oxidative damage, protein imprinting analysis and quantitative detection of Nrf2 were employed. The results are as follows: Figure 4 It was found that JQYG-1 exerts its antioxidant effect by activating the Keap 1 / Nrf2 signaling pathway.

[0088] Finally, it should be noted that the above-described embodiments are merely one specific example of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. An antioxidant tuna bone oligopeptide, characterized in that The amino acid sequence of the oligopeptide is Gln-Glu-His-Glu, and the ESI-MS measured molecular weight is 541.1 Da. The amino acid sequence of the oligopeptide is Gln-Glu-His-Glu, and the ESI-MS measured molecular weight is 541.1 Da. The amino acid sequence of the oligope

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