Sea cucumber polypeptide as well as preparation method and application thereof in treatment of atherosclerosis

Abstract

The invention provides a sea cucumber polypeptide, a preparation method thereof and application of the sea cucumber polypeptide in treatment of atherosclerosis, and the polypeptide with the function of treating and / or preventing atherosclerosis is SCP-1 and SCP-2 and is derived from sea cucumber or artificially synthesized. The preparation method comprises the following steps: pretreating a sea cucumber raw material, carrying out double-enzyme step-by-step enzymolysis by adopting neutral protease and papain, and carrying out enzyme deactivation, centrifugal filtration, nanofiltration desalination, gel filtration chromatography, preparative high performance liquid chromatography purification and the like. The sea cucumber polypeptide can be used for preparing and / or preventing and treating atherosclerosis, can also be prepared into pharmaceutical compositions containing pharmaceutically acceptable carriers or auxiliary materials, provides a new material basis and an effective way for treatment of atherosclerosis, and has wide application prospects in the fields of medicines, health care products and foods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of polypeptides and biotechnology, and particularly relates to a sea cucumber polypeptide, a preparation method thereof and application thereof in treating atherosclerosis. BACKGROUND

[0002] Atherosclerosis (AS) is a very common disease in cardiovascular system diseases, which is characterized by the formation of fibrous or atheromatous plaques in the intima of blood vessels, mainly involving large and medium-sized arteries, resulting in hardening of the vessel wall, stenosis of the lumen and reduced elasticity, and further causing ischemic changes in the corresponding organs. The incidence of AS continues to rise, and it is common in middle-aged and elderly people, and in recent years there has been a tendency towards younger people. Its complications are extremely harmful, such as aortic atherosclerosis, which can form an aortic aneurysm, and even face the risk of rupture and bleeding, once ruptured, the patient can quickly shock and die; coronary atherosclerosis can lead to angina pectoris, myocardial infarction, heart failure, and sudden cardiac death; carotid and cerebral atherosclerosis can cause transient ischemic attack (TIA), cerebral infarction, cerebral hemorrhage, etc.; renal atherosclerosis can affect kidney function, leading to serious consequences such as kidney atrophy and refractory hypertension. However, the current treatment methods for atherosclerosis have many limitations. Existing treatment methods, such as thermal physical treatment, can kill diseased tissue while causing serious damage to the endothelial layer of the blood vessel, resulting in insufficient long-term patency of the blood vessel after treatment, and problems such as restenosis of the blood vessel.

[0003] Sea cucumber is a marine organism rich in various bioactive components, and sea cucumber polypeptide has attracted much attention. In the prior art, the preparation methods of sea cucumber polypeptide mainly include protease enzymolysis and hydrolysis. Hydrolysis usually uses acid or alkali to hydrolyze proteins, and this method is violent and requires strict reaction equipment. Protease enzymolysis is a commonly used method for preparing sea cucumber polypeptide at present, because it has mild reaction conditions, is safe and easy to control, and can obtain active peptides with specific physiological functions by selecting the type of enzyme, controlling the reaction time, enzyme addition amount and substrate concentration. There are few reports on the active peptide segments and mechanisms of sea cucumber polypeptide, and most of them are functional verification of polypeptide mixtures, which is not conducive to the application and promotion of sea cucumber active polypeptide. Although some studies have shown that sea cucumber polypeptide has certain effects on anti-fatigue, physical enhancement, immune enhancement, cell variation prevention, internal circulation improvement, blood pressure regulation, anti-aging, beauty and skin care, etc., the application of sea cucumber polypeptide in the treatment of atherosclerosis still needs to be further improved. How to prepare sea cucumber polypeptide with high purity, high yield and effective treatment of atherosclerosis has become a problem to be solved. SUMMARY

[0004] In order to solve the problems in the prior art, the present application provides a sea cucumber polypeptide, a preparation method thereof and application thereof in treating atherosclerosis, specifically, polypeptides with the function of treating atherosclerosis are obtained by extraction from sea cucumbers, high-quality proteins in sea cucumbers are scientifically utilized, important support can be provided for the development of sea cucumber protein functional products and food-derived polypeptides, and the application of sea cucumbers in the fields of medicines, health products and food is expanded.

[0005] To this end, the present application adopts the following technical solutions:

[0006] The present application provides polypeptides SCP-1 and SCP-2 with the function of treating atherosclerosis.

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

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

[0009] The present application also provides a preparation method of the polypeptide, comprising the following steps:

[0010] (1) raw material pretreatment: fresh sea cucumbers are taken, internal organs are removed, the sea cucumbers are washed three times with sterile water, the sea cucumbers are cut into small pieces and then freeze-dried, the freeze-dried sea cucumbers are ground into powder, sodium acetate buffer solution with pH 7.0 is added according to a solid-liquid ratio of 1:2-10, ultrasonic-assisted soaking is performed, and the filtrate is discarded after filtration;

[0011] (2) double-enzyme step-by-step enzymolysis: the pH of the system is first adjusted to 9.0 for first-time enzymolysis, the pH is adjusted to 5.0 after the first-time enzymolysis is completed, and second-time enzymolysis is performed, so as to obtain an enzymolysis product;

[0012] (3) enzyme inactivation: the enzymolysis product is subjected to water bath enzyme inactivation;

[0013] (4) centrifugal filtration: after cooling to room temperature, the supernatant is obtained by centrifugation, and the supernatant is filtered through a 0.45 μm microporous filter membrane;

[0014] (5) nanofiltration desalination: the filtrate is subjected to desalination treatment using a nanofiltration membrane with a molecular weight cut-off of 1000 Da, and the operation is cycled 2-5 times, and the filtrate is collected;

[0015] (6) gel filtration chromatography: the filtrate is loaded onto a Sephacryl S-200HR gel filtration chromatography column, deionized water is used as an eluent, the flow rate is 0.5 mL / min, and elution components are collected;

[0016] (7) Purification: the collected elution components are purified by preparative high performance liquid chromatography, mobile phase A is 99.9% water and 0.1% formic acid mixture, mobile phase B is 0.05% trifluoroacetic acid in acetonitrile solution, elution program: 0-12min, 8-12%B; 12-28min, 12-25%B; 28-38min, 25-35%B; 38-45min, 35-48%B; flow rate 1.2mL / min, detection wavelength 218nm, collect the elution peak, use LC-TOF-MS to identify the amino acid sequence of the elution peak, obtain the polypeptide with amino acid sequence of SEQ ID NO. 1-2.

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

[0018] Further, the enzyme added in the second enzymolysis is papain, the enzyme is added in an amount of 3800U / g, the enzymolysis temperature is 35-45℃, and the enzymolysis time is 2-8h; preferably, the enzymolysis temperature is 40℃, and the enzymolysis time is 3h.

[0019] The application also provides the polypeptide prepared by the preparation method.

[0020] The application also provides application of the polypeptide in preparation of a medicine for treating and / or preventing atherosclerosis.

[0021] The application also provides application of the polypeptide in preparation of a medicine for treating and / or preventing cardiovascular diseases.

[0022] The application also provides a pharmaceutical composition of the polypeptide.

[0023] Further, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0024] The application also provides food, health products or medicines containing the polypeptide.

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

[0026] The application extracts and prepares sea cucumber polypeptides with treatment of atherosclerosis from sea cucumber by adopting environment-friendly biological enzymatic hydrolysis method, and the obtained polypeptides are SCP-1 with amino acid sequence FAGITQSGR and SCP-2 with amino acid sequence LPVGQDIGMG. The preparation process of the application is scientific and reasonable, simple to operate, and has strong industrial implementation. The polypeptide sequence is clear and the number of amino acid residues is small, so that the polypeptide can be conveniently artificially synthesized on a large scale, has no toxic and side effects on human cells, and has good safety. The sea cucumber polypeptide can promote the autophagy of macrophages, promote the outflow of cholesterol through the autophagy-lysosome system to inhibit the formation of foam cells, can significantly improve the plaque of atherosclerotic blood vessels, in addition, can significantly improve the concentration of TG, TC, LDL-C and HDL-C in serum, and down-regulate the expression of TLR4 and NF-kB proteins in TLR4 / NF-kB signal pathway. The above results all show that the sea cucumber polypeptide can prevent and treat atherosclerosis, has good improvement effect on cardiovascular diseases, and has broad market prospect in many fields such as health food and medicine. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Structure diagram of SCP-1;

[0028] Figure 2 Structure diagram of SCP-2;

[0029] Figure 3 Effect of sea cucumber polypeptide on survival rate of RAW264.7 macrophages;

[0030] Figure 4 LC3II / LC3I value of RAW264.7 macrophages;

[0031] Figure 5 Effect of sea cucumber polypeptide on proportion of carotid artery plaque area of new southwest rabbits;

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

[0033] Figure 7The results of detecting the protein expression of TLR4 and NF-kB in the carotid artery of rabbits: A is the electrophoretogram of the control group, the model group, the SCP-1 group and the SCP-2 group; B is the relative expression amount chart of TLR4 and NF-kB protein (compared with the control group, # p<0.05, ## p<0.01; compared with the model group, * p<0.05, ** p<0.01) DETAILED DESCRIPTION

[0034] To make the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects achieved by the present application clear, the following will be described in detail in combination with the specific embodiments listed and the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0035] In this article, the term "embodiment" means that the specific features, structures or characteristics described in combination with the embodiment can be included in at least one embodiment of the present application. The term "embodiment" appearing at various places in the specification does not necessarily refer to the same embodiment, and does not particularly limit the independence or association between other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the technical features mentioned in each embodiment can be combined in any way to form a corresponding implementable technical solution.

[0036] Unless otherwise defined, the meanings of the technical terms used herein are the same as those commonly understood by those skilled in the art to which the present application belongs; the use of related terms in this article is only for the purpose of describing specific embodiments, and is not intended to limit the present application.

[0037] Without more limitations, in the present application, the use of "includes", "contains", "has" or other similar expressions in the sentence is intended to cover non-exclusive inclusion, and these expressions do not exclude the presence of other elements in the process, method or product including the described elements, so that the process, method or product including a series of elements can not only include those limited elements, but also include other elements not explicitly listed, or also include the elements inherent to such process, method or product.

[0038] As the same as the understanding in the "Examination Guidelines", in the present application, "greater than", "less than", "exceed" and the like are understood as not including the number; "above", "below", "within" and the like are understood as including the number. In addition, in the description of the embodiments of the present application, the meaning of "multiple" is two or more (including two), and similar expressions related to "multiple" are also understood in this way, for example, "multiple groups", "multiple times" and the like, unless otherwise explicitly and specifically limited.

[0039] Statistical analysis: SPSS20.0 statistical software was used for statistical analysis of the data, and the difference between multiple samples was analyzed by one-way ANOVA, and the difference between groups was compared by Dunnett-t method. p<0.05 and p<0.01 indicate that the difference is statistically significant.

[0040] The raw materials, reagents or devices used in the following examples, unless otherwise specified, can be obtained from conventional commercial channels, or can be obtained by existing known methods.

[0041] Example 1

[0042] A preparation method of a polypeptide with the function of treating atherosclerosis, comprising the following steps:

[0043] (1) Raw material pretreatment: 800g of fresh and live sea cucumber (purchased from Wuxi Sunan Agricultural and sideline Products Aquatic City, address: No. 517 Datong Road, Binhu District, Wuxi City, Jiangsu Province) was taken, the internal organs were removed, washed with 6℃ sterile water for three times, cut into small pieces, then freeze-dried, ground into powder, added with pH 7.0 sodium acetate buffer solution according to the solid-liquid ratio of 1:6, ultrasonic-assisted soaking for 2 hours, filtered, and the filtrate was discarded;

[0044] (2) Double-enzyme step-by-step enzymolysis: first, adjust the pH of the system to 9.0 for the first enzymolysis, the enzyme added in the first enzymolysis is neutral protease, the enzyme addition amount is 3200U / g, the enzymolysis temperature is 35℃, and the enzymolysis time is 5h; after the end, adjust the pH to 5.0 for the second enzymolysis, the enzyme added in the second enzymolysis is papain, the enzyme addition amount is 3800U / g, the enzymolysis temperature is 40℃, and the enzymolysis time is 3h, to obtain an enzymolysis product;

[0045] (3) Enzyme inactivation: the enzymolysis product was placed in a 98℃ water bath for 10min for enzyme inactivation;

[0046] (4) Centrifugal filtration: after cooling to room temperature, centrifugation was performed at a speed of 4500r / min for 15min, the supernatant was taken, and then filtered through a 0.45μm microporous filter membrane;

[0047] (5) Nanofiltration desalination: the filtrate was subjected to desalination treatment using a nanofiltration membrane with a molecular weight cut-off of 1000Da, and the operation was repeated for 4 times, and the filtrate was collected;

[0048] (6) Gel filtration chromatography: the filtrate was loaded into a Sephacryl S-200HR gel filtration chromatography column, deionized water was used as the eluent, the flow rate was 0.5 mL / min, and the elution fractions were collected;

[0049] (7) Purification: the collected elution fractions were subjected to preparative high performance liquid chromatography purification, a C18 reversed-phase analysis column (50 mm x 15 cm x 3 μm), mobile phase A was a mixture of 99.9% water and 0.1% formic acid, mobile phase B was 0.05% trifluoroacetic acid in acetonitrile, 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; the flow rate was 1.2 mL / min, the detection wavelength was 218 nm, the elution peaks were collected, and LC-TOF-MS was used to separate and identify the elution peaks;

[0050] (8) Sequence identification: ESI+ mode, data-dependent scanning mode, full scan acquisition (m / z 200-1600) in an orbitrap with a resolution of 70000. The first 8 separated peptide signals (charge state > +1) parent ions were broken down by high-energy collision (HCD) with a normalized collision energy (NCE) of 28.0. The temperature of the capillary was 275°C, and the spray voltage was 1900V. The daughter ions were measured on an orbitrap with a resolution of 17500. The maximum filling time for full scan and MS-MS scan was set to 50 ms and 50 ms respectively, and the dynamic exclusion time was set to 30 s.

[0051] LC-TOF-MS analysis showed that the active peptide structure was FAGITQSGR (SCP-1, SEQ ID NO: 1, molecular weight 936.02 Da, isoelectric point 10.386, structure diagram as shown in Figure 1 ) and LPVGQDIGMG (SCP-2, SEQ ID NO: 2, molecular weight 985.49 Da, isoelectric point 3.539, structure diagram as shown in Figure 2 ).

[0052] Based on the identified amino acid sequence of the active polypeptide, it can be prepared by sea cucumber enzymatic separation, or it can be prepared by chemical synthesis. Regardless of which preparation method, the peptide sequence composition is unchanged. The active sea cucumber polypeptides SCP-1 and SCP-2 of the present application are synthesized by Shanghai Jilsheng Biochemical Co., Ltd. and used, and the purity is more than 98%.

[0053] Example 2

[0054] RAW264.7 macrophages (purchased from BoHui Biotechnology (Guangzhou) Co., Ltd., product model: BH-C415) were cultured in a DMEM medium containing 10% fetal bovine serum (FBS), 1% double antibodies, in a 37°C, 5% CO2 constant-temperature incubator. The macrophages were divided into four groups, namely a control group, an SCP-1 group, an SCP-2 group, and an SCP-1 / SP-2 group. The SCP-1 group, the SCP-2 group, and the SCP-1 / SP-2 group in the experimental groups were treated with a 10 ug / L sea cucumber polypeptide aqueous solution overnight. After 8 hours, the cell survival rate was determined by using a cell counting kit-8 (CCK-8) kit (purchased from Yixing Biotechnology (Shanghai) Co., Ltd., product item number: 40203ES76) according to the kit instructions. The cell survival rate was greater than 98%, indicating that the sea cucumber polypeptide was non-toxic. The cell survival rate is shown in Table 1. Figure 3 The absorbance values (OD values) of the wells were measured by using an enzyme-linked immunoassay instrument at a wavelength of 450 nm. The cell survival rate was calculated according to the average absorbance values of the experimental groups according to the following formula:

[0055]

[0056] The total cell protein was extracted, and the levels of autophagy markers LC3I and LC3II were determined by immunoblotting. LC3 initially exists in the form of LC3I in the cytoplasm, and after being processed at the C-terminal, it is combined with phosphatidylethanolamine to form LC3II. LC3II can be specifically located on the autophagosome membrane and is an important marker of autophagosome formation. As the main cellular component in the development process of arterial plaques, macrophages can promote cholesterol efflux through the autophagy-lysosome system, thereby inhibiting the formation of foam cells, and can inhibit the activation of inflammasomes and reduce the secretion of IL-1β, thereby reducing the inflammatory response of plaques.

[0057] The results of LC3II / LC3I in the test groups are shown in Table 2. Figure 4 Compared with the control group, the LC3II levels in the macrophages treated by the sea cucumber polypeptide groups SCP-1, SCP-2, and SCP-1 / SP-2 of the present application had significant differences (p<0.05), the LC3II levels were obviously improved, and the LC3II / LC3I ratio was significantly up-regulated. The results show that the sea cucumber polypeptides SCP-1 and SCP-2 prepared by the present application can enhance the autophagy ability of macrophages, thereby reducing the inflammatory infiltration of the vascular wall, reducing the formation and expansion of the lipid core in the plaque, and thus alleviating the formation and development of arterial plaques.

[0058] Example 3

[0059] Test animals: New Zealand rabbits, body weight 2.5-3.0 kg, half male and half female, provided by Pizhou Dongfang Breeding Co., Ltd., experimental animal license number: SCXK(Su)2022-0004, quarantine and adaptive feeding for 1 week, no abnormalities were found after use.

[0060] Modeling: New Zealand rabbits were used as materials, and atherosclerotic plaque models were induced by feeding high-fat feed (standard feed added with 1% cholesterol, 7.5% egg yolk powder, 8% lard, 0.5% sodium cholate, and 0.1% propylthiouracil) for 10 weeks, and intraperitoneal injection of vitamin D3 once every 7 days. The normal group of rabbits was fed with standard feed at the same time, and was injected with the same volume of normal saline once every 7 days.

[0061] Drug administration: After successful modeling, each drug group was continuously administered by gavage once a day for 8 weeks. 40 New Zealand white rabbits were divided into 5 groups, 8 in each group, including a blank group, a model group, and SCP-1, SCP-2 and SCP-1 / SP-2 groups in the experimental group. The blank control group and the model control group were fed with normal saline for 8 weeks; the experimental groups were administered by gavage with SCP-1, SCP-2 and SCP-1 / SP-2 (the mass ratio of SCP-1 to SCP-2 is 1:1) at a dose of 1 g / kg / day, for 8 weeks.

[0062] Test results

[0063] (1) Plaque area detection: After 8 weeks of administration, the carotid artery was cut and paraffin-embedded, sectioned, and stained with 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 polypeptide on the carotid plaque area ratio of New Zealand rabbits is shown in Table 1. Figure 5 After feeding with high-fat feed, the carotid plaque area of the model group rapidly expanded, and the modeling was successful. The plaque area of the SCP-1, SCP-2 and SCP-1 / SP-2 groups in the experimental group was significantly reduced compared with the model group (p<0.05) after gavage with sea cucumber polypeptide, indicating that the sea cucumber polypeptide of the present application has a significant improvement effect on carotid atherosclerosis when administered alone or in combination.

[0066] (2) Serum biochemical index detection: on the last day of administration for 8 weeks, 2h after the end of administration, the blood of each rabbit was taken from the tail vein, and after standing at room temperature for 30min, it was centrifuged at 5000rpm for 20min at 4℃, and the serum was taken. According to the instructions of TG, TC, LDL-C, HDL-C kit (purchased from Beijing Puli Lei Gene Technology Co., Ltd., serial numbers are E1003, E1005, E1018, E1017 respectively), the concentrations of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in serum were detected.

[0067] Results as shown in Figure 6 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) of the model group rabbits were all increased; compared with the model group, the serum TC, TG, HDL-C and LDL-C of the SCP-1 group, the SCP-2 group and the SCP-1 / SP-2 group were significantly reduced, and there was a significant difference, indicating that the sea cucumber polypeptide of the application can prevent and treat atherosclerosis, and has a good improvement effect on cardiovascular diseases.

[0068] (3) Western blot experiment: Western-blot method was used to detect the expression of TLR4 and NF-кB proteins in the carotid arteries of rabbits in each group. Half of the frozen tissues were taken out, thawed (-20℃, 4℃), and then washed with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood, and then ground with liquid nitrogen. Add protein lysis buffer, cool on ice for 10min, and ultrasonic breakage. Centrifuge at 12000rpm for 15min at 4℃, and take the supernatant. According to the instructions of the kit (purchased from Beijing Baoer Laboratory Technology Co., Ltd., serial number: WE0298), prepare the working solution and standard curve, and detect the absorbance value at 570nm wavelength, and calculate the concentration of each sample. After SDS-PAGE electrophoresis and PVDF membrane transfer, the corresponding primary antibody and secondary antibody were incubated respectively, and the ECL luminescent liquid was prepared under dark conditions, and the image was photographed. Image J software was used to analyze the gray value of each group of target protein bands.

[0069] Results as shown in Figure 7As shown, compared with the control group, the expression of TLR4 and NF-кB 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-кB 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, and its mechanism can be related to downregulating the TLR4 / NF-кB signaling pathway and improving inflammatory response and oxidative stress.

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

Claims

1. A polypeptide having a function of treating atherosclerosis, characterized by comprising an amino acid sequence represented by SEQ ID NO: 1 or a functional fragment thereof. The polypeptide is selected from SCP-1 with the amino acid sequence of SEQ ID NO. 1 and SCP-2 with the amino acid sequence of SEQ ID NO.

2.

2. The polypeptide of claim 1, wherein, The polypeptide is derived from a sea cucumber or artificially synthesized.

3. A method of producing the polypeptide of any one of claims 1-2, comprising, The method comprises the following steps: (1) raw material pretreatment: fresh sea cucumber is taken, the internal organs are removed, the sea cucumber is washed with sterile water, cut into small pieces, and then freeze-dried, ground into powder, and then added into a sodium acetate buffer solution with pH 7.0 according to a solid-liquid ratio of 1:2-10, ultrasonic-assisted soaking, and then filtered to discard the filtrate; (2) double-enzyme step-by-step enzymolysis: first-time enzymolysis is performed by adjusting the pH of the system to 9.0, and then second-time enzymolysis is performed by adjusting the pH to 5.0 after the first-time enzymolysis is completed, to obtain an enzymolysis product; (3) enzyme inactivation: the enzymolysis product is subjected to water bath enzyme inactivation; (4) centrifugal filtration: after cooling to room temperature, the supernatant is obtained by centrifugation, and then filtered through a 0.45 μm microporous filter membrane; (5) nanofiltration desalination: the filtrate is subjected to desalination treatment by using a nanofiltration membrane with a molecular weight cut-off of 1000 Da, and the operation is repeated for 2-5 times to collect the filtrate; (6) gel filtration chromatography: the filtrate is loaded into a Sephacryl S-200HR gel filtration chromatography column, deionized water is used as an eluent, the flow rate is 0.5 mL / min, and elution components are collected; (7) purification: the collected elution components are subjected to preparative high performance liquid chromatography purification, the mobile phase A is a mixture of 99.9% water and 0.1% formic acid, the mobile phase B is a 0.05% trifluoroacetic acid acetonitrile solution, the elution program is 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 is 1.2 mL / min, the detection wavelength is 218 nm, elution peaks are collected, and LC-TOF-MS is used to identify the amino acid sequences of the elution peaks to obtain polypeptides with the amino acid sequences of SEQ ID NO. 1-2.

4. The production method according to claim 3, characterized by, In the step (2), the enzyme added in the first-time enzymolysis is neutral protease, the enzyme addition amount is 3200 U / g, the enzymolysis temperature is 30-40 ℃, and the enzymolysis time is 2-8 h.

5. The preparation method according to claim 3, characterized in that In the step (2), the enzyme added in the second-time enzymolysis is papain, the enzyme addition amount is 3800 U / g, the enzymolysis temperature is 35-45 ℃, and the enzymolysis time is 2-8 h.

6. The polypeptide obtained by the preparation method in any one of claims 3-5.

7. The polypeptide in any one of claims 1-2 for use in the preparation of a drug for treating and / or preventing atherosclerosis.

8. The polypeptide in any one of claims 1-2 for use in the preparation of a drug for treating and / or preventing cardiovascular diseases.

9. A pharmaceutical composition containing the polypeptide in any one of claims 1-2, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

10. A food, health product or drug containing the polypeptide in any one of claims 1-2.

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