Antioxidant selenium-enriched peptide with neuron protection effect as well as preparation method and application of antioxidant selenium-enriched peptide

Abstract

The invention discloses an antioxidant selenium-enriched peptide with a neuron protection effect as well as a preparation method and application thereof, and belongs to the technical field of bioactive peptides. The amino acid sequence of the antioxidant selenium-rich peptide with the neuron protection effect is as shown in SEQ ID NO.1, and the sulfur element in the fourth amino acid cysteine Cys is substituted by selenium. The method comprises the following steps: dialyzing tuna blood by using a phosphate buffer solution, pre-freezing after dialyzing, and then performing vacuum freeze-drying to prepare tuna blood freeze-dried powder; the preparation method comprises the following steps: dissolving selenium-enriched peptide in water, adding protease for enzymolysis, performing enzyme deactivation treatment after enzymolysis to obtain enzymatic hydrolysate, and performing separation, purification and freeze-drying to obtain the antioxidant selenium-enriched peptide with the neuron protection effect. The antioxidant selenium-rich peptide with the neuron protection effect is clear in structure, high in selenium content, high in antioxidant activity and capable of protecting oxidative damaged cells, can be used for preparing antioxidant drugs or health care products to improve neurodegenerative diseases, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to an antioxidant selenium-enriched peptide with neuroprotective effects, its preparation method, and its application. Background Technology

[0002] Neurodegenerative diseases (NDDs) are a group of neurological disorders that affect memory, cognition, learning, and other brain functions through the progressive loss of neuronal structure or function. Studies have shown that oxidative damage is one of the main causes of synaptic loss and neuronal death in NDDs. Oxidative damage occurs due to excessive production of free radicals and insufficient antioxidant defense systems in the body, leading to oxidation reactions of substances such as lipids, proteins, and DNA, which have a significant negative impact on normal central nervous system function. Reactive oxygen species (ROS) are among the major free radicals in living organisms. Most ROS are caused by physiological processes or harmful exogenous factors, while endogenous ROS are mostly produced by mitochondria. Excessive ROS damages mitochondria, and the resulting mitochondrial dysfunction can induce apoptosis and neurodegeneration. Therefore, maintaining proper mitochondrial homeostasis is crucial for nerve cells.

[0003] Selenium (Se) is an essential trace element for the human body. Se deficiency reduces the activity of selenium-dependent antioxidant enzymes in the brain, increases oxidative stress, and leads to neurodegenerative diseases. Se is a component of selenocysteine ​​(SeCys) and is incorporated into selenoproteins during translation. Glutathione peroxidase (GPx) is one of the earliest discovered selenoprotein antioxidant enzymes. Selenium supplementation can effectively prevent the chain reaction of free radicals by enhancing the expression of GPx in the body, reducing oxidative stress damage to cells, and lowering the incidence of neurodevelopmental and neurodegenerative diseases. Selenium in selenium-enriched peptides exists primarily in the form of selenomethionine and SeCys and their derivatives within the polypeptide chain. These peptides possess both selenium and peptide bioactivity while enhancing selenium bioavailability, making their development as an antioxidant dietary supplement for improving neurodegenerative diseases (NDDs) a promising application.

[0004] Yellowfin tuna ( Thunnus albacaresYellowfin tuna has high commercial value internationally and is the second most produced economic fish species in the world's tuna fishery, after skipjack tuna. The processing of yellowfin tuna generates a large number of byproducts, among which the utilization rate of blood is extremely low, with the vast majority being discarded as processing waste, resulting in significant resource waste and environmental pollution. It is worth noting that the selenium content in tuna blood is 20.20 ± 0.12 mg / kg (wet weight), far exceeding the selenium content standard (0.10-0.50 mg / kg) for selenium-enriched aquatic products in the "Classification Requirements for Selenium Content of Selenium-Enriched Agricultural Products" (DB45 / T 1061-2014). Furthermore, it is rich in high-quality protein, making it a natural selenium-enriched product with good research value and development prospects, and a potential source for the preparation of selenium-enriched bioactive peptides. Summary of the Invention

[0005] To address the problems in the prior art, this invention provides an antioxidant selenium-enriched peptide with neuroprotective effects, its preparation method, and its application. The antioxidant selenium-enriched peptide with neuroprotective effects is obtained from tuna blood and has a protective effect against H2O2-induced oxidative damage to PC12 cells. It can be used to prepare antioxidant drugs or health products (dietary supplements) to improve neurodegenerative diseases.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides an antioxidant selenium-enriched peptide with neuroprotective effects, wherein the amino acid sequence of the antioxidant selenium-enriched peptide with neuroprotective effects is shown in SEQ ID NO.1; The sulfur element in the fourth amino acid cysteine ​​(Cys) of the antioxidant selenium-rich peptide with neuroprotective effects is replaced by selenium.

[0007] In a second aspect, the present invention provides a method for preparing the antioxidant selenium-enriched peptide with neuroprotective effects, comprising the following steps: (1) Tuna blood was dialyzed with phosphate buffer solution, pre-frozen after dialyzing, and then freeze-dried under vacuum to produce tuna blood freeze-dried powder. (2) Dissolve the tuna blood freeze-dried powder obtained in step (1) in water, add protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, perform enzyme inactivation treatment to obtain the hydrolysate. (3) The enzymatic hydrolysate in step (2) is separated, purified and freeze-dried to obtain an antioxidant selenium-rich peptide with neuroprotective effect.

[0008] Furthermore, in step (1), the pre-freezing temperature is -70~-80℃ and the pre-freezing time is 24~48 h; in step (1), the molecular weight cutoff of the dialysis bag is 1 kDa, the dialysis temperature is 0~4℃, and the dialysis time is 24~48 h.

[0009] Furthermore, in step (1), the pH of the phosphate buffer solution is 7 and the concentration is 0.02 mol / L; in step (2), the mass ratio of tuna blood freeze-dried powder to water is 1:4~5.

[0010] Further, the protease mentioned in step (2) is one or more of trypsin, neutral protease, papain, pepsin and alkaline protease; the amount of protease added is 5000~6000 U / g tuna blood freeze-dried powder; the enzyme inactivation method is boiling water inactivation for 5~10 min.

[0011] Furthermore, the enzymatic hydrolysis in step (2) is performed at a pH of 3-8, a temperature of 45-60°C, and a time of 3-4 hours; When the protease is trypsin, the enzymatic hydrolysis temperature is 55℃, the pH is 7.5, and the time is 3 h. When the protease is a neutral protease, the enzymatic hydrolysis temperature is 45℃, the pH is 7, and the time is 3 h; When the protease is papain, the enzymatic hydrolysis temperature is 65℃, the pH is 6.5, and the time is 3 h. When the protease is pepsin, the enzymatic hydrolysis temperature is 60℃, the pH is 3, and the time is 3 h; When the protease is an alkaline protease, the enzymatic hydrolysis temperature is 55℃, the pH is 8, and the time is 3 h.

[0012] Furthermore, in step (3), ultrafiltration membrane is used for separation, and reversed-phase high-performance liquid chromatography is used for purification.

[0013] Furthermore, the ultrafiltration membranes have molecular weights of 10 kDa and 3 kDa, and the centrifugation conditions for ultrafiltration are 4000 g, 30 min, and 4 °C. The chromatographic conditions for the reversed-phase high-performance liquid chromatography are as follows: column: Agilent Prep-C18 column, 20 × 250 mm, 10 μm; mobile phase A is water, mobile phase B is acetonitrile, and the elution program is gradient elution: 0-2 min 4-8% mobile phase B, 2-45 min 8-28% mobile phase B, 45-55 min 28-40% mobile phase B, 55-56 min 40-95% mobile phase B, and 56-66 min 95% mobile phase B.

[0014] In a third aspect, the present invention provides the application of the antioxidant selenium-rich peptide with neuroprotective effects in the preparation of neuroprotective products.

[0015] Furthermore, the neuron protection product is a pharmaceutical or health supplement.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: (1) The antioxidant selenium-rich peptide with neuroprotective effect provided by the present invention reduces ROS generation, decreases the content of malondialdehyde (MDA) and lactate dehydrogenase (LDH), increases the content of glutathione (GSH) and the activity of glutathione peroxidase (GPx) and superoxide dismutase (SOD) by optimizing the vitality and morphology of oxidatively damaged PC12 cells, and improves mitochondrial function to alleviate oxidative stress damage in PC12 cells.

[0017] (2) The present invention isolates and purifies an antioxidant selenium-rich peptide with neuroprotective effect from tuna blood. The antioxidant selenium-rich peptide with neuroprotective effect has a clear structure, high selenium content, and high antioxidant activity. It also has a protective effect on oxidatively damaged cells and can be used to prepare antioxidant drugs or health products (dietary supplements) to improve neurodegenerative diseases (drugs for treating neurodegenerative diseases), which has important application value. Attached Figure Description

[0018] Figure 1 The images show the degree of hydrolysis, sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis results, and antioxidant activity assay results for different enzymatic hydrolysates. Figure 1 A shows the results of the hydrolysis degree test. Figure 1 B shows the results of SDS-PAGE gel electrophoresis. Figure 1 C represents the results of the antioxidant activity assay; Figure 2 The graph shows the results of antioxidant activity determination of different ultrafiltration components; Figure 3 The graph shows the purification and antioxidant activity test results of different purified components by reversed-phase high-performance liquid chromatography. Figure 3 A is the elution curve. Figure 3 B shows the results of the antioxidant activity assay. Figure 4 Total ion current chromatogram (TIC) of component F5; Figure 5 This is the secondary mass spectrum of the antioxidant selenium-enriched peptide; Figure 6 Figure showing the effect of antioxidant selenium-enriched peptides with neuroprotective effects on the proliferation of PC12 cells; Figure 7 The effect of antioxidant selenium-enriched peptides on H2O2-induced PC12 cell viability; Figure 8 The effect of antioxidant selenium-enriched peptides on H2O2-induced PC12 cell morphology; Figure 9 To investigate the effect of antioxidant selenium-enriched peptides on H2O2-induced intracellular ROS production in PC12 cells, Figure 9 Figure A shows the ROS results from flow cytometry. Figure 9 B is a bar chart of average ROS fluorescence intensity; Figure 10 To investigate the effects of antioxidant selenium-enriched peptides on H2O2-induced oxidative stress indices in PC12 cells, Figure 10 A is the LDH activity result graph. Figure 10 B is the result graph of MDA content. Figure 10 C represents the SOD content results. Figure 10 D is the GPx activity result graph. Figure 10 E represents the GSH content results; Figure 11 The effect of antioxidant selenium-enriched peptides on H2O2-induced mitochondrial membrane potential (MMP) in PC12 cells was investigated. Figure 11 A is the flow cytometry result of MMP detection. Figure 11 B shows the red / green fluorescence ratio results for JC-1. Figure 12 The effect of antioxidant selenium-enriched peptides on the cytochrome C (Cyt-C) content in the cytoplasm of H2O2-induced PC12 cells; Figure 13 The effect of antioxidant selenium-enriched peptides on the content of H2O2-induced mitochondrial respiratory chain complexes I, III, IV, and V in PC12 cells; Figure 14 The effect of antioxidant selenium-enriched peptides on the content of adenosine triphosphate (ATP) in the mitochondria of PC12 cells induced by H2O2. Figure 15 To investigate the effect of antioxidant selenium-enriched peptides on the content of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the mitochondria of H2O2-induced PC12 cells; Figure 16 The effect of antioxidant selenium-enriched peptides on H2O2-induced mitochondrial DNA (mtDNA) copy number in PC12 cells. Detailed Implementation

[0019] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0021] The tuna blood used in this invention was obtained from the heart of fresh yellowfin tuna after dissection and stored at -20°C for later use. Trypsin, neutral protease, papain, pepsin, and alkaline protease were purchased from Shanghai Yuanye Biotechnology Co., Ltd. DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) was purchased from Shanghai Maclean Biotechnology Co., Ltd. Total antioxidant capacity (ABTS method) and ferric ion reducing capacity (FRAP method) test kits were purchased from Nanjing Jiancheng Biotechnology Institute. DMEM high glucose culture medium was purchased from Sewell Biotechnology Co., Ltd. Fetal bovine serum was purchased from Wuhan Pronosei Life Science Co., Ltd. MTT, LDH, and GSH test kits were purchased from Nanjing Jiancheng Biotechnology Institute. ROS, SOD, MDA, GPx, BCA, and cell mitochondrial isolation kits were purchased from Beyotime Biotechnology Co., Ltd. Mitochondrial membrane potential, ATP content, and 8-OHdG test kits were purchased from Wuhan Elairite Biotechnology Co., Ltd. Cytochrome C test kits were purchased from Wuhan Beinlai Biotechnology Co., Ltd. Cell genomic DNA extraction kits and ultrathin DNA product purification kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0022] The relevant detection methods involved in this invention are as follows: (1) Test method for degree of hydrolysis The degree of hydrolysis of the enzymatic hydrolysate was determined using the OPA method. 3 mL of OPA reagent was added to each test tube. 400 μL of serine standard solution was then added to the test tube, vortexed for 5 s, and the reaction was timed precisely for 2 min. The absorbance (OD) was then measured at 340 nm. stand Add 400 μL of distilled water to the test tube and measure the absorbance (OD) at 340 nm using the same method. blank Finally, 400 μL of the sample solution before and after hydrolysis were taken and added to the test tube, and the absorbance OD at 340 nm was measured using the same method. sample Calculate the degree of hydrolysis of the sample using the following formula: (1) (2) (3) X: Sample mass; P: Protein content in the sample; h0: Degree of hydrolysis before hydrolysis; h1: Degree of hydrolysis after hydrolysis. α, β, and h values ​​of fish protein. tot The values ​​are 1, 0.4, and 8.6, respectively.

[0023] (2) Method for determining DPPH free radical scavenging rate Weigh 2 mg of DPPH powder and add anhydrous ethanol to make up to 50 mL, preparing a 0.10 mmol / L DPPH-anhydrous ethanol working solution. Sample group: 100 μL sample solution + 100 μL working solution; Anhydrous ethanol group: 100 μL sample solution + 100 μL anhydrous ethanol. Control group: 100 μL distilled water + 100 μL working solution. Mix well, let stand at room temperature (25℃) in the dark for 30 min, and then measure the absorbance value A of each tube at a wavelength of 517 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the clearance rate according to formula (4): (4) (3) ABTS + Methods for determining free radical scavenging rate Prepare ABTS working and application solutions: Sample group: 10 µL sample solution + 20 µL application solution + 170 µL ABTS working solution; Blank group: 10 µL distilled water + 20 µL application solution + 170 µL ABTS working solution. Mix well and incubate at room temperature for 6 min. Measure the absorbance (A) of each tube using a microplate reader at 405 nm. + The free radical scavenging rate is calculated according to formula (5): (5) (4) Method for determining ferric ion reducing power (FRAP) Prepare FRAP working solution and standard solutions at concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mM according to the kit instructions; Sample group: 5 µL sample solution + 180 µL FRAP working solution; Blank group: 5 µL distilled water + 180 µL FRAP working solution; Standard group: 5 µL standard solutions of different concentrations + 180 µL FRAP working solution. Mix well and incubate at 37°C for 3-5 min. Measure the absorbance (A) of each tube using a microplate reader at a wavelength of 593 nm. Subtract the blank (A) from each group, and plot a standard curve with A (standard) as the x-axis and the corresponding standard concentrations as the y-axis. The curve formula is then obtained. 样品 Subtract A 空白 Substitute the curve formula into the equation and obtain the result.

[0024] (5) Methods for determining selenium content The selenium content in the sample was determined by inductively coupled plasma mass spectrometry (ICP-MS) in accordance with GB65009.268.

[0025] Example 1 (1) Raw material pretreatment: Tuna blood was dialyzed at 4°C with a phosphate buffer solution of 0.02 mol / L and pH 7 for 24 h. The dialyzed blood was pre-frozen in a freezer at -80°C for 24 h and then freeze-dried under vacuum to produce tuna blood freeze-dried powder. (2) Mix the tuna blood freeze-dried powder from step (1) with distilled water at a mass ratio of 1:4, prepare 5 portions, add 6000 U / g (tuna blood freeze-dried powder) of trypsin, neutral protease, papain, pepsin and alkaline protease respectively, and enzymatically hydrolyze for 3 h under their optimal enzyme activity reaction conditions. The enzyme hydrolysis conditions are shown in Table 1. After the enzymatic hydrolysis is completed, quickly boil water to inactivate the enzyme for 10 min. The optimal protease is screened with the degree of hydrolysis and antioxidant activity as the main indicators. Table 1. Enzymatic hydrolysis conditions for proteases The degree of hydrolysis, gel electrophoresis, and antioxidant activity of the five enzymatic hydrolysates obtained in step (2) were tested, and the results are as follows: Figure 1 As shown in Figure 1A, the results of the hydrolysis degree test show that the hydrolysis degree of tuna blood protein hydrolysate prepared by neutral protease is the highest, followed by papain, pepsin, alkaline protease, and pepsin. Figure 1B shows the results of SDS-PAGE gel electrophoresis, which shows that the major protein bands in the neutral protease hydrolysate have disappeared. Figure 1C shows the results of the antioxidant activity assay, which shows that the neutral protease has the highest antioxidant activity. Neutral protease hydrolysates will be screened for further separation and purification.

[0026] (3) The enzymatic hydrolysate obtained from the neutral protease hydrolysis in step (1) was separated into three components with different molecular weights using 3 kDa and 10 kDa ultrafiltration membranes, namely component 1 (<3 kDa), component 2 (3-10 kDa), and component 3 (>10 kDa). These components were freeze-dried, and their antioxidant activities were measured. The results are as follows: Figure 2 As shown; by Figure 2 It can be seen that, compared with components 2 and 3, the ultrafiltration fraction of component 1 (molecular weight <3 kDa) has the highest antioxidant activity, so component 1 was selected for the next purification step. (4) Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used to separate component 1 into eight purified fractions: F1, F2, F3, F4, F5, F6, F7, and F8. The chromatographic column was an Agilent Prep-C18 (20×250 mm, 10 μm). Mobile phase A was deionized water, and mobile phase B was acetonitrile. The elution program (gradient elution) was as follows: 0-2 min, 4-8% mobile phase B; 2-45 min, 8-28% mobile phase B; 45-55 min, 28-40% mobile phase B; 55-56 min, 40-95% mobile phase B; 56-66 min, 95% mobile phase B. The fractions of each preparative RPI were collected, freeze-dried, and their antioxidant activity was determined at the same concentration (1 mg / mL). The fraction with the strongest antioxidant activity was collected and freeze-dried to obtain the fraction containing the target active selenopeptide. Figure 3 The graph shows the purification and antioxidant activity test results of different purified components by reversed-phase high-performance liquid chromatography. Figure 3 A is the elution curve. Figure 3 Figure B shows the results of the antioxidant activity assay, which indicates that purified component 5 (F5) has the strongest antioxidant activity.

[0027] Example 2 Amino acid sequence analysis of F5 component (1) Amino acid sequence search analysis was performed using LC-MS / MS technology. Secondary mass spectrometry analysis confirmed the presence of the target selenopeptide. The total ion current chromatogram (TIC) of F5 is shown below. Figure 4 As shown, the secondary mass spectrum is as follows: Figure 5 As shown, a tuna blood antioxidant selenium-enriched peptide was obtained, with the amino acid sequence (SEQ ID NO.1) being ALHCPDALKGNKP (Ala-Leu-His-(Se)Cys-Pro-Asp-Ala-Leu-Lys-Gly-Asn-Lys-Pro), where the sulfur element in the fourth amino acid cysteine ​​(Cys) was replaced by selenium; (2) The tuna blood antioxidant selenium-enriched peptide in step (1) was synthesized by chemical synthesis method. The purity was greater than 95% by high performance liquid chromatography analysis. It was stored at -20℃ and became an antioxidant selenium-enriched peptide with neuroprotective effect.

[0028] Example 3 Protective effect of antioxidant selenium-enriched peptides with neuroprotective properties against H2O2-induced oxidative damage in PC12 cells (1) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on PC12 cell viability With 1.2×10 5PC12 cells were seeded into 96-well plates at a density of 100 μL per well and incubated at 37°C for 24 h in a 5% CO2 incubator until the cells were fully adhered. The old culture medium was then discarded. The experiment was divided into a control group and an experimental group (with 6 concentration groups). The control group was given 100 μL of DMEM medium. The experimental groups were given 100 μL of DMEM medium containing a series of concentrations of 50, 100, 200, 400, 800, and 1000 μmol / L of antioxidant selenium-enriched peptides with neuroprotective effects (10 replicates per group). The cells were cultured for 24 h. Cell viability was determined using the MTT assay (200 μL of culture medium containing 0.5% MTT was added to each well, cultured for 4 h, the culture medium was discarded, 150 μL of DMSO was added to each well, the plate was shaken for 10 min, and the OD value was measured at 490 nm using a microplate reader). The concentration of antioxidant selenium-enriched peptides with neuroprotective effects and a cell viability greater than 90% was selected as the experimental drug concentration.

[0029] Figure 6 The figure shows the effect of antioxidant selenium-enriched peptides with neuroprotective effects on the proliferation of PC12 cells. The results show that different concentrations of antioxidant selenium-enriched peptides with neuroprotective effects have no toxic effect on cells, and that antioxidant selenium-enriched peptides with neuroprotective effects at concentrations of 100, 200, 400, 800, and 1000 μmol / L have a promoting effect on the proliferation of PC12 cells, with the best proliferation effect at a concentration of 400 μmol / L. 100, 200, and 400 μmol / L were selected as the concentrations for subsequent experiments.

[0030] (2) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced PC12 cell viability With 1.2×10 5PC12 cells were seeded into 96-well plates at a density of 100 μL per well and incubated at 37°C for 24 h in a 5% CO2 incubator until complete cell adhesion. The old culture medium was then discarded. The experiment consisted of 5 groups (1 Control group, 1 Model group, and 3 experimental groups (SeP-100, SeP-200, and SeP-400)), with 10 replicates per group. The Control group was cultured in DMEM medium. The Model group was cultured in DMEM medium for 24 h, then the old medium was discarded, and 800 μmol / L H2O2 solution was added for stimulation for 2 h. The experimental groups were cultured in DMEM medium containing 100 (SeP-100), 200 (SeP-200), and 400 (SeP-400) μmol / L of antioxidant selenium-enriched peptides with neuroprotective effects for 24 h, then the old medium was discarded, and 800 μmol / L H2O2 solution was added for stimulation for 2 h. h; Cell viability was determined using the MTT assay (200 μL of culture medium containing 0.5% MTT was added to each well, and after culturing for 4 h, the culture medium was aspirated, 150 μL of DMSO was added to each well, and the plate was shaken for 10 min. The OD value was then measured at 490 nm using a microplate reader).

[0031] Figure 7 The figure shows the effect of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced PC12 cell viability. The results show that different concentrations of antioxidant selenium-enriched peptides with neuroprotective effects can improve oxidative damage in PC12 cells to a certain extent, and the protective effect on PC12 cells gradually increases with the increase of the concentration of antioxidant selenium-enriched peptides with neuroprotective effects.

[0032] (3) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced PC12 cell morphology Following the method described in (2) above, PC12 cells were seeded, grouped, and cultured. After stimulation with DMEM medium containing 800 μmol / L H2O2 for 2 h, the 96-well plates were removed and placed under a microscope to observe the cell morphology of each group and take pictures.

[0033] Figure 8This figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on H2O2-induced PC12 cell morphology. The results indicate that pretreatment with different concentrations of neuroprotective antioxidant selenium-enriched peptides significantly improved cell damage, restored cell morphology, increased cell number, reduced intercellular spaces, and increased protrusions. Most cells remained adherent, significantly reversing H2O2-induced PC12 cell morphological damage. The figure clearly shows that the 400 μmol / L (SeP-400) group exhibited the best cell condition, with the smallest difference in cell number and density compared to the Control group. Cells in this group were predominantly spindle-shaped and elongated with slender protrusions, and showed tight intercellular connections. This indicates that the neuroprotective antioxidant selenium-enriched peptides can effectively reduce oxidative damage to PC12 cells, with the best effect observed at a concentration of 400 μmol / L.

[0034] (4) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced intracellular ROS generation in PC12 cells With 2×10 5 PC12 cells were seeded into 6-well plates at a density of 2 mL per well. After cell adhesion, grouping, and culture (cell grouping and culture method, and H2O2 induction were the same as in (2)), the culture medium was discarded. DCFH-DA was diluted with serum-free culture medium at a ratio of 1:1000 (final concentration of 10 μmol / L) and added to the well plates. The plates were then incubated at 37°C for 40 min. After incubation, the culture medium was aspirated and the cells were washed 3 times with serum-free culture medium to remove any DCFH-DA that had not entered the cells. The cells were then digested and collected and analyzed using a flow cytometer.

[0035] Figure 9 The figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on H2O2-induced intracellular ROS content in PC12 cells. 9A is the ROS flow cytometry histogram, and 9B is the ROS mean fluorescence intensity bar chart. The results showed that the ROS content in PC12 cells treated with H2O2 (Model group) was significantly higher than that in the Control group (P<0.05). Adding different concentrations of neuroprotective antioxidant selenium-enriched peptides could effectively inhibit intracellular ROS generation. Among them, the 200 μmol / L neuroprotective antioxidant selenium-enriched peptide (SeP-200) group showed the best inhibitory effect, with its ROS generation only 50% of that in the Model group. This is likely related to the antioxidant effect of selenium in the neuroprotective antioxidant selenium-enriched peptides, which can neutralize excess oxygen free radicals in cells, reduce oxidative stress, and thus help protect cells from free radical damage.

[0036] (5) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced oxidative stress indices in PC12 cells With 1.5×10 5 The density of cells / mL was determined by seeding PC12 cells into 12-well plates, 1 mL per well. After the cells adhered, the medium was replaced with phenol red-free DMEM. After the cells adhered, were grouped, and cultured (the cell grouping and culture method, as well as H2O2 induction, were the same as in (2)), the cell culture medium was collected, centrifuged at 4000 rpm for 5 min, and the supernatant was taken and measured using an LDH kit. After washing the cells twice with 1 mL PBS, a certain amount of lysis buffer was added directly to the well plate, and the cells were placed on ice for 10 min to lyse. The cells were scraped off with a cell scraper and aspirated, and then vortexed for another 30 min to continue lysis. After centrifugation at 15,000 rpm for 15 min at 4 °C, the supernatant was collected for analysis. The protein content in each group was detected using the BCA protein kit, and the relevant indicators were measured using the MDA, SOD, GPx and GSH kits.

[0037] Figure 10 This figure shows the effect of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced oxidative stress indices in PC12 cells. 10A represents LDH activity, 10B represents MDA content, 10C represents SOD content, 10D represents GPx activity, and 10E represents GSH content. Figure 10 It was found that pretreatment with antioxidant selenium-enriched peptides, which have neuroprotective effects, can reduce MDA content and LDH release, and increase GSH content and SOD and GPx enzyme activities. Specifically, the SOD activity in the SeP-100 and SeP-200 groups was not significantly different from the Control group, while the SOD activity in the SeP-400 group was significantly higher than that in the Control group. Similarly, the GPx activity in the SeP-200 group was not significantly different from that in the Control group, while the GPx activity in the SeP-100 and SeP-400 groups was significantly better than that in the Control group. This indicates that antioxidant selenium-enriched peptides with neuroprotective effects can slow down lipid peroxidation, effectively protect cell membrane integrity, and their outstanding antioxidant properties can neutralize excess oxygen free radicals in cells and increase the activity of antioxidant enzymes to protect neurons.

[0038] (6) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced MMP in PC12 cells Experiments were conducted using antioxidant selenium-enriched peptides with neuroprotective effects at concentrations of 200 and 400 μmol / L. PC12 cells were seeded in 6-well plates and allowed to adhere completely before the old culture medium was discarded. The experiment was divided into 4 groups (Control group 1, Model group 1, and Experimental groups 2 (SeP-200 and SeP-400)), with 10 replicates per group. DMEM medium was added to the Control group. After adding DMEM medium to the Model group for 24 h, the old culture medium was discarded, and 800 μmol / L H2O2 solution was added for stimulation for 2 h. The experimental groups were added to DMEM medium containing 200 (SeP-200) and 400 (SeP-400) μmol / L of antioxidant selenium-rich peptides with neuroprotective effects for 24 h, respectively. After discarding the old culture medium, DMEM medium containing 800 μmol / L H2O2 was added for stimulation for 2 h. The cells were centrifuged at 1000 rpm for 5 min at room temperature, the supernatant was removed, and 500 μL of 1×JC-1 was added for resuspending. The cells were incubated at 37°C for 30 min. After incubation, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were washed once with 1×JC-1 Assay Buffer. The cells were then resuspended in 500 mL of 1×Assay Buffer. The relative ratio of red and green fluorescence in the cells was measured by flow cytometry to assess the proportion of mitochondrial depolarization.

[0039] Figure 11 The figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on H2O2-induced MMP in PC12 cells. Figure 11A shows the MMP results from flow cytometry, and Figure 11B shows the JC-1 red / green fluorescence ratio results. The results show that after H2O2 treatment, the MMP in PC12 cells in the Model group significantly decreased (P<0.05), and the red / green fluorescence ratio was 5.51% of that in the Control group. Adding 200 and 400 μmol / L concentrations of neuroprotective antioxidant selenium-enriched peptides effectively increased the MMP in oxidatively damaged cells, by 3.42 and 4.13 times, respectively, compared to the Model group. This indicates that the antioxidant effect of neuroprotective antioxidant selenium-enriched peptides can protect the membrane integrity of damaged cells' mitochondria and the electron transport function of the respiratory chain, thereby maintaining membrane potential homeostasis.

[0040] (7) Effect of antioxidant selenium-enriched peptides with neuroprotective effects on the concentration of Cyt-C in the cytoplasm of H2O2-induced PC12 cells PC12 cells were loaded at 5 × 10 5Cells were seeded at a density of 6 mL per well in 100 mm culture dishes. After cell adhesion, grouping, and culture (cell grouping and culture methods and H2O2 induction methods were the same as in (6)), the culture medium was discarded, and the cells were digested and collected. The cytoplasm and mitochondria were separated using a cell-mitochondrial separation kit. The Cyt-C concentration in the cytoplasm was detected using a Cyt-C enzyme-linked immunosorbent assay kit.

[0041] Figure 12 The figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on the concentration of Cyt-C in the cytoplasm of H2O2-induced PC12 cells. The results showed that the concentration of Cyt-C in the cytoplasm of the Model group cells induced by H2O2 was significantly increased (P<0.05), which was 2.93 times that of the Control group. Adding different concentrations of neuroprotective antioxidant selenium-enriched peptides reduced the concentration of Cyt-C in the cytoplasm, and the difference was statistically significant compared with the Model group (P<0.05). This indicates that the neuroprotective antioxidant selenium-enriched peptides can improve mitochondrial membrane permeability and inhibit the release of Cyt-C into the cytoplasm.

[0042] (8) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced mitochondrial CⅠ, CⅢ, CⅣ, and CⅤ content in PC12 cells PC12 cells were seeded into 6-well plates. After cell adhesion, grouping, and culture (cell grouping and culture methods and H2O2 induction methods were the same as in (6)), the culture medium was discarded, 1 mL of TrizoL was added for direct digestion, and the cells were pipetted several times to transfer them to 2 mL centrifuge tubes and stored at -80℃ for later testing. The contents of mitochondrial CI (mt-ND5), CⅢ (mt-CYTB), CⅣ (mt-COX2), and CⅤ (mt-ATP6) were detected by real-time quantitative PCR (qRT-PCR). The internal reference gene used was β-actin. Ct The relative expression levels were calculated using the method shown in Table 2, and the primer sequences for each gene are shown in Table 2.

[0043] Table 2 Primer sequences for quantitative PCR detection of mitochondrial CI (mt-ND5), CⅢ (mt-CYTB), CⅣ (mt-COX2), and C Ⅴ (mt-ATP6) levels. Figure 13The figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on the levels of CⅠ, CⅢ, CⅣ, and C Ⅴ in the mitochondria of PC12 cells induced by H₂O₂. The results showed that the levels of CⅠ, CⅢ, CⅣ, and C Ⅴ in the mitochondria of cells treated with H₂O₂ were all significantly downregulated (P<0.05). CⅠ was extremely sensitive to H₂O₂-induced ROS production, therefore the downregulation of CⅠ was the most significant, only 8.9% of the control group; followed by CⅢ and CⅣ, at 18.1% and 25.1% of the control group, respectively; the inhibitory effect of ROS on C Ⅴ was relatively small, at 46.8% of the control group. Therefore, pretreatment with neuroprotective antioxidant selenium-enriched peptides can effectively improve the reduction in respiratory chain complex content, and the improvement effect is best at a concentration of 400 μmol / L (SeP-400). This may be because the neuroprotective antioxidant selenium-enriched peptides can inhibit ROS generation, thereby reducing the damage of ROS to the respiratory chain complex.

[0044] (9) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced mitochondrial ATP content in PC12 cells The ATP content in the mitochondria extracted from (7) was detected using an ATP content kit.

[0045] Figure 14 The effect of antioxidant selenium-enriched peptides on H2O2-induced ATP content in PC12 cell mitochondria is shown in the figure. The results show that compared with the control group, the ATP content in PC12 cell mitochondria in the model group was significantly decreased (P<0.05), only 11.43% of that in the control group. Pretreatment with antioxidant selenium-enriched peptides with neuroprotective effects can effectively increase the ATP content in cell mitochondria. The effect was best at 200 μmol / L (SeP 200), with an ATP content 1.6 times that of the model group. Combined with the results of (6)-(8), the antioxidant selenium-enriched peptides with neuroprotective effects maintain mitochondrial oxidative phosphorylation by increasing the membrane potential gradient generated by the electron transport chain in the mitochondria of oxidatively damaged cells, reducing the release of Cyt-C into the cytoplasm, and increasing the content of mitochondrial respiratory chain complexes I, III, IV, and V, ultimately driving ATP synthesis and promoting cellular energy production.

[0046] (10) Effects of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced 8-OHdG concentration in PC12 cell mitochondria: The concentration of 8-OHdG in the mitochondria extracted from (7) was detected using an 8-OHdG enzyme-linked immunosorbent assay kit.

[0047] Figure 15The figure shows the effect of neuroprotective antioxidant selenium-enriched peptides on H2O2-induced 8-OHdG concentration in PC12 cell mitochondria. The results showed that, compared to the Control group, the 8-OHdG concentration in PC12 cell mitochondria of the Model group was significantly increased (P<0.05), being 2.45 times higher. Adding different concentrations of neuroprotective antioxidant selenium-enriched peptides effectively downregulated the concentration of 8-OHdG in cell mitochondria, with statistically significant differences compared to the Model group (P<0.05). Since excessive ROS attacks mtDNA, leading to strand breaks and altering DNA base structure, the resulting 8-OHdG further disrupts DNA repair mechanisms and DNA-protein cross-linking, indicating that neuroprotective antioxidant selenium-enriched peptides can repair H2O2-induced mtDNA damage in PC12 cells.

[0048] (11) Effect of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced mtDNA copy number in PC12 cells PC12 cells were seeded in 6-well plates. After cell adhesion, grouping, and culture (cell grouping and culture methods and H2O2 induction methods were the same as in (6)), the culture medium was discarded, the cells were digested and collected in 2 mL centrifuge tubes, centrifuged at 1000 rpm for 5 min at 4℃, the supernatant was discarded, and the precipitate was stored at -80℃. The relative contents of mitochondrial DNA and nuclear DNA were detected by qRT-PCR. The Ct values ​​of the ND1 gene encoded by mitochondrial DNA and the HBB gene encoded by nuclear DNA were measured. The relative expression levels were calculated using the method; the primer sequences for detecting ND1 and HBB genes are shown in Table 3.

[0049] Table 3 Primer sequences for detecting ND1 and HBB genes Figure 16The figure shows the effect of antioxidant selenium-enriched peptides with neuroprotective effects on H2O2-induced mtDNA copy number in PC12 cells. The results showed that the mtDNA copy number in PC12 cells induced by H2O2 decreased significantly (P<0.05), approximately 43.6% of that in the Control group. Compared with the Model group, the mtDNA copy number in the experimental group increased significantly (P<0.05). The effect was best at 400 μmol / L (SeP 400), restoring the mtDNA copy number to 70.5%. Increased oxidative stress levels lead to mtDNA damage, which in turn affects mtDNA replication and expression; decreased mtDNA copy number may lead to mitochondrial dysfunction, thereby triggering cellular metabolic disorders and oxidative stress, forming a vicious cycle. This indicates that antioxidant selenium-enriched peptides with neuroprotective effects can effectively repair oxidatively damaged cellular mtDNA and increase mitochondrial numbers.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A selenium-enriched antioxidant peptide with neuroprotective effects, characterized in that, The amino acid sequence of the antioxidant selenium-enriched peptide with neuroprotective effects is shown in SEQ ID NO.1; The sulfur element in the fourth amino acid cysteine ​​(Cys) of the antioxidant selenium-rich peptide with neuroprotective effects is replaced by selenium.

2. A method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect as described in claim 1, characterized in that, The preparation method is either chemical synthesis or separation from tuna blood.

3. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 2, characterized in that, The method for isolating and preparing neuroprotective antioxidant selenium-enriched peptides from tuna blood includes the following steps: (1) Tuna blood was dialyzed with phosphate buffer solution, pre-frozen after dialyzing, and then freeze-dried under vacuum to produce tuna blood freeze-dried powder. (2) Dissolve the tuna blood freeze-dried powder obtained in step (1) in water, add protease for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, perform enzyme inactivation treatment to obtain the hydrolysate. (3) The enzymatic hydrolysate in step (2) is separated, purified and freeze-dried to obtain an antioxidant selenium-rich peptide with neuroprotective effect.

4. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 3, characterized in that, In step (1), the pre-freezing temperature is -70~-80℃ and the pre-freezing time is 24~48 h; in step (1), the molecular weight cutoff of the dialysis bag is 1 kDa, the dialysis temperature is 0~4℃, and the dialysis time is 24~48 h.

5. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 3, characterized in that, In step (2), the mass ratio of tuna blood freeze-dried powder to water is 1:4~5.

6. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 3, characterized in that, The protease mentioned in step (2) is one or more of trypsin, neutral protease, papain, pepsin and alkaline protease; the amount of protease added is 5000~6000 U / g tuna blood freeze-dried powder; the enzyme inactivation method is boiling water for 5~10 min.

7. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 3, characterized in that, The enzymatic hydrolysis in step (2) is performed at a pH of 3-8, a temperature of 45-60℃, and a time of 3-4 hours.

8. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 3, characterized in that, In step (3), ultrafiltration membrane is used for separation, and reversed-phase high-performance liquid chromatography is used for purification.

9. The method for preparing the antioxidant selenium-enriched peptide with neuroprotective effect according to claim 7, characterized in that, The ultrafiltration membrane has a molecular weight of 10 kDa and / or 3 kDa, and the centrifugation conditions for ultrafiltration are 4000 g, 30 min, and 4℃. The chromatographic conditions for reversed-phase high-performance liquid chromatography are as follows: column: Agilent Prep-C18 column, 20 × 250 mm, 10 μm; mobile phase A is water, mobile phase B is acetonitrile, and the elution program is gradient elution: 0-2 min 4-8% mobile phase B, 2-45 min 8-28% mobile phase B, 45-55 min 28-40% mobile phase B, 55-56 min 40-95% mobile phase B, and 56-66 min 95% mobile phase B.

10. The application of the antioxidant selenium-enriched peptide with neuroprotective effect as described in claim 1 in the preparation of neuroprotective products, characterized in that, The neuron protection product described herein is used to treat, prevent, or improve neurodegenerative diseases.

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