A sea cucumber polypeptide, a preparation method thereof and use thereof in treating atherosclerosis

Abstract

This invention provides a sea cucumber polypeptide, its preparation method, and its use in the treatment of atherosclerosis. The polypeptides with therapeutic and / or preventive functions for atherosclerosis are SCP-1 and SCP-2, derived from sea cucumbers or synthesized artificially. The preparation method includes pretreatment of sea cucumber raw materials, followed by stepwise enzymatic hydrolysis using neutral protease and papain, and purification via enzyme inactivation, centrifugation, nanofiltration desalting, gel filtration chromatography, and preparative high-performance liquid chromatography. The sea cucumber polypeptide can be used to prepare and / or prevent and treat atherosclerosis, and can also be formulated into pharmaceutical compositions containing pharmaceutically acceptable carriers or excipients, providing a new material basis and effective approach for the treatment of atherosclerosis, with broad application prospects in the pharmaceutical, health product, and food fields.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide and biotechnology, and particularly relates to a sea cucumber polypeptide, its preparation method, and its use in the treatment of atherosclerosis. Background Technology

[0002] Atherosclerosis (AS) is a very common cardiovascular disease characterized by the formation of fibrous or atherosclerotic plaques in the vascular endothelium, primarily affecting large and medium-sized arteries. This leads to hardening of the vessel walls, narrowing of the lumen, and decreased elasticity, ultimately causing ischemic changes in the corresponding organs. The incidence of AS continues to rise, commonly affecting middle-aged and elderly individuals, and in recent years showing a trend towards affecting younger people. Its complications are extremely dangerous. For example, severe aortic atherosclerosis can lead to aortic aneurysms, even rupturing and bleeding, which can cause rapid shock and death. Coronary atherosclerosis can cause angina, myocardial infarction, heart failure, sudden cardiac death, and other hemodynamic disorders. Carotid and cerebral artery atherosclerosis can trigger transient ischemic attacks (TIAs), cerebral infarction, and cerebral hemorrhage. Renal artery atherosclerosis can affect kidney function, leading to serious consequences such as renal atrophy and refractory hypertension. However, current treatments for atherosclerosis have many limitations. Existing treatments, such as thermophysical therapy, can cause serious damage to the vascular endothelium while killing diseased tissue, leading to insufficient long-term vascular patency and frequent restenosis.

[0003] Sea cucumber, a marine organism rich in various bioactive components, has attracted considerable attention for its sea cucumber peptides. Current technologies primarily employ enzymatic hydrolysis and other methods to prepare sea cucumber peptides. Hydrolysis typically uses acids or alkalis to hydrolyze proteins; this method is violent and requires sophisticated equipment. Enzymatic hydrolysis, due to its mild, safe, and easily controllable reaction conditions, has become a commonly used method for preparing sea cucumber peptides. Furthermore, by selecting the type of enzyme, controlling the reaction time, enzyme dosage, and substrate concentration, specific physiologically active peptides can be obtained. Currently, there are few reports on the active peptides and mechanisms of action of sea cucumber peptides; most studies focus on functional verification of peptide mixtures. Such mixtures with unclear composition are not conducive to the application and promotion of sea cucumber active peptides. Although existing research has shown that sea cucumber peptides have certain effects in anti-fatigue, enhancing physical strength, improving immunity, preventing cell mutation, improving internal circulation, regulating blood pressure, delaying aging, and beautifying the skin, research on the application of sea cucumber peptides in the treatment of atherosclerosis still needs further in-depth study and refinement. How to prepare sea cucumber polypeptides with high purity, high yield and effective treatment of atherosclerosis has become an urgent problem to be solved. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a sea cucumber polypeptide, its preparation method, and its use in the treatment of atherosclerosis. Specifically, by extracting polypeptides with therapeutic effects on atherosclerosis from sea cucumbers, and scientifically utilizing the high-quality proteins in sea cucumbers, this invention can provide important support for the development of functional sea cucumber protein products and food-derived polypeptides, and expand the application of sea cucumbers in the pharmaceutical field.

[0005] To address this, the present invention adopts the following technical solution:

[0006] This invention provides polypeptides SCP-1 and SCP-2, which have therapeutic functions for atherosclerosis.

[0007] Furthermore, the amino acid sequences of the peptides are SCP-1 of FAGITQSGR (SEQ ID NO.1) and SCP-2 of LPVGQDIGMG (SEQ ID NO.2), respectively.

[0008] Furthermore, the polypeptide is derived from sea cucumber or is artificially synthesized.

[0009] The present invention also provides a method for preparing the polypeptide, comprising the following steps:

[0010] (1) Raw material pretreatment: Take fresh sea cucumbers, remove the internal organs, wash them three times with sterile water, cut them into small pieces and freeze-dry them, grind them into powder, add sodium acetate buffer solution with pH 7.0 according to the material-liquid ratio of 1:2-10, soak them with ultrasonic assistance, filter and discard the filtrate.

[0011] (2) Two-enzyme stepwise hydrolysis: First, adjust the pH of the system to 9.0 for the first hydrolysis, and after the first hydrolysis, adjust the pH to 5.0 for the second hydrolysis to obtain the hydrolysis product;

[0012] (3) Enzyme inactivation: Inactivate the enzymes by bathing the enzymatic hydrolysis products in water;

[0013] (4) Centrifugal filtration: After cooling to room temperature, centrifuge to collect the supernatant, and then filter it through a 0.45μm microporous membrane;

[0014] (5) Nanofiltration desalination: The filtrate is desalted using a nanofiltration membrane with a molecular weight cutoff of 1000 Da. The process is repeated 2-5 times, and the filtrate is collected.

[0015] (6) Gel filtration chromatography: Load the filtrate onto a Sephacryl S-200 HR gel filtration chromatography column, use deionized water as the eluent, and a flow rate of 0.5 mL / min. Collect the eluent.

[0016] (7) Purification: The collected elution fraction was purified by preparative high performance liquid chromatography. Mobile phase A was a mixture of 99.9% water and 0.1% formic acid, and mobile phase B was an acetonitrile solution of 0.05% trifluoroacetic acid. The elution program was as follows: 0-12 min, 8-12% B; 12-28 min, 12-25% B; 28-38 min, 25-35% B; 38-45 min, 35-48% B; flow rate 1.2 mL / min, detection wavelength 218 nm. The elution peaks were collected, and the amino acid sequences of the elution peaks were identified by LC-TOF-MS to obtain a polypeptide with the amino acid sequence SEQ ID NO.1-2.

[0017] Furthermore, the enzyme added in the first enzymatic hydrolysis is a neutral protease, the amount of enzyme added is 3200 U / g, the enzymatic hydrolysis temperature is 30-40℃, and the enzymatic hydrolysis time is 2-8 h; preferably, the enzymatic hydrolysis temperature is 35℃ and the enzymatic hydrolysis time is 5 h.

[0018] Furthermore, the enzyme added in the second enzymatic hydrolysis is papain, with an enzyme addition amount of 3800 U / g, an enzymatic hydrolysis temperature of 35-45℃, and an enzymatic hydrolysis time of 2-8 hours; preferably, the enzymatic hydrolysis temperature is 40℃, and the enzymatic hydrolysis time is 3 hours.

[0019] The present invention also provides polypeptides prepared by the preparation method described above.

[0020] The present invention also provides the use of the described polypeptide in the preparation of drugs for treating and / or preventing atherosclerosis.

[0021] The present invention also provides the use of the described polypeptide in the preparation of medicaments for the treatment and / or prevention of cardiovascular diseases.

[0022] The present invention also provides a pharmaceutical composition of the aforementioned polypeptide.

[0023] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0024] The present invention also provides a medicine containing the said polypeptide.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] This invention utilizes an environmentally friendly enzymatic hydrolysis method to extract and prepare sea cucumber polypeptides with therapeutic effects on atherosclerosis. The obtained polypeptides are SCP-1 with the amino acid sequence FAGITQSGR and SCP-2 with the amino acid sequence LPVGQDIGMG. The preparation process of this invention is scientifically sound, simple to operate, and highly industrially feasible. The polypeptide sequences are well-defined and have a small number of amino acid residues, allowing for convenient large-scale artificial synthesis. It exhibits no toxic side effects on human cells and demonstrates good safety. The sea cucumber polypeptides of this invention can promote autophagy in macrophages, thereby promoting cholesterol efflux and inhibiting foam cell formation through the autophagy-lysosome system. This significantly improves plaque formation in atherosclerotic vessels. Furthermore, it can significantly improve the concentrations of TG, TC, LDL-C, and HDL-C in serum and downregulate the expression of TLR4 and NF-κB proteins in the TLR4 / NF-κB signaling pathway. All these results indicate that the sea cucumber polypeptides of this invention can prevent and treat atherosclerosis and have a good effect on improving cardiovascular diseases, showing broad market prospects in many fields such as medicine. Attached Figure Description

[0027] Figure 1 SCP-1 structural diagram;

[0028] Figure 2 SCP-2 structural diagram;

[0029] Figure 3 Effects of sea cucumber polypeptides on the survival rate of RAW264.7 macrophages;

[0030] Figure 4 RAW264.7 macrophage LC3II / LC3I ratio;

[0031] Figure 5 The effect of sea cucumber polypeptides on the proportion of carotid artery plaque area in rabbits from Southwest China;

[0032] Figure 6 Results of detection of lipid factors TG, TC, LDL-C, and HDL-C in rabbits: A represents serum total cholesterol (TC), B represents serum triglyceride (TG) concentration, C represents low-density lipoprotein cholesterol (LDL-C) concentration, and D represents high-density lipoprotein cholesterol (HDL-C) concentration (compared to the control group). # p < 0.05 ## p<0.01; compared with the model group, * p < 0.05 ** p<0.01);

[0033] Figure 7Results of TLR4 and NF-κB protein expression detection in rabbit carotid arteries: A shows electrophoresis images of the control group, model group, SCP-1 group, and SCP-2 group; B shows the relative expression levels of TLR4 and NF-κB proteins (compared to the control group). # p < 0.05 ## p<0.01; compared with the model group, * p < 0.05 ** p<0.01). Detailed Implementation

[0034] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0035] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0036] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0037] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0038] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.

[0039] Statistical analysis: Data were analyzed using SPSS 20.0 software. Mean ± SD is expressed as mean ± SD. One-way ANOVA was used to analyze differences among multiple groups. Dunnett's t-test was used for comparisons between groups. p < 0.05 and p < 0.01 were considered statistically significant.

[0040] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0041] Example 1

[0042] A method for preparing a polypeptide with therapeutic function for atherosclerosis includes the following steps:

[0043] (1) Raw material pretreatment: Take 800g of fresh sea cucumber (purchased from Wuxi Sunan Agricultural and Sideline Products and Aquatic Products City, address: No. 517, Datong Road, Binhu District, Wuxi City, Jiangsu Province), remove the internal organs, wash three times with sterile water at 6℃, cut into small pieces and freeze-dry, grind into powder, add sodium acetate buffer solution with pH 7.0 at a material-to-liquid ratio of 1:6, soak for 2 hours with ultrasonic assistance, filter and discard the filtrate;

[0044] (2) Stepwise enzymatic hydrolysis with two enzymes: First, adjust the pH of the system to 9.0 for the first enzymatic hydrolysis. The enzyme added for the first enzymatic hydrolysis is a neutral protease, with an enzyme addition amount of 3200 U / g, an enzymatic hydrolysis temperature of 35℃, and an enzymatic hydrolysis time of 5 h. After the hydrolysis, adjust the pH to 5.0 for the second enzymatic hydrolysis. The enzyme added for the second enzymatic hydrolysis is papain, with an enzyme addition amount of 3800 U / g, an enzymatic hydrolysis temperature of 40℃, and an enzymatic hydrolysis time of 3 h to obtain the enzymatic hydrolysis product.

[0045] (3) Enzyme inactivation: The enzyme hydrolysis product is placed in a 98℃ water bath and heated for 10 min to inactivate the enzyme;

[0046] (4) Centrifugal filtration: After cooling to room temperature, centrifuge at 4500 r / min for 15 min, take the supernatant, and then filter it through a 0.45 μm microporous membrane;

[0047] (5) Nanofiltration desalination: The filtrate is desalted using a nanofiltration membrane with a molecular weight cutoff of 1000 Da. The process is repeated 4 times, and the filtrate is collected.

[0048] (6) Gel filtration chromatography: Load the filtrate onto a Sephacryl S-200 HR gel filtration chromatography column, use deionized water as the eluent, and a flow rate of 0.5 mL / min. Collect the eluent.

[0049] (7) Purification: The collected elution fraction was purified by preparative high performance liquid chromatography using a C18 reversed-phase analytical column (50 mm × 15 cm × 3 μm). Mobile phase A was a mixture of 99.9% water and 0.1% formic acid, and mobile phase B was an acetonitrile solution of 0.05% trifluoroacetic acid. The elution program was as follows: 0-12 min, 8-12% B; 12-28 min, 12-25% B; 28-38 min, 25-35% B; 38-45 min, 35-48% B. The flow rate was 1.2 mL / min, and the detection wavelength was 218 nm. The elution peaks were collected and separated and identified by LC-TOF-MS.

[0050] (8) Sequence identification: ESI+ mode, using data-dependent scanning mode, full scan acquisition was performed in an orbital trap with a resolution of 70,000 (m / z 200-1600). The first 8 peptide signal precursor ions (charge state ≥ +1) were broken up by high-energy collision (HCD), with a normalized collision energy (NCE) of 28.0. The capillary temperature was 275℃, and the spray voltage was 1900V. Daughter ions were measured in orbitals with a resolution of 17,500. The maximum fill time for full scan and MS-MS scan was set to 50ms and 50ms, respectively, and the dynamic exclusion time was set to 30s.

[0051] LC-TOF-MS analysis revealed the active peptide structure to be FAGITQSGR (SCP-1, SEQ ID NO: 1, molecular weight 936.02 Da, isoelectric point 10.386, structural diagram shown). Figure 1 ) and LPVGQDIGMG (SCP-2, SEQ ID NO:2, molecular weight 985.49 Da, isoelectric point 3.539, structural diagram see Figure 2 ).

[0052] Based on the amino acid sequences of the identified active peptides, they can be prepared by enzymatic hydrolysis and separation from sea cucumbers, or by chemical synthesis. Regardless of the preparation method, the peptide sequence composition remains unchanged. The active sea cucumber peptides SCP-1 and SCP-2 of this invention were synthesized by Shanghai Jier Biochemical Co., Ltd. for future use, with a purity of over 98%.

[0053] Example 2

[0054] RAW264.7 macrophages (purchased from Bio-Tech (Guangzhou) Co., Ltd., product model: BH-C415) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin antibiotics in a 37°C, 5% CO2 incubator. The macrophages were divided into four groups: control group, SCP-1 group, SCP-2 group, and SCP-1 / SP-2 group. The SCP-1, SCP-2, and SCP-1 / SP-2 groups (SCP-1 to SCP-2 mass ratio 1:1) were treated overnight with a 10 μg / L sea cucumber polypeptide aqueous solution for 8 h. After 8 h, cell viability was determined using the Cell Counting Kit-8 (CCK-8) kit (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., product code: 40203ES76), following the kit instructions. A cell viability greater than 98% in each experimental group indicated that the sea cucumber polypeptide was non-toxic. Cell viability is shown in the table below. Figure 3 The absorbance (OD value) of each well was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader. Cell viability was calculated based on the mean absorbance of each experimental group using the following formula:

[0055] .

[0056] Total cellular protein was extracted, and the levels of autophagy markers LC3I and LC3II were determined by immunoblotting. LC3 initially exists in the cytoplasm as LC3I; its C-terminus is processed and binds to phosphatidylethanolamine to form LC3II. LC3II can specifically localize to the autophagosome membrane and is an important marker of autophagosome formation. As a major cellular component in the development and progression of arterial plaques, macrophages can promote cholesterol efflux through the autophagy-lysosome system, thereby inhibiting foam cell formation. Simultaneously, they can inhibit inflammasome activation and reduce IL-1β secretion, thus alleviating the inflammatory response of plaques.

[0057] The LC3II / LC3I results for each group of experiments are as follows: Figure 4 As shown, compared with the control group, the LC3II levels in macrophages treated with the sea cucumber polypeptide groups SCP-1, SCP-2, and SCP-1 / SP-2 of this invention were significantly different (p<0.05), with significantly increased LC3II levels and a significantly upregulated LC3II / LC3I ratio. These results indicate that the sea cucumber polypeptides SCP-1 and SCP-2 prepared in this invention can enhance the autophagy capacity of macrophages, thereby reducing inflammatory infiltration of the vascular wall and decreasing the formation and expansion of lipid cores within plaques, thus alleviating the formation and development of arterial plaques.

[0058] Example 3

[0059] Experimental animals: New Zealand rabbits, weighing 2.5-3.0 kg, half male and half female, provided by Pizhou Oriental Breeding Co., Ltd., experimental animal license number: SCXK (Su) 2022-0004, quarantined and acclimatized for 1 week, and used after no abnormalities were observed.

[0060] Model establishment: New Zealand rabbits were used as the model material. They were fed a high-fat diet (standard diet supplemented with 1% cholesterol, 7.5% egg yolk powder, 8% lard, 0.5% sodium cholate, and 0.1% propylthiouracil) for 10 consecutive weeks, and injected intraperitoneally with vitamin D3 every 7 days to induce the formation of atherosclerotic plaques. Rabbits in the normal group were fed a standard diet simultaneously and injected with the same volume of physiological saline every 7 days.

[0061] Administration: After successful model establishment, each treatment group was administered the drug via gavage once daily for 8 consecutive weeks. Forty New Zealand white rabbits were divided into 5 groups of 8 rabbits each, including a control group, a model group, and the experimental groups: SCP-1, SCP-2, and SCP-1 / SP-2. The control and model groups were routinely fed saline for 8 weeks; the experimental groups were administered SCP-1, SCP-2, and SCP-1 / SP-2 (SCP-1 to SCP-2 mass ratio 1:1) via gavage for 8 weeks.

[0062] Test results

[0063] (1) Plaque area detection: Eight weeks after drug administration, carotid artery vessels were cut, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). After mounting, the morphology was observed under a microscope, and the plaque area was calculated using Image Pro Plus image processing software. The plaque area ratio was calculated according to the following formula:

[0064] .

[0065] The effect of sea cucumber polypeptides on the proportion of carotid artery plaque area in rabbits from Southwest China, as follows: Figure 5 As shown, after being fed a high-fat diet, the carotid artery plaque area in the model group rapidly expanded, indicating successful model establishment. In the experimental groups, the SCP-1, SCP-2, and SCP-1 / SP-2 groups all showed a significant reduction in plaque area compared to the model group after gavage administration of sea cucumber polypeptides (p<0.05), demonstrating that the sea cucumber polypeptides of this invention have a significant ameliorative effect on carotid atherosclerosis when administered alone or in combination.

[0066] (2) Serum biochemical index detection: On the last day of the 8-week drug administration, 2 hours after the end of drug administration, blood was collected from the tail vein of each rabbit. After standing at room temperature for 30 minutes, the blood was centrifuged at 5000 rpm for 20 minutes at 4°C, and the serum was collected. The concentrations of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the serum were detected according to the instructions of the TG, TC, LDL-C and HDL-C kits (purchased from Beijing Pulilai Gene Technology Co., Ltd., catalog numbers: E1003, E1005, E1018 and E1017 respectively).

[0067] The results are as follows Figure 6 As shown, compared with the control group, the serum TC (p<0.01), TG (p<0.01), HDL-C (p<0.01), and LDL-C (p<0.01) levels in the model group rabbits were all increased; compared with the model group, the serum TC, TG, HDL-C, and LDL-C levels in the SCP-1 group, SCP-2 group, and SCP-1 / SP-2 group were all significantly decreased, and the differences were statistically significant, indicating that the sea cucumber polypeptide of the present invention can prevent and treat atherosclerosis and has a good effect on improving cardiovascular diseases.

[0068] (3) Western blot experiment: The expression of TLR4 and NF-κB proteins in the carotid arteries of rabbits in each group was detected by Western blot. Half of the frozen tissues were taken out, thawed (-20℃, 4℃), rinsed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood, and ground with liquid nitrogen. Protein lysis buffer was added, cooled on ice for 10 min, and sonicated. Centrifuged at 12000rpm for 15 min at 4℃, and the supernatant was collected. Working solution and standard curve were prepared according to the kit (purchased from: Beijing Bio-Lab Technology Co., Ltd., catalog number: WE0298). The absorbance value was detected at a wavelength of 570nm, and the concentration of each sample was calculated. After SDS-PAGE electrophoresis and PVDF transfer, the samples were incubated with the corresponding primary and secondary antibodies, respectively. ECL luminescent solution was prepared under light-protected conditions, and images were captured. ImageJ software was used to analyze the gray value of the target protein bands in each group.

[0069] The results are as follows Figure 7As shown, compared with the control group, the expression of TLR4 and NF-κB proteins in the carotid artery tissue of the model group was significantly increased (p<0.01); while compared with the model group, the expression of TLR4 and NF-κB proteins in the carotid artery tissue of rabbits in the experimental groups SCP-1 and SCP-2 was significantly decreased (p<0.01, P<0.05). Based on the above, the sea cucumber polypeptide of the present invention has the effect of alleviating the pathological damage and lipid metabolism abnormalities of atherosclerosis. Its mechanism can be related to downregulating the TLR4 / NF-κB signaling pathway and improving inflammatory response and oxidative stress.

[0070] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, various simple deductions or substitutions can be made without departing from the concept of the present invention, and all such modifications and substitutions should be considered within the scope of protection of the present invention.

Claims

1. A polypeptide with therapeutic function for atherosclerosis, characterized in that, The polypeptide is selected from SCP-1 with the amino acid sequence SEQ ID NO.1 and SCP-2 with the amino acid sequence SEQ ID NO.

2.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is derived from sea cucumber or is artificially synthesized.

3. A method for preparing the polypeptide according to any one of claims 1-2, characterized in that, Includes the following steps: (1) Raw material pretreatment: Take fresh sea cucumbers, remove the internal organs, wash with sterile water, cut into small pieces and freeze dry, grind into powder, add sodium acetate buffer solution with pH 7.0 according to the material-liquid ratio of 1:2-10, soak with ultrasonic assistance, filter and discard the filtrate; (2) Two-enzyme stepwise hydrolysis: First, adjust the pH of the system to 9.0 for the first hydrolysis, and after the first hydrolysis, adjust the pH to 5.0 for the second hydrolysis to obtain the hydrolysis product; (3) Enzyme inactivation: Inactivate the enzymes by bathing the enzymatic hydrolysis products in water; (4) Centrifugal filtration: After cooling to room temperature, centrifuge to collect the supernatant, and then filter it through a 0.45μm microporous membrane; (5) Nanofiltration desalination: The filtrate is desalted using a nanofiltration membrane with a molecular weight cutoff of 1000 Da. The process is repeated 2-5 times, and the filtrate is collected. (6) Gel filtration chromatography: Load the filtrate onto a Sephacryl S-200 HR gel filtration chromatography column, use deionized water as the eluent, and a flow rate of 0.5 mL / min. Collect the eluent. (7) Purification: The collected elution fraction was purified by preparative high performance liquid chromatography. Mobile phase A was a mixture of 99.9% water and 0.1% formic acid, and mobile phase B was an acetonitrile solution of 0.05% trifluoroacetic acid. The elution program was: 0-12 min, 8-12% B; 12-28 min, 12-25% B; 28-38 min, 25-35% B; 38-45 min, 35-48% B; flow rate 1.2 mL / min, detection wavelength 218 nm. The elution peaks were collected, and the amino acid sequences of the elution peaks were identified by LC-TOF-MS to obtain the polypeptide with the amino acid sequence SEQ ID NO.1-2.

4. The preparation method according to claim 3, characterized in that, In step (2), the first enzymatic hydrolysis uses a neutral protease at a concentration of 3200 U / g, a hydrolysis temperature of 30-40℃, and a hydrolysis time of 2-8 hours.

5. The preparation method according to claim 3, characterized in that, In step (2), the second enzymatic hydrolysis involves adding papain at a dosage of 3800 U / g, at a hydrolysis temperature of 35-45℃, and for 2-8 hours.

6. The polypeptide prepared by the method according to any one of claims 3-5.

7. The use of the polypeptide according to any one of claims 1-2 in the preparation of a drug for treating atherosclerosis.

8. A pharmaceutical composition comprising the polypeptide of any one of claims 1-2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

9. A drug comprising the polypeptide of any one of claims 1-2.

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