Mussel peptide, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a mussel peptide and a preparation method and application thereof. The mussel peptide comprises one or more of the following peptide segments: (1) Ile-Leu-Thr-Glu-Arg (2) Leu-Asp-Leu-Ala-Gly-Arg (3) Val-Thr-Ile-Met-Pro-Lys (4) Met-Gln-Ile-Phe-Val-Lys (5) Leu-Phe-Asp-Ala-Val-Ile-Arg. The mussel peptide is prepared by adopting a complex enzyme-membrane separation-dextran gel column chromatography purification coupling technology. The peptide not only has the effect of relieving spontaneous hypertension, but also has the effect of improving various target organ damages caused by spontaneous hypertension. The application provides the possibility of application of the mussel peptide in improving various target organ damages caused by spontaneous hypertension or other diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mussel peptide, its preparation method, and its application. Background Technology

[0002] Hypertension is a chronic disease and a major risk factor for cardiovascular disease, stroke, kidney failure, and peripheral artery disease. From a pathogenesis perspective, the renin-angiotensin system (RAS) is the main pathway for regulating blood pressure, vascular tone, and maintaining cardiovascular function. Angiotensin-converting enzyme (ACE) is a key enzyme in the RAS, converting angiotensin I (Ang I) to angiotensin II (Ang II). Ang II binds to its cell surface receptors, triggering the adrenal glands to secrete aldosterone, leading to increased salt and water reabsorption and elevated blood pressure due to arterial constriction. Therefore, ACE has long been considered a primary target for hypertension treatment. Currently, most ACE inhibitors are chemically synthesized drugs, such as captopril, lisinopril, and enalapril. While these chemically synthesized drugs have good antihypertensive effects, long-term use can cause significant side effects, such as cough, rash, acute kidney failure, and angioedema. Furthermore, these complications caused by hypertension are untreatable and tend to worsen over time. Therefore, there is an urgent need to research and develop a safer, more effective, and non-toxic food-derived ACE inhibitory peptide that can improve the damage to multiple target organs caused by hypertension while treating hypertension.

[0003] Various ACE inhibitory peptides have been isolated from natural plants and animals such as Antarctic krill, oysters, walnuts, and soybeans. However, studies have not found that they can improve hypertension symptoms while simultaneously mitigating damage to multiple target organs (such as the heart, kidneys, and blood vessels) caused by hypertension. Research reports that converting proteins from aquatic animals into smaller peptide molecules through enzymatic hydrolysis is more beneficial for human absorption and physiological activity. Mussels are molluscs that attach to their shells via byssal threads. Widely distributed in China, Spain, Thailand, and other regions, they are one of my country's major economic shellfish. Mussels are rich in high-quality protein, vitamins, and minerals, earning them the nickname "eggs of the sea." Currently, mussels produced in my country are mainly consumed fresh, with a small amount processed into frozen or dried products. Compared to other seafood, mussels have extremely low added value, resulting in significant resource waste. Therefore, in-depth research into mussel processing technologies is of great importance to the mussel industry.

[0004] Currently, no studies have found any application of mussel peptides in lowering blood pressure, nor have they found any application of mussel peptides in improving target organ damage (such as heart, kidneys, and blood vessels) caused by spontaneous hypertension. Summary of the Invention

[0005] The purpose of this invention is to solve the aforementioned problems in the prior art, and to propose a mussel peptide, its preparation method, and its application. A mussel peptide is prepared using a coupled technology of complex enzyme-membrane separation-dextran gel column chromatography purification, adding a novel food-derived ACE peptide to the field of blood pressure reduction. This peptide not only alleviates spontaneous hypertension symptoms but also improves target organ damage (such as brain, heart, kidneys, and blood vessels) caused by spontaneous hypertension; thus providing possibilities for the application of mussel peptide in improving various target organ damages caused by spontaneous hypertension or other diseases.

[0006] The technical solution of this invention is:

[0007] A mussel peptide comprising one or more of the following peptides with high ACE inhibitory activity:

[0008] (1)Ile-Leu-Thr-Glu-Arg (SEQ ID NO.1)

[0009] (2)Leu-Asp-Leu-Ala-Gly-Arg (SEQ ID NO.2)

[0010] (3)Val-Thr-Ile-Met-Pro-Lys (SEQ ID NO.3)

[0011] (4)Met-Gln-Ile-Phe-Val-Lys (SEQ ID NO.4)

[0012] (5) Leu-Phe-Asp-Ala-Val-Ile-Arg (SEQ ID NO. 5);

[0013] Among them, peptide IR-5, with an amino acid sequence as shown in SEQ ID NO.1, has a molecular weight of 631.38 Da and an ACE inhibition rate of 87.81%; peptide LR-6, ​​with an amino acid sequence as shown in SEQ ID NO.2, has a molecular weight of 644.37 Da and an ACE inhibition rate of 77.03%; peptide VK-6, with an amino acid sequence as shown in SEQ ID NO.3, has a molecular weight of 704.40 Da and an ACE inhibition rate of 57.30%; peptide MK-6, with an amino acid sequence as shown in SEQ ID NO.4, has a molecular weight of 765.43 Da and an ACE inhibition rate of 85.24%; and peptide LR-7, with an amino acid sequence as shown in SEQ ID NO.5, has a molecular weight of 833.49 Da and an ACE inhibition rate of 80.59%.

[0014] The present invention also provides a method for preparing the mussel peptide, comprising the following steps:

[0015] (1) Pretreatment: Mussel meat and water are mixed in a certain ratio and then set aside for use;

[0016] (2) Enzymatic hydrolysis: Using the mussel meat paste obtained in step (1) as the substrate, at natural pH, set the enzymatic hydrolysis temperature to 50-55℃, add the complex protease and stir for 3-6 hours to obtain the enzymatic hydrolysate. After the enzymatic hydrolysis is completed, raise the temperature of the enzymatic hydrolysate to above 85℃. Specifically, any temperature within the range of 85℃-90℃ can be raised to 85℃-90℃ and kept for 10-20 minutes to inactivate the enzyme.

[0017] (3) Centrifugation: Cool the enzymatic hydrolysate from step (2) to below 40°C. Specifically, it can be cooled to any temperature within the range of 10°C to 40°C, and then centrifuged using a tube centrifuge.

[0018] (4) Membrane filtration: The centrifuged liquid in step (3) is filtered through microfiltration and ultrafiltration membranes in sequence to obtain a peptide liquid with an average relative molecular mass of less than 5000 Da;

[0019] In step (4), the peptide solution obtained by membrane filtration contains more than 80% peptides with an average relative molecular mass of less than 5000 Da.

[0020] (5) Purification: The peptide solution obtained in step (4) was further purified by Sephadex G-25 gel filtration chromatography to obtain peptides with a molecular weight of 600-1800 Da, which exhibited stronger biological activity.

[0021] (6) Concentration and drying: The peptide solution obtained in step (4) is concentrated by nanofiltration and then sent to a drying tower for instantaneous drying into powder. The mussel peptide obtained after the above preparation steps contains one or more peptide segments with amino acid sequences as shown in SEQ ID NO.1-5.

[0022] Furthermore, in step (1), the fresh mussel meat is washed with water and drained, then homogenized at a material-to-liquid ratio of 1:2 to 1:3 and set aside; the dried mussels are crushed and homogenized at a material-to-liquid ratio of 1:8 to 1:10.

[0023] In step (2), the amount of compound protease added is 0.2-0.4% of the weight of fresh mussel meat, or 0.7-1.0% of the weight of dried mussels.

[0024] Furthermore, in step (2), the formulation of the complex protease is trypsin: neutral protease: alkaline protease: flavor protease: papain = (1.5~2.0):(1.8~2.2):(0.5~0.7):(0.3~0.5):(1~1.5); the ratio of trypsin, neutral protease, alkaline protease, flavor protease and papain in the complex protease can be any numerical ratio within the above ratio range, for example, it can be 1.5:1.8:0.5:0.3:1, it can be 2.0:2.2:0.7:0.5:1.5, it can also be 1.5:2.2:0.5:0.5:1, it can also be 2.0:1.8:0.7:0.3:1.5, etc.

[0025] Preferably, the ratio of trypsin, neutral protease, alkaline protease, flavor protease, and papain in the complex protease is 2:2:0.6:0.4:1.

[0026] Furthermore, in step (5), the sample concentration for Sephadex G-25 gel filtration chromatography is 100-150 mg / mL, and the elution flow rate is 2.8-3.2 mL / min; preferably, the sample loading temperature is 120 mg / mL, and the elution flow rate is 3 mL / min.

[0027] This invention employs a coupled enzyme-membrane separation-dextran gel column chromatography technique to prepare mussel peptides. The composite protease used is a proportional mixture of trypsin, neutral protease, alkaline protease, flavor protease, and papain. This composite protease preparation has multiple cleavage sites, exhibiting strong specificity and high efficiency in the enzymatic hydrolysis of mussel meat, resulting in products with enhanced biological activity. Microfiltration-ultrafiltration technology is used to obtain peptides with a molecular weight of 5 kDa, which exhibit strong antihypertensive activity. Sephadex G-25 dextran gel column chromatography is used for purification, yielding peptides with a molecular weight of 600–1800 Da, which exhibit even stronger antihypertensive activity. The final prepared mussel peptides have an average molecular weight of 1065 Da, with over 98% having a molecular weight less than 2000 Da.

[0028] The present invention also provides the application of the mussel peptide in the preparation of antihypertensive drugs or antihypertensive products.

[0029] Furthermore, the aforementioned antihypertensive drugs or products have a hypotensive effect on spontaneous hypertension, and can also improve vascular damage, dyslipidemia, renal function damage and cardiac damage caused by spontaneous hypertension.

[0030] Furthermore, the aforementioned antihypertensive drugs or products are suitable for improving vascular damage, dyslipidemia, kidney damage, and heart damage caused by other diseases.

[0031] Furthermore, the blood pressure lowering products include blood pressure lowering functional foods or health foods.

[0032] The present invention also provides a medicine comprising the mussel peptide of claim 1 and pharmaceutically acceptable excipients or carriers.

[0033] The beneficial effects of this invention are:

[0034] (1) The mussel peptide provided by the present invention has been shown in vivo to have a hypotensive effect on spontaneous hypertension and to alleviate the symptoms of spontaneous hypertension. It can also improve the target organ damage caused by spontaneous hypertension, including lipid metabolism abnormalities, vascular damage, kidney function damage and heart damage.

[0035] (2) The present invention uses a composite enzyme-membrane separation-dextran gel column chromatography coupling technology to prepare a mussel peptide. The composite enzyme technology makes the obtained mussel peptide have a small molecular weight and is easy to absorb, and has high biological activity. The average molecular weight of the peptide is 1065 Da. The membrane separation-dextran gel column chromatography technology makes the active peptide more pure, and the proportion of peptides with a molecular weight of 600-1800 Da reaches more than 80%.

[0036] (3) This invention opens up new medical and health care applications for mussel peptides. Mussel peptides are characterized by high safety and easy absorption. They can be taken for a long time, providing a range of drugs and health foods for the prevention and relief of symptoms for patients with hypertension. While helping to lower blood pressure, they also alleviate complications caused by hypertension and other diseases. The application of mussel peptides provided by this invention is not limited to vascular damage, dyslipidemia, renal damage and heart damage caused by hypertension, but is also applicable to complications such as vascular damage, dyslipidemia, renal damage and heart damage caused by other diseases. Attached Figure Description

[0037] Figure 1 This is a graph showing the activity results of the ACE inhibitory peptides separated by ultrafiltration in Example 1;

[0038] Figure 2 This is a graph showing the results of purifying component B using gel column chromatography in Example 1;

[0039] Figure 3 This is a graph showing the ACE inhibition rate results of the gel filtration separation of components B-1, B-2, and B-3 in Example 1;

[0040] Figure 4 This is the mass spectrum of component B-2 in Example 1;

[0041] Figure 5 The graph shows the ACE inhibition rate results for the five peptides in Example 1;

[0042] Figure 6 The effect of MEPs-E on blood pressure in SHR rats was investigated. (A) showed changes in SBP and DBP in rats within 24 hours after the first gavage; (B) showed changes in SBP and DBP during the 4-week experimental period. All data are expressed as mean ± standard deviation. Compared with the control group... # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01;

[0043] Figure 7 The results of comparing the levels of TG, TC, LDL-C, and HDL-C in the serum of rats in each group; compared with the control group, # p<0.05, ## p < 0.01; compared with the model group, *p < 0.05, **p < 0.01;

[0044] Figure 8 The effects of MEPs-E on vascular structure and function in SHR rats were investigated. (A) HE staining was used to observe vascular pathological morphological changes, with a scale bar of 50 μm; (B) Masson staining was used to observe the degree of vascular fibrosis, with a scale bar of 50 μm.

[0045] Figure 9 The effects of MEPs-E on vascular structure and function in SHR rats were investigated. (C)-(D) represent comparisons of vascular WCSA / LCSA and WT / ID; (E)-(F) represent comparisons of vascular collagen fiber area / muscle fiber area and CVF; (G)-(H) represent serum NO and ET-1 levels in each group of SHR rats. All data are expressed as mean ± standard deviation. Compared with the control group, [the results were as follows]. # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01;

[0046] Figure 10 The effects of MEPs-E on the structure and function of the kidneys in SHR rats were investigated. (A) HE staining was used to observe the pathological morphological changes of the kidneys, with a scale bar of 20 μm; (B) Masson staining was used to observe the degree of renal fibrosis, with a scale bar of 20 μm.

[0047] Figure 11 This study investigated the effects of MEPs-E on the renal structure and function of SHR rats. (C)-(D) represent the comparison of renal collagen fiber area / muscle fiber area and CVF among the groups; (E) represents the serum levels of BUN, CR, and UA in each group. All data are expressed as mean ± standard deviation. Compared with the control group... # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01;

[0048] Figure 12 The effects of MEPs-E on cardiac structure and function in SHR rats; (A) HE staining to observe cardiac pathological morphological changes, scale bar 50 μm; (B) Masson staining to observe the degree of cardiac fibrosis, scale bar 50 μm;

[0049] Figure 13 (C)-(D) Comparison of renal collagen fiber area / muscle fiber area and CVF; (E) Serum levels of CK, CK-MB, and LDH in each group of rats; All data are expressed as mean ± standard deviation; Compared with the control group # p<0.05, ## p<0.01; compared with the model group, *p<0.05, **p<0.01. Detailed Implementation

[0050] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] To further understand the present invention, it will be further described in conjunction with the accompanying drawings and embodiments.

[0052] Example 1

[0053] Fresh mussel meat was collected, washed, and drained. Water was added at a material-to-liquid ratio of 1:2 to form a homogenate. The enzymatic hydrolysis temperature was set at 55℃, and a complex protease (trypsin: neutral protease: alkaline protease: flavor protease: papain = 2:2:0.6:0.4:1) was added at 0.3% of the mussel meat weight. Hydrolysis was carried out for 4 hours. After hydrolysis, the hydrolysate was heated to 87℃ and held for 15 minutes to inactivate the enzyme. The hydrolysate was then cooled to 25℃ and centrifuged in a tube to obtain mussel peptide solution A. Ultrafiltration was performed to obtain three components based on molecular weight: component B (<5kDa), component C (5–50kDa), and component D (>5kDa). The ACE inhibition rate of mussel peptide solution A and components B, C, and D was determined.

[0054] The method for determining the ACE inhibition rate was based on the literature with appropriate modifications. The experiment was divided into a sample group (a), a control group (b), and a blank group (c), with three replicates for each group. 100 μL of 5 mmol / L HCl was added to groups a, b, and c respectively. Group a was then supplemented with 30 μL of the sample, while groups b and c were not supplemented. After vortexing and mixing, the mixture was incubated at 37°C for 10 min. After removal, 10 μL of 0.1 N ACE enzyme solution was added to groups a and b respectively, while group c was not supplemented. After vortexing and mixing, the mixture was incubated at 37°C for 30 min. The reaction was then terminated by adding 250 μL of 1 mol / L HCl solution to groups a, b, and c respectively. Then, 30 μL of the sample was added to group b alone, and the mixture was mixed. 1.2 mL of pre-cooled ethyl acetate was added to each of the three mixed groups, and the mixture was vortexed for 30 s, centrifuged at 3500 rpm for 5 min, and allowed to stand for 2 min. Take 1 mL of supernatant from each group and put it into a 5 mL centrifuge tube. Place the tube in a 90℃ oven for 1 h. After cooling to room temperature, add 4 mL of distilled water, zero the tube with the distilled water, vortex to mix, and measure the absorbance at 228 nm. Calculate the ACE inhibition rate of the enzyme hydrolysate according to the following formula.

[0055] ACE inhibition rate (%) = (A b -A a ) / (A b -A c )×100

[0056] In the formula: A a —The absorbance values ​​of the sample solution and ACE reacting simultaneously with HHL.

[0057] A b —Absorbance values ​​of the ACE and HHL reactions without sample solution.

[0058] A c —Absorbance value when neither the sample solution nor ACE is present.

[0059] Statistical analysis: SPSS 18.0 was used for statistical analysis. Quantitative data are expressed as mean ± standard deviation. One-way ANOVA was used to compare differences between groups. p < 0.05 and p < 0.01 indicated statistically significant and highly significant differences, respectively. The ACE inhibitory activities of components A, B, C, and D are as follows: Figure 1 As shown in the figure, the results indicate that component B exhibits strong ACE inhibitory activity.

[0060] Component B was further purified by Sephadex G-25 gel column chromatography at a loading concentration of 120 mg / mL and an elution flow rate of 3.0 mL / min, resulting in the separation of three main components, such as... Figure 2As shown, components B-1, B-2, and B-3 were used to determine their ACE inhibition rates using the aforementioned method. The ACE inhibition activities of components B-1, B-2, and B-3, with B-2 exhibiting the following results: Figure 3 As shown, the results indicate that component B-2 exhibits stronger ACE inhibitory activity, at 71.79%, and the molecular weight of component B-2 is 600–1800 Da.

[0061] Mussel peptides can be obtained by drying component B-2. The amino acid sequence of component B-2 was identified by LC-MS / MS, and the molecular weight of B-2 was determined by LC-MS / MS as follows: Figure 4 As shown in the figure. Five peptides were identified through comparison with protein databases: Ile-Leu-Thr-Glu-Arg (IR-5, 631.38 Da), Leu-Asp-Leu-Ala-Gly-Arg (LR-6, ​​644.37 Da), Val-Thr-Ile-Met-Pro-Lys (VK-6, 704.40 Da), Met-Gln-Ile-Phe-Val-Lys (MK-6, 765.43 Da), and Leu-Phe-Asp-Ala-Val-Ile-Arg (LR-7, 833.49 Da). The ACE inhibition rates of the five peptides are shown in the figure. Figure 5 As shown, all five peptides exhibited high ACE inhibitory activity. All data are expressed as mean ± standard error.

[0062] Example 2

[0063] Fresh mussel meat was taken, washed, and drained. Water was added at a material-to-liquid ratio of 1:3 to form a homogenate. The enzymatic hydrolysis temperature was set at 50°C. A complex protease (trypsin: neutral protease: alkaline protease: flavor protease: papain = 1.5:2.2:0.5:0.5:1.0) was added at 0.4% of the mussel meat weight. Hydrolysis was carried out for 3 hours. After hydrolysis, the hydrolysate was heated to 85°C and held for 10 minutes to inactivate the enzyme. The hydrolysate was then cooled to 10°C and subjected to tubular centrifugation and ultrafiltration to obtain peptide solutions with a molecular weight <5 kDa (the ACE inhibition rate of the peptide solutions was determined using the same method as in Example 1; the results showed that components in this molecular weight range had good ACE inhibitory activity). The peptides were then analyzed using Sephadex. Further purification was achieved using G-25 gel column chromatography, with a loading concentration of 100 mg / mL and an elution flow rate of 2.8 mL / min. Peptide solutions with molecular weights ranging from 600 to 1800 Da were obtained. (The ACE inhibition rate of each peptide solution obtained after purification was determined using the same method as in Example 1. The results showed that the components with molecular weights ranging from 600 to 1800 Da exhibited the best ACE inhibition activity.) The mussel peptides were then dried to obtain the final product.

[0064] Example 3

[0065] Fresh mussel meat was taken, washed, and drained. Water was added at a material-to-liquid ratio of 1:2 to form a homogenate. The enzymatic hydrolysis temperature was set at 53℃. A complex protease (trypsin: neutral protease: alkaline protease: flavor protease: papain = 2.0:1.8:0.6:0.4:1.5) was added at 0.2% of the mussel meat weight. Hydrolysis was carried out for 6 hours. After hydrolysis, the hydrolysate was heated to 90℃ and held for 20 minutes to inactivate the enzyme. The hydrolysate was then cooled to 40℃ and subjected to tubular centrifugation and ultrafiltration to obtain peptide solutions with a molecular weight <5kDa (the ACE inhibition rate of the peptide solutions was determined using the same method as in Example 1; the results showed that components in this molecular weight range had good ACE inhibitory activity). The peptides were then analyzed using Sephadex. Purification was performed using G-25 gel column chromatography with a loading concentration of 150 mg / mL and an elution flow rate of 3.2 mL / min to obtain peptide solutions with molecular weights ranging from 600 to 1800 Da (the ACE inhibition rate of each peptide solution obtained after purification was determined using the same method as in Example 1, and the results showed that the components with molecular weights ranging from 600 to 1800 Da had the best ACE inhibition activity). The peptides were then dried to obtain mussel peptides.

[0066] Example 4

[0067] Alternatively, dried mussels can be crushed and mixed with water at a ratio of 1:8, 1:9, or 1:10, with the remaining steps the same as in Example 1.

[0068] Experimental Example 1

[0069] Mussel peptide efficacy experiment

[0070] 1 Experimental Methods

[0071] 1.1 Test Drugs: Mussel peptide and captopril provided in Example 1

[0072] 1.2 Animal experimental protocol:

[0073] 12-week-old female SHR rats (n=18) and 12-week-old female Wistar yoto (WKY) rats (n=6) were provided by Vital River Experimental Technology Co., Ltd. (SCXK 2016-0006, Beijing, China).

[0074] All experimental animals were housed in a room with a temperature of 19–25℃, humidity of 60%–70%, and artificial light and darkness for 12 hours each. All groups had free access to water and food. After one week of acclimatization, SHR rats were randomly divided into 3 groups (n=6), and WKY rats (n=6) were used as the normal control group. Different drugs were administered daily: (1) Normal control group: 1 mL·kg physiological saline. -1 ·d -1(2) Model group, 1 mL / kg physiological saline -1 ·d -1 (3) Positive drug group, captopril 8 mg / kg -1 ·d -1 (4) Mussel peptides (MEPs-E) group, mussel peptide powder 400 mg·kg -1 ·d -1 .

[0075] The systolic blood pressure (SBP) and diastolic blood pressure (DBP) of rats were measured using the BP-2010A non-invasive animal blood pressure testing system. Short-term single-dose blood pressure data were obtained at 0, 2, 4, 6, 8, and 24 hours after the first administration. For long-term intervention, blood pressure was measured weekly, with at least three successful measurements taken for each rat, and the average blood pressure was obtained. After four weeks of continuous gavage, rats were fasted for 12 hours after the last administration and weighed. Rats were euthanized after anesthesia, and blood was collected from the abdominal aorta. The blood was centrifuged at 3000 rpm for 15 minutes, and serum was separated. Heart, kidney, and thoracic aorta tissues were collected and weighed. A portion of the thoracic aorta, kidney, and heart were preserved in 4% paraformaldehyde solution for HE staining and Masson staining. All serum and tissue samples were stored at -80°C before use.

[0076] 1.3 Serum sample testing:

[0077] Serum triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), blood urea nitrogen (BUN), creatinine (CR), uric acid (UA), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH) were measured using a fully automated biochemical analyzer. Serum nitric oxide (NO) and endothelin-1 (ET-1) in rats were detected using an ELISA kit.

[0078] 1.4 Histopathological analysis:

[0079] Paraffin-embedded tissue sections were sliced ​​to a thickness of 5 μm and subjected to histopathological analysis using HE staining and Masson staining, respectively. The vascular lumen cross-sectional area (LCSA), vascular wall cross-sectional area (WCSA), vascular inner diameter (ID), and vascular wall thickness (WT) were measured using Image-pro plus software. The ratio of LCSA to LCSA, the ratio of wall thickness to inner diameter (WT / ID), and the ratios of myofibrils and myofibrils in blood vessels, kidneys, and heart, as well as the collagen volume fraction (CVF), were further calculated and analyzed.

[0080] 2 Experimental Results

[0081] 2.1 Blood pressure reduction test after drug administration in spontaneously hypertensive rats

[0082] Results of the effect of mussel peptides (MEPs-E) on blood pressure in SHR rats:

[0083] The short-term single-dose antihypertensive experimental data were obtained at 0, 2, 4, 6, 8, and 24 hours after the first administration, as shown in Table 1 below:

[0084] Table 1. Changes in SBP and DBP in rats within 24 hours after the first gavage.

[0085]

[0086] During the long-term intervention, blood pressure was measured weekly, and the average blood pressure was obtained after at least three successful measurements for each rat. After four weeks of continuous gavage, the experimental data are shown in Table 2 below:

[0087] Table 2. Changes in SBP and DBP in rats during the 4-week experimental period after continuous gavage.

[0088]

[0089]

[0090] (Compared with the control group, #p<0.05, ##p<0.01; compared with the model group, *p<0.05, **p<0.01.)

[0091] Meanwhile, in the results of the effect of mussel peptides (MEPs-E) on blood pressure in SHR rats, the short-term single-dose antihypertensive experiment evaluated the antihypertensive effect of mussel peptides as follows: Figure 6 As shown in (A), compared with the model group, both the captopril group and the MEPs-E group showed the maximum reduction in systolic and diastolic blood pressure 8 hours after gavage. The captopril group showed a 41.7% decrease in systolic blood pressure and a 38.3% decrease in diastolic blood pressure; the MEPs-E group showed a 32.7% decrease in systolic blood pressure and a 22.8% decrease in diastolic blood pressure. Blood pressure recovered after 24 hours. The results of blood pressure changes in rats during the 4-week experiment are shown below. Figure 6 (B) shows that SBP and DBP in the model group were significantly higher than those in the control group (p<0.01). Compared with the model group, SBP and DBP were significantly reduced after 1 week of gavage administration of captopril and MEPs-E and remained so until the end of the experiment. After 4 weeks of gavage administration, SBP in the positive drug group decreased by 34.7% and DBP decreased by 37.9%; SBP in the MEPs-E group decreased by 21.6% and DBP decreased by 12.4%.

[0092] 2.2 Tests on the regulation of lipid metabolism after drug administration in spontaneously hypertensive rats

[0093] The levels of lipid markers TG, TC, LDL-C, and HDL-C in serum samples of hypertensive rats are as follows: Figure 7As shown, compared with the control group, the serum levels of TG, TC, and LDL-C in the model group were significantly increased (p < 0.01), while the HDL-C level was significantly decreased (p < 0.01). Compared with the model group, the serum levels of TG, TC, and LDL-C in the MEPs-E group were significantly decreased (p < 0.01), while the serum HDL-C levels in the yang-drug group and the MEPs-E group were increased (p > 0.05).

[0094] Hypertension and hyperlipidemia are two major risk factors for cardiovascular disease, with elevated blood pressure often accompanied by hyperlipidemia. Dyslipidemia is mainly characterized by elevated levels of TG, TC, and LDL-C, and decreased levels of HDL-C. These results indicate that serum TG, TC, and LDL-C levels were significantly reduced and HDL-C levels were increased in SHR rats after intervention with mussel peptides, suggesting that MEPs-E can regulate lipid metabolism and alleviate the syndrome caused by dyslipidemia.

[0095] 2.3 Tests to improve vascular injury after drug administration in spontaneously hypertensive rats

[0096] Results of HE staining of rat aortic arch as follows Figure 8 As shown in (A), the aorta of the normal control group rats had normal structure and morphology, a smooth intima, and relatively uniform wall thickness; the tunica media fibers were neatly arranged without thickening, and the vascular smooth muscle cells were evenly and orderly arranged; the adventitia was intact, and the layers of the vessel wall were clearly distinguishable, with clear differentiation between the intima, media, and adventitia. In the model group, the aorta of the hypertensive rats showed significant lesions, with a rough and uneven intima, disordered arrangement of tunica media fibers, hypertrophic vascular smooth muscle cells, adventitia damage, and thickened vessel wall. After long-term treatment, compared with the model group, the aortic tissue structure of the Yangyao group and the MEPs-E group showed significant improvement, with a smooth intima, neatly arranged tunica media fibers, restored vascular smooth muscle cells, and significantly reduced vessel wall thickness. Masson staining results are as follows. Figure 8 As shown in (B), collagen fibers were stained blue and muscle fibers were stained red. In the control group, the area of ​​collagen fibers in the aorta of rats was significantly less, and the muscle fibers were neatly arranged. In the model group, the area of ​​collagen fibers in the aorta of rats was significantly increased, while the area of ​​muscle fibers was decreased, and the arrangement of muscle fibers was disordered. After long-term treatment with captopril and mussel peptides, the degree of vascular fibrosis in rats was significantly improved, the area of ​​collagen fibers was significantly reduced, and the muscle fibers were neatly arranged. Vascular WCSA / LCSA and WT / ID results are shown in [Figure 1]. Figure 9As shown in (C)(D), compared with the control group, the WCSA / LCSA and WT / ID ratios in the model group rats were significantly increased (p < 0.01), with statistically significant differences. After MEPs-E treatment, compared with the model group, the WCSA / LCSA ratio in rats decreased, while the WT / ID ratio decreased significantly (p < 0.01), indicating that MEPs-E can improve aortic vascular remodeling in SHR rats. Vascular collagen fiber area / muscle fiber area and CVF results are shown below. Figure 9 As shown in (E) and (F), compared with the control group, the cardiovascular collagen fiber / muscle fiber area ratio and CVF of rats in the model group were significantly increased (p < 0.01); after treatment, compared with the model group, the cardiovascular collagen fiber / muscle fiber area ratio and CVF of rats in the Yangyao group and MEPs-E group were significantly decreased (p < 0.01). This indicates that MEPs-E can significantly improve aortic collagen deposition in SHR rats.

[0097] Serum ET-1 and NO levels in each group of SHR rats are as follows: Figure 9 As shown in (G)(H), compared with the control group, the NO content in the serum of SHR rats decreased and the ET-1 content increased. However, the above phenomena were alleviated to some extent after MEPs-E intervention, indicating that MEPs-E may protect endothelial cell function and improve vascular remodeling by maintaining the secretion balance between NO and ET-1.

[0098] Hypertension is closely related to vascular remodeling. Vascular remodeling is a significant pathological feature of hypertension and one of the main causes of many systemic complications such as atherosclerosis and heart and kidney failure. The above results indicate that after treatment with mussel peptides, the aortic tissue structure was significantly improved, the vessel wall thickness was significantly reduced, the WT / ID ratio was significantly decreased, and the ratio of collagen fibers to muscle fibers and CVF were significantly reduced. This suggests that mussel peptides can improve vascular remodeling in hypertensive rats.

[0099] 2.4 Tests to improve kidney damage after drug administration in spontaneously hypertensive rats

[0100] Results of HE staining of rat kidneys as follows Figure 10 (A) shows that, compared with the control group, the model group rats exhibited significant glomerular atrophy and vacuolar deformation, with some showing necrosis, and marked congestion of the renal interstitial matrix. After treatment with mussel peptide, the degree of renal lesions in rats improved to varying degrees, with no congestion of the renal interstitial matrix and no significant deformation of the glomerular and tubular epithelial cells. Masson staining results of rat kidneys are as follows. Figure 10 As shown in (B), compared with the control group, the model group rats showed a significant increase in renal collagen fiber area and a decrease in muscle fiber area, with disordered muscle fiber arrangement. After treatment with mussel peptides, the degree of renal fibrosis in rats was significantly improved, the collagen fiber area was significantly reduced, and the muscle fiber arrangement became more regular. Furthermore, as shown in [the original text]... Figure 11 As shown in (C)(D), compared with the control group, the collagen fiber / muscle fiber area and CVF in the kidneys of rats in the model group were significantly increased (p<0.01); compared with the model group, the collagen fiber / muscle fiber area and collagen volume fraction (CVF) in the kidneys of rats in the MEPs-E group were significantly decreased (p<0.01). This indicates that MEPs-E can significantly improve collagen deposition in the kidneys of SHR rats.

[0101] Metabolic indicators related to kidney damage, such as Figure 11 As shown in (E), compared with the control group, the serum levels of BUN and UA in the model group were significantly increased (p<0.01) and the CR level was significantly increased (p<0.05). Compared with the model group, the serum levels of BUN and CR in the Yangyao group and the MEPs-E group were significantly decreased (p<0.01) and the UA level was significantly decreased (p<0.05), indicating that mussel peptides improved kidney damage while lowering blood pressure.

[0102] 2.5 Tests to improve cardiac injury after drug administration in spontaneously hypertensive rats

[0103] The protective effect of MEPs-E on cardiac structure in SHR rats was observed by staining results as follows: Figure 12 (A)-(B) show that, compared with the control group, the cardiomyocytes of rats in the SHR model group exhibited significant deformation, irregular morphology, uneven thickness, and disordered arrangement, with varying degrees of swelling, nuclear lysis, myocardial necrosis, and a significant increase in collagen fibers. After MEPs-E treatment, the morphology of rat cardiomyocytes improved to varying degrees, with a relatively orderly arrangement and a reduction in collagen fibers. Simultaneously, the myofibril / myofibril area ratio and CVF were measured using Image-proplus software, and the results are as follows: Figure 13 As shown in (C)(D), compared with the control group, the collagen fiber / muscle fiber area and collagen volume fraction (CVF) of the heart in the model group rats were significantly increased (p<0.01); after treatment, compared with the model group, the collagen fiber / muscle fiber area and collagen volume fraction (CVF) of the heart in the positive control group and the MEPs-E group rats were significantly decreased (p<0.01). This indicates that MEPs-E can significantly improve collagen deposition in the heart of SHR rats. The detection results of markers related to myocardial injury, CK, CK-MB, and LDH, are as follows: Figure 13 As shown in (E), compared with the control group, the serum levels of CK and LDH in the model group were significantly increased (p<0.01), and the serum level of CK-MB was significantly increased (p<0.05); compared with the model group, the serum level of CK in the MEPs-E group was significantly decreased (p<0.05); the serum levels of CK-MB and LDH were also decreased.

[0104] The above results indicate that MEPs-E can improve cardiac deformity in SHR rats, reduce the area of ​​myocardial fibrosis, and lower the levels of serum CK, CK-MB, and LDH in hypertensive rats, suggesting that MEPs-E also has a significant protective effect against myocardial injury.

[0105] In summary, the mussel peptide of the present invention exhibits a good antihypertensive effect in spontaneously hypertensive model rats. Specifically, after oral administration of mussel peptide to spontaneously hypertensive model rats for one week, both systolic and diastolic blood pressure were significantly reduced and remained stable after the reduction.

[0106] It exerts a good regulatory effect on lipid metabolism in spontaneously hypertensive rats, especially on dyslipidemia caused by elevated serum triglyceride (TG), cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) levels and decreased low-density lipoprotein cholesterol (LDL-C) levels.

[0107] It exhibits good effects in improving vascular damage in spontaneously hypertensive rat models, especially in vascular damage caused by vascular fibrosis, aortic remodeling, and aortic collagen deposition.

[0108] It exhibits good effects in improving kidney damage in spontaneously hypertensive rat models, especially kidney damage caused by glomerular atrophy, renal fibrosis, renal collagen deposition, and elevated blood urea nitrogen (BUN), uric acid (UA), and creatinine (CR).

[0109] It exhibits good effects in improving cardiac damage in spontaneously hypertensive rat models, especially cardiac damage caused by cardiac collagen deposition and elevated levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH).

[0110] A comparative experiment was conducted on the blood pressure lowering effects of the mussel peptide provided by this invention and related products in the prior art. Table 3 below shows the data results obtained from the comparative experiment.

[0111] Table 3 compares the blood pressure-lowering effects of the mussel peptide provided by this invention with those of related products in the prior art.

[0112]

[0113]

[0114] The above results indicate that the mussel peptide prepared by this invention has a good antihypertensive effect on spontaneous hypertension compared with related products in the prior art, and can be applied to antihypertensive drugs or health foods.

[0115] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, alterations, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mussel peptide, characterized in that, The amino acid sequence of the mussel peptide is Leu-Phe-Asp-Ala-Val-Ile-Arg.

2. The method for preparing mussel peptide according to claim 1, characterized in that, Includes the following steps: (1) Pretreatment: Mussel meat and water are homogenized at a certain material-to-liquid ratio and then set aside for use; (2) Enzymatic hydrolysis: Using homogenate as substrate, set the enzymatic hydrolysis temperature to 50~55℃, add complex protease and stir for 3~6 h to obtain enzymatic hydrolysate. After enzymatic hydrolysis, heat the enzymatic hydrolysate to 85℃~90℃ and keep it for 10~20 min to inactivate the enzyme. The complex protease includes trypsin, neutral protease, alkaline protease, flavor protease and papain in the ratio of (1.5~2.0):(1.8~2.2):(0.5~0.7):(0.3~0.5):(1~1.5). (3) Centrifugation: Cool the enzyme hydrolysate to 10℃~40℃ and centrifuge; (4) Membrane filtration: The centrifuged liquid is filtered through microfiltration and ultrafiltration membranes in sequence to obtain a peptide solution with an average relative molecular mass of less than 5000 Da; (5) Purification: The obtained peptide solution was further purified by Sephadex G-25 gel filtration chromatography to obtain peptides with a molecular weight of 600~1800 Da; (6) Concentration and drying: The obtained peptides are concentrated by nanofiltration and then instantaneously dried into powder.

3. The preparation method according to claim 2, characterized in that, In step (1), fresh mussel meat is homogenized with water at a ratio of 1:2 to 1:3, and dried mussels are pulverized and homogenized with water at a ratio of 1:8 to 1:

10. In step (2), the amount of compound protease added is 0.2 to 0.4% of the mass of fresh mussel meat or 0.7 to 1.0% of the mass of dried mussels.

4. The preparation method according to claim 2, characterized in that, In step (2), the ratio of trypsin, neutral protease, alkaline protease, flavor protease and papain is 2:2:0.6:0.4:

1.

5. The preparation method according to claim 2, characterized in that, In step (5), the sample concentration for Sephadex G-25 gel filtration chromatography is 100~150 mg / mL, and the elution flow rate is 2.8~3.2 mL / min.

6. The use of the mussel peptide according to claim 1 in the preparation of antihypertensive drugs.

7. A medicament comprising the mussel peptide of claim 1 and a pharmaceutically acceptable excipient or carrier.

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