Myocardial active peptide capable of relieving heart failure and protecting heart and preparation method thereof

Abstract

The invention relates to the technical field of active peptides, in particular to a myocardial active peptide capable of relieving heart failure and protecting heart and a preparation method thereof. The method takes pig hearts as raw materials, and sequentially comprises the following steps: pretreating the pig hearts, adding water, and homogenizing; adding alkaline protease to carry out first constant-temperature enzymolysis; adding neutral protease and flavourzyme to carry out second constant-temperature enzymolysis; then lipase is added for third-time constant-temperature enzymolysis, and enzyme deactivation and cooling are carried out; and finally, adding activated carbon into the enzymatic hydrolysate, filtering with diatomite, and spray-drying to obtain the myocardial active peptide. According to the method, the yield and the purity of the active peptide are remarkably improved through a synergistic process of three-step enzymolysis and activated carbon purification, the obtained product is rich in small molecule peptide with a definite heart protection function, and a new way is provided for high-value utilization of pig heart resources and development of functional products for preventing and treating heart failure.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide technology, and in particular to a myocardial bioactive peptide that can alleviate heart failure and protect the heart, and its preparation method. Background Technology

[0002] Myocardial dysfunction can lead to diseases such as myocarditis and myocardial infarction, affecting the normal function of the heart and potentially causing heart failure in severe cases, resulting in persistently high morbidity and mortality rates from cardiovascular diseases. In alleviating heart failure and protecting the heart, clinical treatments mainly focus on chemically synthesized small-molecule drugs. While these methods have some effect, they suffer from significant side effects and poor stability, failing to meet the demand for safe and effective products that can alleviate heart failure and protect the heart. Bioactive peptides are easily absorbed by the gastrointestinal tract, thus exerting their biological activity in the human body and exhibiting higher bioavailability. They have potential application value in alleviating heart failure and protecting the heart. Existing technologies include research on extracting bioactive peptides from animal tissues, but these mainly focus on functions such as antioxidation or lowering blood pressure. Therefore, developing myocardial bioactive peptides with cardioprotective functions for alleviating heart failure is of great significance.

[0003] In addition, pig hearts, as a by-product of livestock and poultry processing, are abundant but have low utilization rates, and their high-value development has significant economic and social implications.

[0004] Therefore, developing a method for preparing myocardial active peptides from pig heart that can alleviate heart failure and protect the heart is of great scientific significance and application prospects. Summary of the Invention

[0005] To address the shortcomings of the prior art, this invention provides a method for preparing a myocardial active peptide capable of alleviating heart failure and protecting the heart, comprising the following steps: S1. After pre-treating the pig heart, add water and homogenize to obtain mixture A; S2. Add alkaline protease to the mixture A for the first isothermal enzymatic hydrolysis to obtain mixture B; S3. Add neutral protease and flavor protease to the mixture B and perform a second isothermal enzymatic hydrolysis to obtain mixture C; S4. Lipase is added to the mixture C for a third isothermal enzymatic hydrolysis. After enzyme inactivation and cooling, mixture D is obtained. S5. Add activated carbon to the mixture D, filter through diatomaceous earth, and spray dry to obtain myocardial active peptides.

[0006] Furthermore, the conditions for the first isothermal enzymatic hydrolysis described in S2 are: temperature 45-65℃, pH 7.5-9.5, time 1-3 h, and finally enzyme inactivation.

[0007] Furthermore, the conditions for the second isothermal enzymatic hydrolysis described in S3 are: temperature 40-60℃, pH 6.0-8.0, time 0.5-1.5 h, and finally enzyme inactivation.

[0008] Furthermore, the conditions for the third isothermal enzymatic hydrolysis described in S4 are: temperature 30-50℃, pH 6.0-8.0, time 0.5-1.5 h, and finally enzyme inactivation.

[0009] Furthermore, the ratio of the mass of the pig heart to the volume of water in S1 is 1:1-5.

[0010] Furthermore, the amount of activated carbon added in S5 is 3%-5% of the weight of the pig heart in S1; the working conditions of the activated carbon are: temperature 50-60℃, time 0.5-1.5 h.

[0011] Furthermore, the amount of alkaline protease added in S2 is 3000-7000 U / g pig heart; The addition amounts of neutral protease and flavor protease mentioned in S3 are 1000-2000 U / g pig heart, respectively; The amount of lipase added in S4 is 4000-8000 U / g pig heart.

[0012] The present invention also provides a myocardial active peptide that can alleviate heart failure and protect the heart, prepared by the preparation method described above.

[0013] Furthermore, the amino acid sequence of the myocardial active peptide is one or more combinations of IPI, IPV, VPL, LPGAL, PYLE, and LMP.

[0014] The present invention also provides a functional product, the components of which include the myocardial active peptides described above that can alleviate heart failure and protect the heart.

[0015] Compared with the prior art, the myocardial bioactive peptide and its preparation method provided by the present invention have the following beneficial effects: This invention utilizes a unique three-step sequential enzymatic hydrolysis and purification process to efficiently prepare cardioprotective peptides from pig heart raw materials. A specific enzymatic hydrolysis sequence—first, alkaline protease opens the higher-order structure of the protein; then, neutral and flavor proteases synergistically refine the protein; and finally, lipase removes lipid interference—constitutes a highly efficient synergistic hydrolysis system. This design significantly improves the yield and extraction efficiency of the target active peptides, overcoming the shortcomings of traditional single-enzyme or two-step enzymatic hydrolysis methods, such as limited hydrolysis sites and insufficient hydrolysis. It also enables the high-value utilization of pig heart by-products. Furthermore, the entire preparation process is simple, mild, stable, and easily scalable, ensuring that the final cardioprotective peptide product not only has clearly defined biological activity but also stable quality, making it suitable as a core functional ingredient for applications in health foods, special medical purpose formula foods, and other fields.

[0016] The myocardial bioactive peptides prepared by this method are rich in specific small molecule bioactive peptides. These peptides are characterized by small molecular weight and easy absorption. They can not only effectively enhance myocardial cell vitality and resist oxidative stress damage, but also significantly reduce the release of myocardial cell damage markers and inhibit the expression of key genes in myocardial fibrosis, thereby playing a multi-target and comprehensive role in alleviating heart failure and protecting cardiac function. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Cell viability of myocardial bioactive peptides at different sample concentrations in specific embodiments of the present invention; Figure 2 This is an example of the effect of myocardial bioactive peptides on H9C2 cell viability in a specific embodiment of the present invention; Figure 3 This invention illustrates the effect of myocardial bioactive peptides on the antioxidant activity index ROS in a specific embodiment. Figure 4 This is an example of the effect of myocardial bioactive peptides on LDH, a myocardial injury-related indicator, in a specific embodiment of the present invention. Figure 5 This is an example of the effect of myocardial bioactive peptides on the content of Ca-Mg-ATPase, a marker related to myocardial injury, in a specific embodiment of the present invention. Figure 6 This is an example of the effect of myocardial bioactive peptides on the mRNA expression of the myocardial fibrosis-related gene Col1a1 in a specific embodiment of the present invention. Figure 7 This is an example of the effect of myocardial bioactive peptides on the mRNA expression of the myocardial fibrosis-related gene Col3a1 in a specific embodiment of the present invention; Figure 8 This is an example of the effect of myocardial bioactive peptides on the mRNA expression of the myocardial fibrosis-related gene α-SMA in a specific embodiment of the present invention. Figure 9 This is an example of the effect of myocardial bioactive peptides on the mRNA expression of the myocardial fibrosis-related gene TGF-β1 in a specific embodiment of the present invention; Figure 10 The results of molecular docking between IPI and ACE, APJ are shown in a specific embodiment of the present invention. Figure 11 The results of molecular docking between IPV and ACE and APJ are shown in a specific embodiment of the present invention. Figure 12 The results of molecular docking between VPL and ACE and APJ are shown in a specific embodiment of the present invention. Figure 13 The results of molecular docking between LPGAL and ACE, APJ in a specific embodiment of the present invention; Figure 14 The results of molecular docking between PYLE and ACE and APJ are shown in a specific embodiment of the present invention. Figure 15 The results show the molecular docking of LMP with ACE and APJ in a specific embodiment of the present invention. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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, 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.

[0020] Example 1 A myocardial bioactive peptide capable of alleviating heart failure and protecting the heart, and its preparation method, comprising the following steps: Step 1: Wash and drain the pig heart (protein content 16.35%), cut it into small pieces, and add water at a ratio of 1:3 (g:mL) to homogenize and obtain a mixture.

[0021] Step 2: Pre-treat the mixture at 55℃ with constant stirring for 30 min. Adjust the pH of the mixture to 8.5 with 1 mol / L sodium hydroxide solution. After the pH stabilizes, add 5000 U / g alkaline protease and enzymatically hydrolyze at 55℃ for 2.0 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 8.5. Then, inactivate the enzyme at high temperature.

[0022] Step 3: Adjust the temperature of the mixture to 50℃, adjust the pH of the mixture to 7.0 with 1 mol / L hydrochloric acid solution, and after stabilization, add 1500 U / g of neutral protease and 1500 U / g of flavor protease. Enzymatically hydrolyze at 50℃ for 1.0 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 7.0, and then inactivate the enzyme at high temperature.

[0023] Step 4: Adjust the temperature of the mixture to 40℃. After stabilization, add 6000 U / g of lipase and enzymatically hydrolyze at 40℃ for 1.0 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 7.0.

[0024] Step 5: After enzymatic hydrolysis, inactivate the enzyme at 85℃ for 20 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0025] Step 6: Add activated carbon to the supernatant at 4% of the weight of the pig heart, keep it at 55℃ for 1.0 h, then pass it through diatomaceous earth and spray dry to obtain powdered myocardial active peptides.

[0026] Example 2 Step 1: Wash and drain the pig heart (protein content 16.35%), cut it into small pieces, and add water at a ratio of 1:1 (g:mL) to homogenize and obtain a mixture.

[0027] Step 2: Pre-treat the mixture at 45℃ with constant stirring for 30 min. Adjust the pH of the mixture to 7.5 with 1 mol / L sodium hydroxide solution. After the pH stabilizes, add 3000 U / g alkaline protease and enzymatically hydrolyze at 45℃ for 1.0 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 7.5. Then, inactivate the enzyme at high temperature.

[0028] Step 3: Adjust the temperature of the mixture to 40℃, adjust the pH of the mixture to 6.0 with 1 mol / L hydrochloric acid solution, and after stabilization, add 1000 U / g of neutral protease and 1000 U / g of flavor protease. Enzymatically hydrolyze at 40℃ for 0.5 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 6.0, and then inactivate the enzyme at high temperature.

[0029] Step 4: Adjust the temperature of the mixture to 30℃. After stabilization, add 4000 U / g of lipase and enzymatically hydrolyze at 30℃ for 0.5 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 6.0.

[0030] Step 5: After enzymatic hydrolysis, inactivate the enzyme at 80℃ for 10 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0031] Step 6: Add activated charcoal to the supernatant at 4% of the myocardial mass, keep at 50°C for 0.5 h, pass through diatomaceous earth, and then spray dry to obtain powdered myocardial active peptides.

[0032] Example 3 Step 1: Wash and drain the pig heart (protein content 16.35%), cut it into small pieces, and add water at a ratio of 1:5 (g:mL) to homogenize and obtain a mixture.

[0033] Step 2: Pre-treat the mixture at 65℃ with constant stirring for 30 min. Adjust the pH of the mixture to 9.5 with 1 mol / L sodium hydroxide solution. After the pH stabilizes, add 7000 U / g alkaline protease and enzymatically hydrolyze at 65℃ for 3.0 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 9.5, and then inactivate the enzyme at high temperature.

[0034] Step 3: Adjust the temperature of the mixture to 60℃, adjust the pH of the mixture to 8.0 with 1 mol / L hydrochloric acid solution, and after stabilization, add 2000 U / g of neutral protease and 2000 U / g of flavor protease. Enzymatically hydrolyze at 60℃ for 1.5 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 8.0, and then inactivate the enzyme at high temperature.

[0035] Step 4: Adjust the temperature of the mixture to 50℃. After stabilization, add 8000 U / g of lipase and enzymatically hydrolyze at 50℃ for 1.5 h. During the enzymatic hydrolysis, continuously add 1 mol / L sodium hydroxide solution to stabilize the pH of the solution at 8.0.

[0036] Step 5: After enzymatic hydrolysis, inactivate the enzyme at 90℃ for 30 min, cool, and centrifuge at 5000 rpm for 5 min to collect the supernatant.

[0037] Step 6: Add activated charcoal to the supernatant at 5% of the myocardial mass, keep at 60°C for 1.5 h, pass through diatomaceous earth, and then spray dry to obtain powdered myocardial active peptides.

[0038] Comparative Example 1 This comparative example involves a three-step hydrolysis process, using papain, neutral protease, flavor protease, and lipase in a stepwise manner. The difference between this comparative example and Example 1 is that the stirring temperature in step two of this comparative example is 55°C, the pH of the mixture is adjusted to 6.0 using 1 mol / L sodium hydroxide solution, the enzyme used is papain, and the hydrolysis conditions are: hydrolysis temperature 55°C, hydrolysis pH 6.0, and 1 mol / L sodium hydroxide solution is continuously added during the hydrolysis process to stabilize the solution pH at 6.0. The remaining operations and processes are the same as in Example 1.

[0039] Comparative Example 2 This comparative example involves a three-step hydrolysis process, employing acidic protease, neutral protease, flavor protease, and lipase in a stepwise manner. The difference between this comparative example and Example 1 is that the stirring temperature in step two of this comparative example is 37°C, the pH of the mixture is adjusted to 2.5 using 1 mol / L hydrochloric acid solution, the enzyme used is acidic protease, and the hydrolysis conditions are: hydrolysis temperature 37°C, hydrolysis pH 2.5, and 1 mol / L hydrochloric acid solution is continuously added during the hydrolysis process to stabilize the solution pH at 2.5. The remaining operations and processes are the same as in Example 1.

[0040] Comparative Example 3 This comparative example involves a three-step hydrolysis process, using bromelain, neutral protease, flavor protease, and lipase for stepwise enzymatic hydrolysis. The difference between this comparative example and Example 1 is that the stirring temperature in step two of this comparative example is 40°C, the pH of the mixture is adjusted to 6.5 using 1 mol / L sodium hydroxide solution, the enzyme used is bromelain, and the enzymatic hydrolysis conditions are: hydrolysis temperature 40°C, hydrolysis pH 6.0, and 1 mol / L sodium hydroxide solution is continuously added during the hydrolysis process to stabilize the solution pH at 6.5. The remaining operations and processes are the same as in Example 1.

[0041] Comparative Example 4 This comparative example involves a two-step hydrolysis process, using alkaline protease and lipase. The difference between this comparative example and Example 1 is that the hydrolysis time in step two is 3.0 h, step three is removed, and the amount of lipase added in step four is modified to 9000 U / g. The remaining operations and processes are the same as in Example 1.

[0042] Comparative Example 5 This comparative example involves a two-step hydrolysis process, using neutral protease and lipase. The difference between this comparative example and Comparative Example 4 is that the stirring temperature in step two of this comparative example is 50℃, the pH of the mixture is adjusted to 7.0 using 1 mol / L sodium hydroxide solution, the enzyme used is neutral protease, and the hydrolysis conditions are: hydrolysis temperature 50℃, hydrolysis pH 7.0, and 1 mol / L sodium hydroxide solution is continuously added during the hydrolysis process to stabilize the solution pH at 7.0. The remaining operations and processes are the same as in Comparative Example 4.

[0043] Comparative Example 6 This comparative example involves a two-step hydrolysis process, using papain and lipase. The difference between this comparative example and Comparative Example 4 is that the stirring temperature in step two of this comparative example is 55℃, the pH of the mixture is adjusted to 6.0 using 1 mol / L sodium hydroxide solution, the enzyme used is papain, and the hydrolysis conditions are: hydrolysis temperature 55℃, hydrolysis pH 6.0, and 1 mol / L sodium hydroxide solution is continuously added during the hydrolysis process to stabilize the solution pH at 6.0. The remaining operations and processes are the same as in Comparative Example 4.

[0044] Comparative Example 7 This comparative example involves a two-step hydrolysis process, using acidic protease and lipase. The difference between this comparative example and Comparative Example 4 is that the stirring temperature in step two of this comparative example is 37°C, the pH of the mixture is adjusted to 2.5 using 1 mol / L hydrochloric acid solution, the enzyme used is acidic protease, and the hydrolysis conditions are: hydrolysis temperature 37°C, hydrolysis pH 2.5, and 1 mol / L hydrochloric acid solution is continuously added during the hydrolysis process to stabilize the pH of the solution at 2.5. The remaining operations and processes are the same as those in Comparative Example 4.

[0045] Performance testing The myocardial bioactive peptides obtained in Examples 1-3 and Comparative Examples 1-7 were subjected to the following performance tests: 1. Cell viability detection H9C2 cell viability analysis was performed on the myocardial bioactive peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, 6, and 7.

[0046] The samples prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, 6, and 7 were respectively formulated into myocardial active peptide solutions with concentrations of 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL. These solutions were then applied to H9C2 cells for 24 h, and cell viability was measured using a CCK8 assay kit.

[0047] The results showed that the myocardial bioactive peptide samples had no significant cytotoxicity to H9C2 cells, and some samples even promoted cell proliferation. Figure 1 As shown, the cell viability was highest when the concentration of myocardial active peptide was 2 mg / mL, so a concentration of 2 mg / mL was selected for subsequent experiments.

[0048] H9C2 cells in good growth condition were seeded into 96-well plates at a cell density of 10,000 cells / well and cultured in a 37°C, 5% CO2 incubator for 24 h before being grouped.

[0049] This experiment was set up with the following four groups: Control group: only complete culture medium was added, and no cells were inoculated.

[0050] Normal group: H9C2 cells were inoculated and cultured in normal complete culture medium throughout the process.

[0051] Model group: H9C2 cells were inoculated and cultured normally for 24 hours, then cultured in a medium containing 10 µM doxorubicin for 24 hours to establish a myocardial injury model.

[0052] Experimental group: H9C2 cells were seeded and pretreated for 24 hours with a medium containing 2 mg / mL of specific myocardial active peptide samples (from Examples 1-3 and Comparative Examples 1-7, respectively). Then, the medium was replaced with a medium containing 10 µM doxorubicin (without peptides) and cultured for 24 hours.

[0053] After observing cell adhesion, the upper culture medium was discarded. Cell-free complete culture medium was added to the blank group, while complete culture medium containing the corresponding sample and concentration was added to the normal group and the model group, respectively. Meanwhile, 2 mg / mL of myocardial active peptide solution was added to the experimental group. The intervention lasted for 24 h.

[0054] After the intervention, all groups except the normal group were replaced with complete culture medium containing 10 µM doxorubicin, while the normal control group had its culture medium changed. Twenty-four hours after the doxorubicin intervention, the absorbance at 450 nm was measured using the CCK-8 assay to calculate cell viability.

[0055] The results showed that myocardial active peptides promoted the proliferation of H9C2 cells treated with doxorubicin and significantly improved cell activity.

[0056] Depend on Figure 2 It can be seen that the cell viability of the model group was significantly reduced compared with the normal group (P<0.05), indicating that the intervention of doxorubicin on H9C2 cells was successful. Compared with the model group, the cell viability of Examples 1-3 and Comparative Examples 1-6 was significantly increased (P<0.05), indicating that the myocardial bioactive peptide prepared in this invention can effectively promote the increase of H9C2 cell activity.

[0057] Furthermore, compared with the cell viability of the model group, no significant change was observed in Comparative Example 7 (P<0.05). This may be because different enzymatic hydrolysis processes resulted in inconsistencies in the enzyme cleavage sites during hydrolysis, leading to different polypeptide compositions and molecular structures, which in turn resulted in inconsistent effects on cardiomyocyte activity.

[0058] 2. Cellular antioxidant capacity analysis The antioxidant activity of myocardial active peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, 6, and 7 was analyzed in H9C2 cells. The grouping in this experiment was the same as that used for cell viability testing.

[0059] H9C2 cells with good growth were divided into groups of 1×10 5 Cells were seeded at a density of 1 cell per well in 96-well plates and cultured in a cell culture incubator at 37°C with 5% CO2 for 24 h to allow for cell adhesion.

[0060] After cell adhesion, the upper culture medium was discarded. Each group was then given complete culture medium containing the corresponding sample and concentration. The control group was given only cell-free complete culture medium, while the experimental groups were given 2 mg / mL of myocardial active peptide solution. The intervention lasted for 24 h. After the intervention, except for the normal group, all other groups were given complete culture medium containing 10 µM doxorubicin. The normal control group had its culture medium changed. The doxorubicin intervention lasted for 24 h.

[0061] ROS was measured according to the kit instructions, and the results are as follows: Figure 3 As shown.

[0062] Depend on Figure 3 As can be seen, the ROS content in the model group was significantly increased compared with the normal group (P<0.05), indicating that the modeling was successful. Among them, compared with the model group, the myocardial active peptides in the examples and each comparative example significantly reduced the ROS content of cells (P<0.05), indicating that myocardial active peptides can improve the oxidative stress of H9C2 cells to a certain extent.

[0063] Examples 1-3 showed significant differences from Comparative Examples 1 and 7 (P<0.05), indicating that the myocardial bioactive peptides prepared by the process of the present invention are significantly superior to alternative processes that use papain-initiated (Comparative Example 1) or acidic protease two-step method (Comparative Example 7) in alleviating myocardial cell oxidative stress.

[0064] 3. Enzyme activity detection The myocardial bioactive peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, 6, and 7 were subjected to myocardial injury marker lactate dehydrogenase (LDH) and myocardial enzyme Ca-Mg-ATPase activity assays. The grouping in this experiment was the same as that used for cell viability assays.

[0065] The activities of LDH and Ca-Mg-ATPase were determined according to the instructions of the enzyme activity kit. The test procedure is as follows: (1) Detection of lactate dehydrogenase (LDH) activity Sample collection: After the cells have been cultured, centrifuge them at 1000×g for 10 minutes to remove particles and polymers.

[0066] Operating steps 1) Mix all reagents thoroughly before use.

[0067] 2) Make 2-3 duplicate holes for each standard sample and blank hole.

[0068] 3) Add 50 μL of diluted standard to the reaction wells and 50 μL of the sample to be tested to the reaction wells. Immediately add 50 μL of biotin-labeled antibody. Cover the membrane plate, gently shake to mix, and incubate at 37°C for 1 hour. 4) Shake off the liquid in the well, fill each well with washing solution, shake for 30 seconds, shake off the washing solution, and pat dry with absorbent paper. Repeat this operation 3 times.

[0069] 5) Add 80 μL of avidin-HRP to each well, gently shake to mix, and incubate at 37°C for 30 minutes. 6) Shake off the liquid in the holes, fill each hole with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper.

[0070] 7) Add 50 μL each of substrate A and B to each well, gently shake to mix, and incubate at 37°C for 10 minutes. Avoid light exposure. 8) Remove the microplate and quickly add 50 μL of stop solution. The results should be measured immediately after adding the stop solution.

[0071] 9) Measure the OD value of each well at a wavelength of 450 nm.

[0072] 3. Data Processing With absorbance OD value as the ordinate (Y) and the corresponding concentration of the analyte standard as the abscissa (X), a curve is plotted. The concentration of the analyte in the sample can be calculated from the standard curve based on its OD value.

[0073] (2) Ca-Mg-ATPase activity detection After the cells were cultured as described above, add 300 μL of Tris-Triton lysis buffer and incubate on ice for 30 minutes for lysis. Transfer the lysis buffer to a 1.5 EP tube, vortex to promote lysis, centrifuge at 12000g at 4℃ for 10 minutes, and collect the supernatant.

[0074] Reagent preparation 1) Coenzyme I solution: Dissolve each vial of powder in 1.3 mL of double-distilled water.

[0075] 2) 0.4 mol / L NaOH: Dilute 4 mol / L NaOH 10 times with double-distilled water and prepare fresh each time.

[0076] 3) 0.2 μmol / mL sodium pyruvate standard solution: Dilute 2 μmol / mL sodium pyruvate standard solution 10 times with double-distilled water and prepare fresh before use.

[0077] 3. Experimental Procedure 1) Set up measurement wells, control wells, standard wells, and blank wells.

[0078] 2) Add in the following order: Sample / Standard / Double-distilled water (16ul), Reagent 1 (Matrix Buffer) (20ul), Reagent 2 (Coenzyme I) (4ul).

[0079] 3) Incubate at 37℃ for 15 minutes.

[0080] 4) Add reagent tris(2,4-dinitrophenylhydrazine) (20 μL) and incubate at 37°C for 15 minutes.

[0081] 5) Add reagent four (0.4 mol / L NaOH) (200 μL) and mix well.

[0082] 6) Let it sit at room temperature for 5 minutes.

[0083] 7) Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 440 nm.

[0084] 4. Calculation formula ; C standard: Standard solution concentration, 0.2 μmol / mL; CPR: Sample protein concentration, g prot / mL (prot refers to protein).

[0085] The results are as follows: Figure 4 and Figure 5 As shown.

[0086] LDH is present in almost all human cells and catalyzes the interconversion between pyruvate and lactate. Cardiac cardiomyocytes contain abundant LDH; when the myocardium is damaged, LDH leaks into the bloodstream, leading to elevated serum LDH concentrations. Ca-Mg-ATPase is a key protein in cardiomyocyte relaxation, responsible for pumping cytoplasmic calcium ions back to the sarcoplasmic reticulum to store calcium for the next contraction. Ca-Mg-ATPase dysfunction is one of the core mechanisms of functional deterioration after cardiac injury; almost all cardiac injuries (myocardial infarction, heart failure, etc.) lead to a decrease in Ca-Mg-ATPase expression and activity, thus making Ca-Mg-ATPase a key target for heart failure treatment.

[0087] like Figure 4 and Figure 5 As shown, compared with the normal group, the cells in the model group had higher LDH and lower Ca-Mg-ATPase content, indicating that the modeling was successful.

[0088] Compared with other experimental groups, the peptide samples of Examples, Comparative Examples 1 and 7 had a more significant positive effect on the LDH content of H9C2 cells (P<0.05), but the Ca-Mg-ATPase content of the peptide sample of Comparative Example 1 was not significantly different from that of the model group; the peptide samples of Examples and Comparative Examples 2-7 significantly increased the Ca-Mg-ATPase content of H9C2 cells (P<0.05), and Examples and Comparative Examples 3-5 were more significant, while Comparative Example 1 had no significant change compared with the model group.

[0089] The effects of peptides prepared by different enzymatic hydrolysis processes on the viability, antioxidant capacity, and myocardial injury markers of H9C2 cells were analyzed. No process with a highly significant advantage was observed in Examples and Comparative Examples 2-6.

[0090] 4. Analysis of genes related to myocardial fibrosis in H9C2 cells The myocardial bioactive peptides prepared in Examples 1, 2, 3, Comparative Examples 1, 2, 3, 4, 5, 6, and 7 were analyzed for myocardial fibrosis-related genes in H9C2 cells to further verify the effects of peptides prepared by different enzymatic hydrolysis processes on H9C2 cell fibrosis.

[0091] The grouping in this experiment was the same as that in the cell viability assay.

[0092] H9C2 cells with good growth were divided into groups of 5 × 10 5Cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h to allow for cell attachment. After cell attachment, the supernatant was discarded, and each group was treated with complete culture medium containing the corresponding sample and concentration, along with 2 mg / mL of myocardial bioactive peptide solution. A control group (containing only complete culture medium without cells) was also included. The intervention lasted for 24 h. After the intervention, except for the normal control group, all other groups were treated with complete culture medium containing 10 µM doxorubicin, while the normal control group underwent a medium change.

[0093] Twenty-four hours after doxorubicin intervention, RNA was extracted from the treated cell samples of each group, and reverse transcription and Real-time qPCR were used to detect myocardial fibrosis-related genes.

[0094] The results are as follows Figure 6 , Figure 7 , Figure 8 and Figure 9 As shown, the expression of myocardial fibrosis-related genes in each embodiment was downregulated to varying degrees compared with the model group, indicating that the addition of myocardial active peptides has a positive preventive effect on myocardial fibrosis in H9C2 cells.

[0095] 5. Screening of myocardial active peptide fragments Mass spectrometry was used to identify peptide sequences, and PeptideRanker, ToxinPred, and BioPepDB were used to further screen for myocardial bioactive peptides that can promote bone growth and development. The specific experimental steps are as follows: The liquid chromatography conditions were as follows: pre-column: PEPMAP NEO C18, 300 μm × 5 mm. Analytical column: 150 μm id × 170 mm, packaging: Reprosil-Pur 120 C18-AQ 1.9 μm. Mobile phase A is 0.1% FA; mobile phase B is 0.1% FA, 80% ACN; flow rate is 600 nL / min; The analysis time for each component is 66 min.

[0096] The mass spectrometry conditions were as follows: full scan range 100-1500 m / z, first-stage mass spectrometry resolution set to 120000, AGC set to Standard, Maximum IT: 20 ms; second-stage mass spectrometry resolution set to Resolution: 15000, AGC set to Standard, Maximum IT: 22 ms, Cycle time: 2 s, peptide fragmentation collision energy set to 30, generating raw mass spectrometry data (.raw), ultimately obtaining more than 4500 peptide fragments.

[0097] Peptides with 2-6 amino acids were screened and sorted according to their relative abundance. ToxinPred was used to predict the toxicity of the top 12 peptides. BioPepDB was used to evaluate the innovativeness of the peptides and to conduct subsequent analysis.

[0098] The final screened peptides are shown in Table 1. The results indicate that these peptides are mainly composed of 3-5 amino acids, with molecular weights between 320-650 Da, consistent with the conclusion that bioactive peptides typically contain 2-20 amino acid residues. All 12 peptides predicted using ToxinPred were non-toxic, and BioPepDB assessments showed they were all innovative, as shown in Table 1.

[0099] Table 1. Screening results of cardiac peptides

[0100] 6. Molecular docking experiment Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin system (RAS), converting the less active angiotensin I (Ang I) into the highly active angiotensin II (Ang II). Ang II causes strong vasoconstriction, increases diastolic pressure, and directly promotes cell proliferation, hypertrophy, and fibrosis, making it a major culprit in the worsening of heart failure. Therefore, inhibiting ACE activity can not only alleviate heart failure symptoms but also fundamentally improve cardiac function, slow the progression of heart failure, and thus protect the heart. APJ receptors belong to the GPCR (G protein-coupled receptor) A family, and their endogenous ligands are currently known to be apelin and ELA. APJ receptors are an important protective receptor in the cardiovascular system; their activation can counteract several key processes leading to heart failure (such as vasoconstriction and cardiac remodeling). In heart failure, blood apelin levels decrease, affecting APJ receptor activation and consequently impacting the protection of cardiac function. ACE inhibitors work by inhibiting the "bad" receptors, while APJ receptor agonists work by activating the "good" receptors, thus improving heart failure. Theoretically, the two can complement each other and protect the heart from different levels.

[0101] Molecular docking software was used to perform molecular docking between the 12 peptides screened in Example 9 and the receptor proteins. The receptor proteins selected were ACE (PDB: 1O86) and APJ (PDB: 5VBL). The results showed that only 10 of the 12 peptides could bind to the two receptor proteins to a certain extent. Among them, 6 peptides with relatively high binding energies were selected, namely IPI, IPV, VPL, LPGAL, PYLE and LMP.

[0102] The molecular docking conformation diagrams of the above six peptides with ACE and APJ receptors are shown below. Figures 10-15 As shown. Figure 10 It can be seen that IPI binds to amino acid residues such as Asp and Ala of ACE and amino acid residues such as Lys and Arg of APJ receptor through hydrogen bonds, with the maximum "-CDOCKER ENERGY" values ​​at docking being 6.0 kJ / mol and 4.47 kJ / mol, respectively. Figure 11 It can be seen that IPV binds to amino acid residues such as Asn and Lys of ACE and amino acid residues such as Met and Glu of APJ receptor through hydrogen bonds. The maximum "-CDOCKER ENERGY" values ​​at docking are 4.78 kJ / mol and 4.01 kJ / mol, respectively. Figure 12 It can be seen that VPL binds to the Asp, Lys and other amino acid residues of ACE and the Lys, Glu and other amino acid residues of APJ receptor through hydrogen bonds. The maximum "-CDOCKER ENERGY" values ​​at docking are 4.83 kJ / mol and 4.07 kJ / mol, respectively. Figure 13 It can be seen that LPGAL binds to amino acid residues such as Asp and Trp of ACE and amino acid residues such as Arg and Glu of APJ receptor through hydrogen bonds, with the maximum "-CDOCKER ENERGY" values ​​at docking being 2.62 kJ / mol and 2.12 kJ / mol, respectively. Figure 14 It can be seen that PYLE binds to the Arg and Arg amino acid residues of ACE and the Arg and Glu amino acid residues of APJ receptor through hydrogen bonds, with the maximum "-CDOCKER ENERGY" values ​​at docking being 2.93 kJ / mol and 1.74 kJ / mol, respectively. Figure 15 It can be seen that LMP binds to amino acid residues such as Leu and Trp of ACE and amino acid residues such as Gln and Glu of APJ receptor through hydrogen bonds. The maximum "-CDOCKER ENERGY" values ​​at docking are 4.74 kJ / mol and 4.53 kJ / mol, respectively.

[0103] In summary, the first objective of this invention is to propose a myocardial active peptide that can alleviate heart failure and protect the heart. This myocardial active peptide has a clear activity in alleviating heart failure and protecting the heart, and it is derived from natural resources, with high safety and good stability. This provides a new idea and approach for developing novel, safe, and effective products for alleviating heart failure and protecting the heart.

[0104] The second objective of this invention is to provide a method for preparing the aforementioned myocardial active peptide, which has low equipment requirements, is simple and easy to operate, facilitates large-scale industrial production, and is conducive to promoting the development of the livestock and poultry processing by-product industry.

[0105] The third objective of this invention is to propose an application of the described myocardial active peptide or the method for preparing the described myocardial active peptide in the research and development of myocardial active peptides, to help researchers better understand the interaction mechanism between peptide sequences and targets, and to provide theoretical support for further optimization of peptide sequences.

[0106] Although this document frequently uses terms such as myocardial active peptide, peptide segment, alkaline protease, and neutral protease, the possibility of using other terms is not excluded. These terms are used merely for the convenience of describing and explaining the essence of this invention; interpreting them as any additional limitation would contradict the spirit of this invention.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a myocardial active peptide capable of alleviating heart failure and protecting a heart, characterized by, The preparation method comprises the following steps: S1, pretreating a pig heart and then adding water to homogenize to obtain a mixed solution A; S2, adding alkaline protease to the mixed solution A to perform first constant-temperature enzymolysis to obtain a mixed solution B; S3, adding neutral protease and flavor protease to the mixed solution B to perform second constant-temperature enzymolysis to obtain a mixed solution C; S4, adding lipase to the mixed solution C to perform third constant-temperature enzymolysis, and then cooling to obtain a mixed solution D; S5, adding activated carbon to the mixed solution D, filtering through diatomite, and then spray drying to obtain a heart muscle active peptide.

2. The method of claim 1, wherein: The first constant-temperature enzymolysis in S2 is performed under the following conditions: a temperature of 45-65 DEG C, a pH of 7.5-9.5, a time of 1-3 h, and finally enzyme inactivation.

3. The method of claim 1, wherein: The second constant-temperature enzymolysis in S3 is performed under the following conditions: a temperature of 40-60 DEG C, a pH of 6.0-8.0, a time of 0.5-1.5 h, and finally enzyme inactivation.

4. The method of claim 1, wherein: The third constant-temperature enzymolysis in S4 is performed under the following conditions: a temperature of 30-50 DEG C, a pH of 6.0-8.0, a time of 0.5-1.5 h, and finally enzyme inactivation.

5. The method of claim 1, wherein: The mass of the pig heart to the volume of water in S1 is 1:1-5.

6. The preparation method according to claim 1, characterized in that: The activated carbon in S5 is added in an amount of 3%-5% of the mass of the pig heart in S1, and the activated carbon is used under the following conditions: a temperature of 50-60 DEG C and a time of 0.5-1.5 h.

7. The method of claim 1, wherein: The alkaline protease in S2 is added in an amount of 3000-7000 U / g of the pig heart; The neutral protease and the flavor protease in S3 are added in amounts of 1000-2000 U / g of the pig heart, respectively; The lipase in S4 is added in an amount of 4000-8000 U / g of the pig heart.

8. A heart muscle active peptide capable of relieving heart failure and protecting the heart, which is prepared by the preparation method in any one of claims 1-7.

9. The cardiomyocyte active peptide according to claim 8, characterized by: The amino acid sequence of the heart muscle active peptide is one or more combinations of IPI, IPV, VPL, LPGAL, PYLE and LMP.

10. A functional product characterized by: The components include the heart muscle active peptide capable of relieving heart failure and protecting the heart according to claim 8 or 9. The amino acid sequence of the heart muscle active peptide is one or more combinations of IPI, IPV, VPL, LPGAL, PYLE and LMP. The components include the heart muscle active peptide capable of relieving heart failure and protecting the heart according to claim 8 or 9.

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