CN114031669A - Mytilus coruscus antioxidant active peptide and preparation and application thereof - Google Patents

Mytilus coruscus antioxidant active peptide and preparation and application thereof Download PDF

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CN114031669A
CN114031669A CN202111451315.4A CN202111451315A CN114031669A CN 114031669 A CN114031669 A CN 114031669A CN 202111451315 A CN202111451315 A CN 202111451315A CN 114031669 A CN114031669 A CN 114031669A
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mytilus coruscus
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王玉倩
孙坤来
尹冰洁
胡倩男
陈洁
高世丹
李佳瑶
卢嘉乐
王斌
陈荫
赵玉勤
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Zhejiang Ocean University ZJOU
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Abstract

The invention provides mytilus coruscus antioxidant peptides Gln-Glu-Thr-Tyr and Tyr-Glu-Leu-His-Asp which activate a Keap1/Nrf2 signal channel, and the polypeptides Gln-Glu-Thr-Tyr and Tyr-Glu-Leu-His-Asp can reduce the generation of Reactive Oxygen Species (ROS) in cells to form a protective effect. The compound can play a role in resisting oxidation and protecting vascular endothelial cells by activating a Keap1/Nrf2 signal channel, and can provide candidate medicines for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure and the like.

Description

Mytilus coruscus antioxidant active peptide and preparation and application thereof
Technical Field
The invention relates to the technical field of polypeptides, and in particular relates to mytilus coruscus antioxidant peptide for activating a Keap1/Nrf2 signal channel.
Background
The living body can generate free radicals automatically, and when the free radicals are generated excessively and exceed a certain amount, the living body can cause damage to the human body. Under normal physiological conditions, the living body's antioxidant defense system can use enzymatic and non-enzymatic antioxidants to remove the attack and impact of harmful molecules on the human body. However, an imbalance between free radicals and the endogenous antioxidant defense system, once generated, causes oxidative stress in cells, thereby causing damage to cells, including deoxyribonucleic acid (DNA), proteins, membrane lipids, and the like. Oxidative damage may cause many chronic diseases such as diabetes mellitus, arthritis, heart disease, alzheimer's disease, and cancer. Synthetic antioxidants (t-butylhydroquinone, butylhydroxyanisole, etc.) are effective in controlling oxidation. However, it has the disadvantages of high cost and potential toxicity. In addition to this, antioxidants we ingest through the diet can also reduce the risk of chronic diseases associated with oxidative stress. Therefore, as the demand of high-nutrition and functional foods is continuously increased, people also pay more attention to natural animals and plants, and the enzymatic hydrolysis of active antioxidant peptides from marine animals and plants becomes a focus of attention. .
Disclosure of Invention
The invention takes the Mytilus coruscus with rich resources as the raw material to prepare antioxidant peptides H5 and H1, which play the role of antioxidation and protecting vascular endothelial cells by activating the Keap1/Nrf2 signal channel, and can provide candidate medicines for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure and the like.
The invention provides two Mytilus coruscus antioxidant active peptides H5 and H1 which activate a Keap1/Nrf2 signal path, wherein the amino acid series of H5 is Gln-Glu-Thr-Tyr, and the molecular weight of the amino acid series measured by ESI-MS is 539.54 Da;
the amino acid sequence of the mytilus coruscus antioxidant active peptide H1 is Tyr-Glu-Leu-His-Asp, and the molecular weight is 675.70Da when determined by ESI-MS.
The invention discloses a preparation method of an antioxidant active peptide of mytilus coruscus, which comprises the following steps:
1) pretreating a thick-shell mussel sample:
unfreezing frozen mytilus coruscus in running water at room temperature, drying the surface moisture at low temperature, dissecting and mincing, adding ethyl acetate to soak for 48h, and continuously stirring in the degreasing process; removing ethyl acetate from the degreased sample by vacuum filtration (rotary evaporation recovery), drying and crushing;
2) enzymolysis:
weighing a proper amount of pretreated mytilus coruscus body wall powder, adding deionized water according to a material-liquid ratio of 1:30, adding neutral protease for enzymolysis in 4%, performing enzymolysis for 4h, and adjusting pH with 0.5mol/L NaOH and 0.1mol/L HCL solution to obtain neutral protease (45 ℃, pH 7.0) for enzymolysis;
3) and inactivating the enzymolysis liquid in a water bath at 100 ℃ by adopting a heating enzyme inactivating method. After enzyme deactivation, cooling to room temperature, centrifuging for 10min at 8000r/min, collecting supernatant, and freeze-drying to detect ACE inhibitory activity of the enzymatic hydrolysate;
4) separating and purifying active peptide in the enzymolysis peptide: taking the scavenging rate of free radicals such as DPPH and the like as evaluation indexes, firstly, carrying out fractional separation on the polypeptide by adopting an ultrafiltration technology, then, further separating the polypeptide by adopting QFF ion exchange chromatography, Sephadex G-15 gel filtration chromatography and high performance liquid chromatography, screening out a separation component with the highest antioxidant capacity, and carrying out separation and purification to obtain mytilus coruscus antioxidant peptide H5 Gln-Glu-Thr-Tyr, the peak-off time of 6.484min, mytilus coruscus antioxidant peptide H1 Tyr-Glu-Leu-His-Asp and the peak-off time of 5.970 min.
Preferably, the addition amount of the ethyl acetate in the step 1) is 5 times of the weight of the dissected and minced mytilus coruscus.
Preferably, the ratio of the thick-shell mussel body wall powder to the deionized water in the step 2) is 1: 30.
Preferably, the enzymolysis temperature in the step 2) is 45 ℃, the enzymolysis time is 4h, and the added enzyme amount is 4%.
The invention discloses antioxidant active peptides H5 and H1: the compound has better scavenging activity on radicals such as DPPH, HO, O2, ABTS and the like, can reduce the generation of Reactive Oxygen Species (ROS) in cells, and forms a protective effect; the mechanism is that the release of Nitric Oxide (NO) is promoted, the levels of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), Catalase (CAT) and total antioxidant capacity (T-AOC) are improved, and the levels of Malondialdehyde (MDA) and Lactate Dehydrogenase (LDH) are reduced. Therefore, H5 and H1 both play a role in protecting HUVEC cells from oxidative damage induced by hydrogen peroxide; h5 and H1 play a role in resisting oxidation and protecting vascular endothelial cells by activating a Keap1/Nrf2 signal channel, and can provide candidate medicines for preventing and treating cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, heart failure and the like.
Drawings
FIG. 1 shows the comparison of the capacity of each interception stage of ultrafiltration to scavenge DPPH free radicals in the substrate.
FIG. 2 is a comparison of the OH radical scavenging ability of the zymolyte of each interception subsection of ultrafiltration in the embodiment of the invention.
FIG. 3 is a comparison of the capacity of each interception segment of ultrafiltration to scavenge the ABTS free radicals of the substrate in the example of the present invention.
FIG. 4 is a QFF anion exchange step elution profile of an embodiment of the present invention.
FIG. 5 is a comparison of the DPPH radical scavenging ability of the components after desalting according to the example of the present invention.
FIG. 6 is a comparison of the HO free radical scavenging ability of the components after desalination according to the example of the present invention.
FIG. 7 is a comparison of the ABTS free radical scavenging ability of the components after desalination according to the examples of the present invention.
FIG. 8 is a Sephadex G-15 gel column chromatography elution profile of an embodiment of the present invention.
FIG. 9 is a comparison of the DPPH radical scavenging ability of the components of the examples of the present invention.
FIG. 10 is a comparison of the scavenging ability of the components of the inventive example for HO free radicals.
FIG. 11 is a comparison of the scavenging ability of the components of the present invention against ABTS free radicals.
FIG. 12 is a graph showing the protective effect of different concentrations of H1 and H5 on oxidative damage of HUVEC cells in accordance with the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 13 is a graph showing the effect of varying concentrations of H1 and H5 on SOD levels in HUVEC cells in accordance with an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 14 is a graph showing the effect of varying concentrations of H1 and H5 on the level of MDA in HUVEC cells, according to an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 15 is a graph showing the effect of varying concentrations of H1 and H5 on GSH-PX levels in HUVEC cells in accordance with the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 16 is a graph showing the effect of varying concentrations of H1 and H5 on NO levels in HUVEC cells in accordance with an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 17 is a graph showing the effect of varying concentrations of H1 and H5 on the levels of iNOS (A) and eNOS (B) in HUVEC cells in accordance with an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 18 is a graph showing the effect of varying concentrations of H1 and H5 on CAT levels in HUVEC cells in accordance with an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 19 is a graph showing the effect of varying concentrations of H1 and H5 on LDH levels in HUVEC cells in accordance with an embodiment of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 20 is a graph showing the effect of varying concentrations of H1 and H5 on T-AOC levels in HUVEC cells according to an example of the present invention.
Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group.
FIG. 21 is a DCFH-DA staining protocol of the present invention to determine intracellular ROS levels.
Wherein, # # P<0.01vs Control group; p<0.01,*P<0.05vs model set. A is a blank group; b is H2O2A model group; c is NAC + H2O2Group (d); d is 50 mu M H1+ H2O2Group (d); e is 100 mu M H1+ H2O2Group (d); f is 200 mu M H1+ H2O2Group (d); g is 50 mu M H5+ H2O2Group (d); h is 100 mu M H5+ H2O2Group (d); i is 200 mu M H5+ H2O2Group (d); j is intracellular ROS content.
FIG. 22 shows the protein protection of polypeptides of the embodiments of the invention.
Wherein, # # P<0.01vs Control group; p<0.01,*P<0.05vs model set; a: electropherograms of polypeptide-protected BSA damage; 1: BSA;2:BSA+H2O2;3:BSA+NAC+H2O2;4:BSA+H1(2.5mM)+H2O2;5:BSA+H1(5mM)+H2O2;6:BSA+H1(10mM)+H2O2;7:BSA+H5(2.5mM)+H2O2;8:BSA+H5(5mM)+H2O2;9:BSA+H5(10mM)+H2O2(ii) a B: and analyzing the relative strength of each strip.
FIG. 23 shows the effect of polypeptides H1 and H5 on the Keap1/Nrf2 pathway-associated proteins of HUVEC cells according to an embodiment of the present invention. Wherein, # # P <0.01vs Control group; p <0.01, P <0.05vs model group; a: western blot analysis of Nrf2, HO-1, NQO1, GCLM and Keap 1; b: quantitative analysis of Nrf 2; c: quantitative analysis of HO-1; d: quantitative analysis of NQO 1; e: quantitative analysis of GCLM; f: quantitative analysis of Keap 1.
Detailed Description
The following examples are intended to further illustrate the present invention, but they are not intended to limit or restrict the scope of the invention.
Examples
1) Pretreating a thick-shell mussel sample:
unfreezing frozen mytilus coruscus in running water at room temperature, drying the surface moisture at low temperature, dissecting and mincing, adding ethyl acetate with the weight 5 times of that of the mytilus coruscus, soaking for 48 hours, and continuously stirring in the degreasing process; removing ethyl acetate from the degreased sample by vacuum filtration (rotary evaporation recovery), drying and crushing;
2) enzymolysis:
weighing a proper amount of pretreated mytilus coruscus body wall powder, adding deionized water according to a material-liquid ratio of 1:30, adding neutral protease for enzymolysis in 4%, performing enzymolysis for 4h, and adjusting pH with 0.5mol/L NaOH and 0.1mol/L HCL solution to obtain neutral protease (45 ℃, pH 7.0) for enzymolysis;
3) and inactivating the enzymolysis liquid in a water bath at 100 ℃ by adopting a heating enzyme inactivating method. After enzyme deactivation, cooling to room temperature, centrifuging for 10min at 8000r/min, collecting supernatant, and freeze-drying to detect ACE inhibitory activity of the enzymatic hydrolysate;
4) separating and purifying active peptide in the enzymolysis peptide: taking the scavenging rate of free radicals such as DPPH and the like as evaluation indexes, firstly, carrying out fractionation on polypeptides by adopting an ultrafiltration technology, wherein the ultrafiltration step comprises the following steps: ultrafiltration is carried out on the mytilus coruscus enzymatic hydrolysate by using ultrafiltration membranes with the molecular weight cut-off of 1kDa, 3kDa, 5kDa and 10kDa, M-I (MW < 1kDa), M-II (MW < 1kDa), M-III (MW < 5kDa), M-IV (MW < 5kDa) and M-V (MW > 10kDa), concentration and freeze-drying are carried out. The sample after the 5 components are freeze-dried and the component without ultrafiltration are respectively prepared into 5mg/mL sample solutions, and the free radical scavenging capacity is compared, and the result is shown in figure 1; the HO radical scavenging ability of the different component zymolytes was different, as shown in fig. 2; the ABTS free radical scavenging ability of the zymolytes with different components is different, and the result is shown in figure 3; the results show that: the M-I component (MW < 1kDa) has the best antioxidant activity, so the M-I component is selected for subsequent separation and purification.
QFF ion exchange chromatography: the M-I component is further separated and purified by QFF ion exchange chromatography. The M-I fraction was prepared into a sample solution of 50mg/mL, and after loading, it was eluted with a Tris-HCl gradient, the results are shown in FIG. 4. As can be seen from FIG. 4, the sample was eluted with 5 peaks in steps of Tris-HCl buffer, Tris-HCl (containing 0.05M NaCl) buffer, Tris-HCl (containing 0.1M NaCl) buffer, Tris-HCl (containing 0.25M NaCl) buffer, Tris-HCl (containing 0.5M NaCl) buffer, which were designated as M-I-1, M-I-2, M-I-3, M-I-4, and M-I-5, respectively. And carrying out rotary evaporation concentration on the obtained unimodal component, carrying out dialysis desalting, and then carrying out rotary evaporation and freeze-drying. The sample is prepared into a sample solution of 1mg/mL, and DPPH free radical, HO free radical and ABTS free radical scavenging activities of 5 components are compared for carrying out the next experiment.
Comparing the DPPH free radical scavenging ability of each component polypeptide after dialysis desalting, the result is shown in FIG. 5; the results of comparing their scavenging ability of HO radicals are shown in FIG. 6; the comparison of the scavenging ability of ABTS free radicals is shown in FIG. 7. The results show that: the M-I-5 component has the best antioxidant activity, so the M-I-5 component is selected for the next experiment.
Sephadex G-15 gel filtration chromatography: subjecting the M-I-5 fraction obtained by QFF chromatography to Sephadex G-15 gel filtration chromatography, detecting peak at 214nm wavelength to obtain fractions M-I-5-1 and M-I-5-2, and performing activity determination on the fractions obtained by separation as shown in FIG. 8.
After the two components are freeze-dried, samples are prepared into a sample solution of 1mg/mL, and the DPPH free radical scavenging capacity is compared, and the result is shown in FIG. 9; the results of comparing their scavenging ability of HO radicals are shown in FIG. 10; the comparison of the scavenging ability of ABTS free radicals is shown in FIG. 11. The results show that: the M-I-5-1 component has the best antioxidant activity, so the M-I-5-1 component is selected for the next experiment.
Purifying and sequencing by high performance liquid chromatography: and (3) further separating, purifying and preparing the component with the best antioxidant activity after passing through a Sephadex G-15 chromatographic column by using HPLC, and entrusting the obtained component to a related scientific research detection mechanism for amino acid sequencing. The component M-I-5-1 is subjected to high performance liquid chromatography analysis and sequencing to obtain 2 polypeptides, and the peak emergence time, the amino acid sequence and the molecular weight of the polypeptides are shown in Table 1.
TABLE 1
Figure BDA0003386120050000051
Determination of radical scavenging Activity: with DPPH free radicals, OH free radicals, ABTS free radicals, O2 -Free radical scavenging rate evaluation of free radical scavenging activity of Mytilus coruscus pure peptide, and determination of EC50The results are shown in Table 2.
TABLE 2
Figure BDA0003386120050000061
Four free radical scavenging Activity assays, EC, were performed on 2 polypeptides50The lower the value, the stronger the antioxidant capacity of the polypeptide.
Different concentrations of polypeptide pairs H2O2Protection of induced injury HUVEC cells: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The sample groups were pre-added at concentrations of (50. mu.M, 100. mu.M, 20. mu.M, respectively)0. mu.M) of the polypeptide (H1, H5) on HUVEC cells, followed by addition of H2O2Inducing cell damage. The MTT method is used for determining the cell survival rate of each experimental group, so as to determine the protective effect of the polypeptide with different concentrations on the damage of Human Umbilical Vein Endothelial Cells (HUVEC). As shown in FIG. 12, the cell viability of the model group was significantly different from that of the blank group (P)<0.01) of 48.58 + -1.16%, indicating that the established oxidative damage model is usable. Cell viability for the polypeptide sample groups (H1, H5) increased with concentration compared to the model group, which was for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein the protective effect of H1 and H5 on HUVEC cell injury under the concentration of 50 mu M and 100 mu M is not obvious compared with that of a model group (P)<0.05). But they can significantly increase menses H at a concentration of 200. mu.M2O2Injured HUVEC cell survival (P)<0.01) to improve the cell survival rate from 48.58 +/-1.16% to 60.34 +/-1.87% and 58.47 +/-1.37%, respectively. From the general trend, the protection effect of the polypeptides H1 and H5 on oxidative damage HUVEC cells is dose-dependent.
Determination of superoxide dismutase (SOD) content: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The SOD content in the cells is used for evaluating the protection effect of the polypeptide with different concentrations on the damage of Human Umbilical Vein Endothelial Cells (HUVEC). As a result, as shown in FIG. 13, the SOD content in the model group was significantly reduced (P) as compared with that in the blank group<0.01), which indicates that the oxidative damage model is successfully established. The SOD content in the cells of the polypeptide sample group (H1, H5) increased with the increase of the polypeptide concentration, compared with the model group, indicating that it was on H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly increase the SOD content of HUVEC cells in comparison with the model group at the concentration of 50 mu M, but have no significant difference. The polypeptide H1, H5 can significantly increase SOD content (P) in oxidative damage cells at a concentration of 100 μ M and 200 μ M<0.01). From the general trend, the polypeptides H1 and H5 can increase the oxidative damage in HUVEC cellsThe SOD content of (A) is in a dose-dependent manner.
Malondialdehyde (MDA) content determination: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The MDA content in the cells is used for evaluating the protective effect of the polypeptide with different concentrations on the damage of Human Umbilical Vein Endothelial Cells (HUVEC). As shown in FIG. 14, the MDA content of the model group was significantly increased compared to that of the blank group (P)<0.01), which indicates that the oxidative damage model is successfully established. The MDA content in the cells of the polypeptide sample group (H1, H5) decreased with increasing polypeptide concentration, compared with the model group, indicating that it was for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly reduce the MDA content in HUVEC cells in comparison with a model group at the concentration of 50 mu M, but have no significant difference. Polypeptides H1, H5 significantly reduced MDA levels in oxidatively damaged cells at 100. mu.M, 200. mu.M concentrations (P)<0.01). From a general trend, the polypeptides H1, H5 can reduce the amount of MDA in oxidatively damaged HUVEC cells and are dose-dependent.
Measuring the content of glutathione peroxidase (GSH-PX): 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The content of GSH-PX in the cells is used for evaluating the protective effect of the polypeptides with different concentrations on the damage of Human Umbilical Vein Endothelial Cells (HUVEC). The results are shown in FIG. 15, where the GSH-PX content of the model group is significantly reduced (P) compared to the blank group<0.01), which indicates that the oxidative damage model is successfully established. The GSH-PX content in the cells of the polypeptide sample group (H1, H5) increased with increasing polypeptide concentration compared to the model group, indicating that it was for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 compare GSH-PX content in HUVEC cells with model at 50 μ M concentrationGroups were slightly increased but there was no significant difference. The polypeptides H1 and H5 can obviously improve the GSH-PX content (P) in oxidative damage cells under the concentration of 100 mu M and 200 mu M<0.01). From a general trend, the polypeptides H1, H5 can increase GSH-PX content in oxidatively damaged HUVEC cells, and are dose-dependent.
Determination of Nitric Oxide (NO): 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The protection effect of the polypeptide with different concentrations on Human Umbilical Vein Endothelial Cell (HUVEC) injury is evaluated according to the content of NO in the cells. As a result, as shown in FIG. 16, the NO content in the model group was significantly reduced (P) as compared with that in the blank group<0.01), which indicates that the oxidative damage model is successfully established. The NO content in the cells of the polypeptide sample group (H1, H5) increased with the increase of the polypeptide concentration, compared with the model group, indicating that it is for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly increase the NO content in HUVEC cells compared with the model group at the concentration of 50 mu M, but have NO significant difference. The polypeptides H1 and H5 can obviously improve the NO content (P) in oxidative damage cells under the concentration of 100 mu M and 200 mu M<0.01). From a general trend, the polypeptides H1, H5 can increase the NO content in oxidatively damaged HUVEC cells and are dose-dependent.
Determination of Nitric Oxide Synthase (NOS) content: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The protective effect of the polypeptide with different concentrations on Human Umbilical Vein Endothelial Cell (HUVEC) injury is evaluated according to the iNOS and eNOS content in the cells. As can be seen from FIG. 17(A), the iNOS content in the model group was significantly increased (P) as compared with that in the blank group<0.01), which indicates that the oxidative damage model is successfully established. Polypeptide sample group (H1, H5) cells compared to model groupThe content of iNOS decreases with the increase of the concentration of the polypeptide, which indicates that the polypeptide is H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly reduce the iNOS content in HUVEC cells in comparison with a model group at the concentration of 50 mu M, but have no significant difference. The polypeptides H1 and H5 can obviously reduce the iNOS content (P) in oxidative damage cells under the concentration of 100 mu M and 200 mu M<0.01). From the general trend, the polypeptides H1 and H5 can reduce the iNOS content in oxidative damage HUVEC cells and are dose-dependent. As can be seen from FIG. 17(B), the eNOS content in the model group was significantly reduced (P) compared to the blank group<0.01), which indicates that the oxidative damage model is successfully established. The eNOS content in the cells of the polypeptide sample group (H1, H5) increased with the increase in the polypeptide concentration, compared with the model group, indicating that it was on H2O2The protective effect of injured HUVEC cells was gradually increased. Among them, H1 and H5 slightly increased the eNOS content in HUVEC cells at a concentration of 50. mu.M compared with the model group, but had no significant difference. The polypeptide H1 and H5 can obviously improve the eNOS content (P) in oxidative damage cells under the concentration of 100 mu M and 200 mu M<0.01). From a general trend, the polypeptides H1, H5 can increase eNOS content in oxidatively damaged HUVEC cells and are dose-dependent.
Determination of Catalase (CAT) content: 500 mu M H2H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The protection effect of the polypeptide with different concentrations on Human Umbilical Vein Endothelial Cell (HUVEC) injury is evaluated according to the CAT content in the cells. As a result, as shown in FIG. 18, the CAT content in the model group was significantly reduced (P) as compared with that in the blank group<0.01), which indicates that the oxidative damage model is successfully established. The CAT content in the cells of the polypeptide sample group (H1, H5) increased with the increase in the concentration of the polypeptide, as compared with the model group, indicating that it was responsible for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly increase the CAT content in HUVEC cells compared with a model group at the concentration of 50 mu M, but have no significant difference. The polypeptides H1 and H5 are at 100. mu.M and 200. mu.MObviously improving the CAT content (P) in oxidative damage cells under the concentration<0.01). From a general trend, the polypeptides H1, H5 can increase the CAT content in oxidatively damaged HUVEC cells and are dose-dependent.
Lactate Dehydrogenase (LDH) content determination: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The LDH content in the cells is used for evaluating the protective effect of the polypeptide with different concentrations on the damage of Human Umbilical Vein Endothelial Cells (HUVEC). The results are shown in FIG. 19, where the LDH content in the model group is significantly increased compared to the blank group (P)<0.01), which indicates that the oxidative damage model is successfully established. The LDH content in the cells of the polypeptide sample group (H1, H5) decreased with increasing polypeptide concentration, compared to the model group, indicating that it was for H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1 and H5 slightly reduce the LDH content in HUVEC cells compared with a model group at the concentration of 50 mu M, but have no significant difference. Polypeptides H1, H5 significantly reduced LDH levels (P) in oxidatively damaged cells at a concentration of 100. mu.M, 200. mu.M<0.01). From a general trend, the polypeptides H1, H5 can reduce LDH content in oxidatively damaged HUVEC cells, and are dose-dependent.
Total antioxidant capacity (T-AOC) assay: 500 mu M H is selected2O2HUVEC cell damage is induced, and a model group is established. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. The samples were prepared by adding polypeptides (H1, H5) at concentrations of 50. mu.M, 100. mu.M, 200. mu.M, respectively, to HUVEC cells, and then adding H2O2Inducing cell damage. The protective effect of the polypeptide on Human Umbilical Vein Endothelial Cell (HUVEC) injury at different concentrations is evaluated according to the T-AOC level in the cells. As a result, as shown in FIG. 20, the T-AOC level of the model group was significantly reduced (P) compared to that of the blank group<0.01), which indicates that the oxidative damage model is successfully established. T-AOC levels in cells of the polypeptide sample group (H1, H5) increased with increasing polypeptide concentration compared to the model group, indicating that they are on H2O2The protective effect of injured HUVEC cells was gradually increased. Wherein H1, H5 at a concentration of 50. mu.M resulted in a slight increase, but no significant difference, in T-AOC levels in HUVEC cells compared to the model group. Polypeptides H1, H5 significantly increased T-AOC levels (P) in oxidatively damaged cells at a concentration of 100. mu.M, 200. mu.M<0.01). From a general trend, the polypeptides H1 and H5 can increase the level of T-AOC in oxidative damage HUVEC cells and are dose-dependent.
DCFH-DA staining: at 500 μ M H2O2And (3) inducing HUVEC cell damage and establishing a HUVEC cell oxidative damage model. Acetylcysteine (NAC, 1mM final concentration) was used as a positive control sample group. HUVEC cells were treated with 50. mu.M, 100. mu.M, 200. mu.M polypeptide (H1, H5) followed by addition of H2O2Inducing cell damage. And observing whether the polypeptide has the effect of reducing active oxygen in oxidative damage cells or not through a fluorescence microscope. The results are shown in FIG. 21. After DCFH-DA staining, the ROS content in HUVEC cells was significantly increased in the model group compared to the normal group (P)<0.01), indicating that the established oxidative damage model is available. The polypeptide sample group (H1, H5) increased with the concentration of the polypeptide sample compared to the model group, such that H2O2ROS levels within the injured HUVEC cells gradually decreased. From the results, the mytilus coruscus polypeptides H1 and H5 can reduce the increase of intracellular ROS caused by oxidative damage and are concentration-dependent.
Protein protection: in order to study the protective effect of the polypeptide on the oxidative damage of Bovine Serum Albumin (BSA), hydrogen peroxide and iron (II) ions are used to generate (Fenton reaction) hydroxyl radicals to damage the BSA. The polypeptide was added to the reaction solution and incubated at 37 ℃ for 60 min. After the incubation is finished, the oxidation damage degree of BSA is detected by adopting a Coomassie brilliant blue R-250 staining method. FIG. 22(A) is a SDS-PAGE graph of Bovine Serum Albumin (BSA) treated under different conditions, from which it can be seen that the bands of the model group damaged by the Fenton reaction are significantly lighter than those of the normal group, and the bands are darker than those of the model group after protection by the addition of 2.5mM, 5mM, 10mM polypeptide (H1, H5), are lighter than those of the normal group, and the more significant the change with the increase of the concentration, indicating that the model is more successful. It can also be seen from the relative intensity analysis of the individual bands in fig. 22(B) that the band intensities are significantly reduced in the model group compared to the normal group (P <0.01), indicating that the model constructed is useful. The polypeptide sample groups (H1, H5) showed a gradual increase in band intensity with increasing concentration of the polypeptide sample compared to the model group. From the results, the mytilus coruscus polypeptides H1 and H5 can protect the effect of hydroxyl radical induced damage Bovine Serum Albumin (BSA), and are concentration-dependent.
Western blot detection of Nrf2 and protein expression related thereto:
(1) polyacrylamide gel electrophoresis (SDS-PAGE): and cleaning the glass plate, drying, aligning and clamping. Preparing 10% of separation gel: ultrapure water, 30% acrylamide, 4 xSDS-PAGE gel buffer, 10% ammonium persulfate and TEMED were added in this order according to the formulation. And pouring the glue into the interlayer of the glass plate along the wall of the glass plate immediately after uniformly mixing, adding a layer of ultrapure water into the glue for liquid sealing, and removing the ultrapure water after the separation glue is solidified. Preparing 5% concentrated glue: ultrapure water, 30% acrylamide, 4 xSDS-PAGE gel concentrate buffer, 10% ammonium persulfate and TEMED were added in this order according to the formulation. After being uniformly mixed, the mixture is poured above the separation glue along the wall of the glass plate, a comb is inserted, and no air bubble can be generated in the glue preparation process. And (4) sampling, wherein the voltage is 80V, the concentration glue layer is run, and the voltage is 120V, the separation glue layer is run.
(2) Film transfer: soaking the PVDF membrane in anhydrous methanol, sequentially placing and clamping the sponge, the filter paper, the gel, the PVDF membrane, the filter paper and the sponge according to the specification, and continuously removing bubbles in the operation process. The clip is placed in a transfer chamber to transfer the protein.
(3) And (3) sealing: washing the PVDF membrane with TBST for 3 times and 15 min/time. And (3) sealing the PVDF membrane for 2 hours by a 5% skimmed milk powder shaking table.
(4) Incubating the primary antibody: the membrane was washed 3 times with TBST for 15 min/time, and incubated overnight at 4 ℃ with primary antibody.
(5) Incubation of secondary antibody: washing the membrane for 3 times and 15 min/time by TBST, adding a secondary antibody, and incubating for 2h at room temperature.
(6) ECL chemiluminescence development: the membrane was washed 3 times with TBST and 15 min/time, and the developer was dropped on the PVDF membrane and photographed by developing with a gel imager.
The results are shown in fig. 23, compared with the blank group, the expression level of Keap1 in the model group is increased, the expression level of Nrf2 protein is reduced, the content of downstream antioxidant protein regulated by Nrf2 is further reduced, the expression of downstream proteins HO-1, NQO1 and GCLM is obviously reduced (P is less than 0.01), which indicates that hydrogen peroxide causes damage to HUVEC cells and inhibits the expression of Nrf2 pathway. After the polypeptides H1 and H5 are added, compared with a model group, the Keap1 expression level is reduced, the Nrf2 protein expression level is obviously increased, the expression of downstream proteins HO-1, NQO1 and GCLM is obviously increased (P is less than 0.01), and the concentration dependence is presented, and the results show that the polypeptides H1 and H5 play a protective effect through a Keap1/Nrf2 pathway.
Finally, it should be noted that the above-mentioned list is only one specific embodiment of the present invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Claims (6)

1. The mytilus coruscus antioxidant active peptide H5 is characterized in that the amino acid series of the antioxidant active peptide H5 is Gln-Glu-Thr-Tyr, and the molecular weight of the antioxidant active peptide H5 is 539.54Da when ESI-MS is used for determination.
2. The preparation of the mytilus coruscus antioxidant peptide H5 as claimed in claim 1, comprising the steps of:
1) pretreating a thick-shell mussel sample:
unfreezing frozen mytilus coruscus in running water at room temperature, drying the surface moisture at low temperature, dissecting and mincing, adding ethyl acetate to soak for 48h, and continuously stirring in the degreasing process; removing ethyl acetate from the degreased sample by vacuum filtration (rotary evaporation recovery), drying and crushing;
2) enzymolysis:
weighing a proper amount of pretreated mytilus coruscus body wall powder, adding deionized water according to a material-liquid ratio of 1:30, adding neutral protease for enzymolysis in 4%, performing enzymolysis for 4h, and adjusting pH with 0.5mol/L NaOH and 0.1mol/L HCL solution to obtain neutral protease (45 ℃, pH 7.0) for enzymolysis;
3) and inactivating the enzymolysis liquid in a water bath at 100 ℃ by adopting a heating enzyme inactivating method. After enzyme deactivation, cooling to room temperature, centrifuging for 10min at 8000r/min, collecting supernatant, and freeze-drying to detect ACE inhibitory activity of the enzymatic hydrolysate;
4) separating and purifying active peptide in the enzymolysis peptide: taking the scavenging rate of free radicals such as DPPH and the like as evaluation indexes, firstly, carrying out fractional separation on the polypeptide by adopting an ultrafiltration technology, then, further separating the polypeptide by adopting QFF ion exchange chromatography, Sephadex G-15 gel filtration chromatography and high performance liquid chromatography, screening out a separation component with the highest antioxidant capacity, and carrying out separation and purification to obtain mytilus coruscus antioxidant peptides H5 Gln-Glu-Thr-Tyr and H1 Tyr-Glu-Leu-His-Asp.
3. The preparation of the mytilus coruscus antioxidant peptide H5 as claimed in claim 2, wherein the amount of ethyl acetate added in step 1) is 5 times of the weight of the mytilus coruscus after anatomical mincing.
4. The preparation of the mytilus coruscus antioxidant peptide H5 as claimed in claim 2, wherein the ratio of mytilus coruscus wall powder to deionized water in step 2) is 1: 30.
5. The preparation method of the mytilus coruscus antioxidant peptide H5 as claimed in claim 2, wherein the enzymolysis temperature in step 2) is 45 ℃, the enzymolysis time is 4H, and the amount of the added enzyme is 4%.
6. The use of the mytilus coruscus antioxidant peptide H5 as claimed in claim 1 in the preparation of a drug candidate for the prevention and treatment of cardiovascular and cerebrovascular diseases such as hypertension, coronary heart disease, cerebral thrombosis, myocardial infarction, atherosclerosis, and heart failure.
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