Application of cardiomodulin in ischemia-reperfusion injury drugs after myocardial infarction intervention

Abstract

The application provides application of a myocardial peptide in ischemia-reperfusion injury drugs after myocardial infarction intervention, and belongs to the technical field of biological medicine. The application provides a preparation method of the myocardial peptide. The myocardial peptide polypeptide prepared through sound wave treatment and electrolysis treatment has higher polypeptide content and small molecule polypeptide ratio, higher yield, and a simplified process. The myocardial peptide provided by the application can improve the heart function of STEMI patients treated by PCI, reduce the myocardial infarction area, and can be used for preparing drugs for treating myocardial infarction.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to ST-segment elevation myocardial infarction, specifically to the application of cardiac peptides in drugs for ischemia-reperfusion injury after interventional myocardial infarction. Background Technology

[0002] For acute ST-segment elevation myocardial infarction (STEMI), primary percutaneous coronary intervention (PPCI) is currently the most effective treatment. It can rapidly open the affected blood vessel, reduce the infarct size, increase the number of viable myocardial cells, and significantly reduce mortality. Although coronary blood flow can return to normal after direct PCI, a significant proportion of patients still experience microvascular obstruction, resulting in increased infarct size, reduced ventricular function, and increased mortality. Therefore, further research is needed to effectively protect the coronary microcirculation, further reduce the infarct size, and improve post-infarction cardiac function.

[0003] Cardiac peptides are small molecule polypeptides first extracted from the ventricular myocardium of piglets by domestic institutions. Numerous animal experiments have confirmed that these peptides possess significant biological activity and exhibit marked preventative and therapeutic effects on myocardial injury caused by various factors, especially acute ischemia. Research results indicate that heat shock protein (HSP)70 has anti-myocardial ischemia effects, suggesting that polypeptides or proteins may be a promising new class of anti-myocardial ischemia drugs. Studies have shown that energy metabolism disorders are the main pathogenesis basis of ischemia-reperfusion injury, and the role of free radicals is a crucial link in its pathogenesis. A series of animal experiments have found that cardiac peptides can reduce the release of myocardial enzymes after ischemia-reperfusion, inhibit intracellular calcium currents in ventricular myocardial cells, reduce the incidence of severe arrhythmias after ischemia-reperfusion, enhance the myocardium's ability to scavenge oxygen free radicals, improve energy metabolism in ischemia-reperfusion-damaged myocardium, increase membrane lipid fluidity, help maintain cell membrane function, and reduce degeneration and necrosis in damaged myocardium, thus demonstrating a preventative and therapeutic effect on myocardial ischemia-reperfusion injury.

[0004] Chinese patent CN1552733A discloses a method for preparing cardiac peptides. The method involves washing and chopping the ventricular myocardium (excluding human) of a healthy mammal, adding sterile distilled water to form a slurry, repeatedly freezing and thawing the slurry 3-4 times, heating it to 65-95℃, filtering to remove residue, filtering with a plate and frame filter to obtain a coarse filtrate, then ultrafiltration with a hollow fiber column to obtain a fine filtrate, ultrafiltration with an ultrafiltration membrane, and concentration by reverse osmosis to obtain a cardiac peptide solution with a molecular weight less than 10000 Da. The solution is then filtered, sterilized, and freeze-dried to obtain the final product.

[0005] Chinese patent CN101012455A discloses a method for inactivating biochemical substances. The method includes taking animal organs or tissues (excluding humans), extracting the bioactive components therein, and heating the solution of the extracted active components at 60-85°C for 10-120 minutes to inactivate the virus, preferably at 75-85°C for 20-80 minutes.

[0006] Cardiac peptides have been approved by the National Medical Products Administration as a Class I chemical drug for use as an adjunct to myocardial protection during the perioperative period of cardiac surgery. Currently, the efficacy of cardiac peptides in improving ischemia-reperfusion in STEMI patients after direct PCI is unclear, and there are no clinical studies using myocardial magnetic resonance imaging (MRI) to more accurately evaluate the preventive and therapeutic effects of cardiac peptides on myocardial ischemia-reperfusion in STEMI patients. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides the application of cardiac peptides in drugs for ischemia-reperfusion injury after interventional myocardial infarction. This invention also provides a method for preparing cardiac peptides, which, through acoustic treatment and electrolysis, yields cardiac peptides with higher polypeptide content and a higher proportion of small molecule peptides, resulting in a higher yield, while simplifying the process.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a method for preparing cardiac peptides, comprising the following steps:

[0010] S1. Take the ventricular muscle of a piglet, mix it with water, and homogenize it to obtain a homogenate.

[0011] S2. The homogenate is subjected to acoustic treatment at a frequency of 30-40Hz for 20-30 minutes.

[0012] S3. Apply alternating current and process for 5-10 minutes. Then, disconnect the power supply and apply constant voltage reverse current for 15-25 minutes. Finally, apply constant voltage forward current for 12-18 minutes to obtain a mixture. The frequency of the alternating current is 38-46Hz and the voltage is 300-360V. The voltage of the constant voltage reverse current and constant voltage forward current is 220-300V.

[0013] S4. The mixture is filtered through gauze to obtain coarse filtrate. The coarse filtrate is then passed through a hollow fiber column to obtain fine filtrate.

[0014] S5. Heat the filtrate to inactivate it, and then filter it through a filter membrane to obtain the inactivated solution;

[0015] S6. Concentrate the inactivation solution to obtain a myocardial peptide solution;

[0016] S7. The myocardial peptide solution is freeze-dried to obtain the finished myocardial peptide product.

[0017] Preferably, step S1 includes the following steps:

[0018] The materials were added according to the mass ratio of ventricular myocardium to water for injection of 1:1-2, and homogenized three times using a colloid mill to obtain a homogenate.

[0019] Preferably, the mass ratio of ventricular myocardium to water for injection is 1:1.

[0020] Preferably, when homogenizing the colloid mill, centrifugation is performed at 3000 rpm / min for 5 minutes each time, with a 5-minute interval between each centrifugation.

[0021] Preferably, step S4 includes the following steps:

[0022] The coarse filtrate was filtered twice under positive pressure through a three-column hollow fiber column connected in series to obtain a fine filtrate with a molecular weight cutoff of ≤12KD.

[0023] Preferably, during positive pressure filtration, the peristaltic pump pressure is 0.2 MPa, the permeate flow rate is 5 ± 3 ml / min, and the initial 50 ml of solution is discarded.

[0024] Preferably, step S5 includes the following steps:

[0025] Heat the filtrate to 75-85℃ and stir in a water bath for 1-2 hours, then filter it through a microporous membrane for sterilization.

[0026] Preferably, the filtrate is heated to 80℃±2℃ and stirred in a water bath for 1 hour, then filtered through a 0.22μm microporous membrane for sterilization.

[0027] Preferably, in step S6, a reverse osmosis concentration column is used for concentration.

[0028] Preferably, freeze drying is used in step S7.

[0029] In some specific embodiments, the freeze-drying process involves the following steps: The shelf temperature inside the drying chamber reaches -20°C within 20 minutes; the product temperature reaches -40°C after another 30 minutes; this temperature is maintained for 2 hours; the condenser temperature is then lowered to -50°C; a vacuum is then applied, reaching a vacuum level of 100 kPa; the drying chamber and condenser are connected, and the refrigeration of the drying chamber is stopped; when the vacuum level in the drying chamber reaches 15 Pa, the temperature is raised at a rate of 3°C / min to 15°C, held for 3 hours, then raised to 22°C at a rate of 10°C / min for 5 hours; then raised to 35°C at a rate of 10°C / min for 2 hours; and finally raised to 45°C at a rate of 5°C / min for 1 hour. The product then enters the cooling phase, where the temperature is lowered to 40°C within 20 minutes and maintained for 10 hours, yielding a freeze-dried myocardial peptide product with acceptable appearance.

[0030] On the other hand, the present invention provides myocardial peptides prepared by the above preparation method.

[0031] On the other hand, the present invention provides the above-described preparation method or the application of the above-described cardiac peptide.

[0032] Preferably, the application includes the preparation of drugs for ischemia-reperfusion injury after interventional myocardial infarction or the preparation of drugs for treating ST-segment elevation myocardial infarction.

[0033] Preferably, the application includes the preparation of a drug that enhances cardiac function.

[0034] Preferably, the application includes the preparation of a drug that reduces the concentration level of BNP in serum.

[0035] The myocardial peptide provided by the present invention may further include an excipient, wherein the mass ratio of the myocardial peptide to the excipient is 15-20:100-375, preferably 18-20:200-375; the excipient is selected from mannitol, trehalose, lactose, sucrose or other freeze-drying excipients, preferably mannitol.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] This invention provides a method for preparing cardiac peptides. Through acoustic treatment and electrolysis, the prepared cardiac peptides have higher content and proportion of small molecule peptides, higher yield, and simplified process. The cardiac peptides provided by this invention significantly improve cardiac function and reduce myocardial infarction area in STEMI patients undergoing PCI treatment, and can be used to prepare drugs for treating myocardial infarction. Detailed Implementation

[0038] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0040] Preparation example: Raw material (pig heart chamber) quality control

[0041] Select hybrid pigs from Guangdong, Guangxi, Hunan, etc. Pigs should be 30 ± 6 days old, weigh less than 10 kg, and have a heart weight of 35 g ± 5 g. They should be raised in a unified facility with standardized feed, feeding times, and purchasing. The following documents should be provided: "Animal Origin Quarantine Certificate" or "Animal Exit Quarantine Certificate," and "Disinfection Certificate for Animal and Animal Product Transport Vehicles."

[0042] Before and after slaughter, pigs shall undergo quarantine by a nationally recognized quarantine station in accordance with the "Quarantine Operation Procedures for Pig Slaughterhouses". After live slaughter, the heart shall be removed and transported under low-temperature conditions. The surface of the pig heart should be smooth, without nodules, and fresh in color, with no abnormalities in the cross-section. One percent of the pig hearts shall be randomly sampled for histological examination, and five consecutive sections of the atria and ventricles shall be prepared. The tissue structure shall be normal under an optical microscope. Volatile basic nitrogen shall be tested according to law (National Standard GB / T5009.44-2003), and shall meet the requirements.

[0043] Preliminary processing: Remove the capsule, blood vessels, heart valves, tendons, and atria from the qualified pig hearts. Clean the ventricular myocardium with distilled water and then chop it into pieces about 1cm in size. 3 The small pieces were washed again with cold water for injection to obtain porcine ventricular muscle.

[0044] Example 1: Preparation of cardiac peptides

[0045] 1) Prepare porcine ventricular myocardium according to the preparation example;

[0046] 2) Preparation of homogenate: Add materials according to the ratio of ventricular myocardium to water for injection = 1:1 (mass ratio), and repeat homogenization three times in a colloid mill (centrifuge at 3000 rpm / min for 5 minutes each time, with a 5-minute interval) to obtain homogenate.

[0047] 3) Acoustic treatment: The homogenate is subjected to acoustic treatment at a frequency of 30Hz. The acoustic treatment interval is 30s for each treatment and 60s for each treatment. The total acoustic treatment time is 30min.

[0048] 4) Electrolysis treatment: Apply alternating current for 5 minutes, then cut off the power supply. Next, apply constant voltage reverse current for 15 minutes, then apply constant voltage forward current for 12 minutes to obtain a mixed solution.

[0049] The frequency of the alternating current is 46Hz and the voltage is 300V;

[0050] The voltage of the constant voltage reverse current and the constant voltage forward current is 300V.

[0051] 5) Preparation of the refined filtrate: After electrolysis, the mixture is filtered through gauze to remove residue, and the filtrate is collected. The coarse filtrate is then filtered twice under positive pressure using a three-column hollow fiber system (molecular weight cutoff ≤12KD, peristaltic pump pressure 0.2MPa, permeate flow rate 5±3ml / min, initial 50ml discarded), and concentrated to obtain the refined filtrate. Quality control standards: The solution should be clear and transparent with a slight yellow tint; the protein test should be negative (tested according to the method under the protein section of the quality standard for injectable cardiac peptides).

[0052] 6) Virus inactivation: Heat the filtrate to 80℃±2℃ and stir in a water bath for 1 hour. After virus inactivation, the solution is filtered through a 0.22μm microporous membrane for sterilization, dispensed into pyrogen-free, sterile stainless steel containers, ≤5000ml / container, sealed, and stored at -20℃.

[0053] 7) Ultrafiltration: The product is concentrated using an ultrafiltration membrane from Millipore to obtain a cardiac peptide solution. After passing a full inspection according to the cardiac peptide solution quality standard, it is then filled into bottles.

[0054] 8) Freeze-drying

[0055] The filled cardiac peptide solution was freeze-dried using a freeze dryer. The specific steps were as follows: The shelf temperature in the drying chamber reached -20°C in 20 minutes, and then the product temperature reached -40°C in 30 minutes. The product temperature was maintained at -40°C for 2 hours. Then, the temperature in the condenser was lowered to -50°C, and a vacuum was drawn. When the vacuum reached 100 kPa, the drying chamber and condenser were connected, and the cooling process was stopped. When the vacuum in the drying chamber reached 15 Pa, the temperature was increased at a rate of 3°C / min to 15°C and held for 3 hours. The temperature was then increased to 22°C at a rate of 10°C / min and held for 5 hours. The temperature was then increased to 35°C at a rate of 10°C / min and held for 2 hours. Finally, the temperature was increased to 45°C at a rate of 5°C / min and held for 1 hour. The product then entered the cooling phase, and the temperature was lowered to 40°C within 20 minutes and held for 10 hours. This yielded a qualified freeze-dried cardiac peptide product, which was then removed and sealed.

[0056] Example 2: Preparation of cardiac peptides

[0057] The difference from the method in Example 1 is that:

[0058] Acoustic treatment: The homogenate was subjected to acoustic treatment at a frequency of 40 Hz. The acoustic treatment interval was 30 seconds for each treatment and 60 seconds for the next treatment. The total acoustic treatment time was 25 minutes.

[0059] Electrolysis: Apply alternating current for 8 minutes, then disconnect the power supply. Next, apply constant voltage reverse current for 20 minutes, then apply constant voltage forward current for 15 minutes to obtain a mixed solution.

[0060] The frequency of the alternating current is 42Hz and the voltage is 330V;

[0061] The voltage of the constant voltage reverse current and the constant voltage forward current is 260V.

[0062] All other preparation methods are the same as in Example 1.

[0063] Example 3: Preparation of cardiac peptides

[0064] The difference from the method in Example 1 is that:

[0065] Acoustic treatment: The homogenate was subjected to acoustic treatment at a frequency of 50 Hz, with an interval of 30 seconds between each acoustic wave and a 60-second pause, for a total duration of 20 minutes.

[0066] Electrolysis: Apply alternating current for 10 minutes, then disconnect the power supply. Next, apply constant voltage reverse current for 25 minutes, and then apply constant voltage forward current for 18 minutes to obtain a mixed solution.

[0067] The frequency of the alternating current is 38Hz and the voltage is 360V;

[0068] The voltage of the constant voltage reverse current and the constant voltage forward current is 220V.

[0069] All other preparation methods are the same as in Example 1.

[0070] Comparative Example 1: Preparation of Cardiac Peptides

[0071] The only difference from Example 1 is that the sound wave frequency is 50Hz and the sound wave processing time is 20min.

[0072] Comparative Example 2: Preparation of Cardiac Peptides

[0073] The only difference from Example 1 is the AC frequency of 50Hz.

[0074] Comparative Example 3: Preparation of Cardiac Peptides

[0075] The only difference from Example 1 is the alternating current processing time of 15 minutes.

[0076] Comparative Example 4: Preparation of Cardiac Peptides

[0077] The only difference from Example 1 is the AC voltage of 400V.

[0078] Comparative Example 5: Preparation of Cardiac Peptides

[0079] The only difference from Example 1 is the constant voltage reverse current voltage of 360V.

[0080] Comparative Example 6: Preparation of Cardiac Peptides

[0081] The only difference from Example 1 is the constant voltage positive current treatment time of 20 min.

[0082] Comparative Example 7: Preparation of Cardiac Peptides

[0083] Replace steps 3), 4), and 5) of Example 1 with the following steps, keeping everything else unchanged:

[0084] Preparation of coarse filtrate: After preparing the homogenate, the homogenate was frozen at -20℃ for 24 hours and then thawed. Then, the homogenate was repeatedly frozen and thawed 3 times. After thawing, it was placed in a 75℃ water bath for 10 minutes. The residue was removed by filtering with gauze, the filtrate was collected, and then filtered once with a 5μm plate and frame filter to obtain coarse filtrate.

[0085] Preparation of the refined filtrate: The coarse filtrate is filtered twice under positive pressure through a three-column series hollow fiber filter (molecular weight cutoff ≤12KD, peristaltic pump pressure 0.2MPa, permeate flow rate 5±3ml / min, initial 50ml discarded) to obtain the refined filtrate. Quality control standards: The solution should be clear and transparent with a slight yellow tint; the protein test should be negative (tested according to the protein section of the quality standard for injectable cardiac peptides).

[0086] Preparation of ultrafiltrate: The fine filtrate is ultrafiltered and purged through a Millipore ultrafiltration membrane (ultrafiltration molecular weight <10KD, purging molecular weight <1KD, pressure 1.5-2.0MPa, permeate flow rate should be 500ml±30ml / min) to obtain ultrafiltrate, and then the ultrafiltrate is subjected to virus inactivation treatment.

[0087] The parameter design statistics for the embodiments and comparative examples are shown in Table 1:

[0088] Table 1

[0089]

[0090] Example 1: Determination of cardiac peptide solution composition

[0091] The myocardial peptide solutions (n=3) obtained in the concentration steps of Examples 1-3 and Comparative Examples 1-7 were analyzed by HPLC to detect the peptide content and the proportion of peptides with a molecular weight less than 1000 Daltons. The yield was calculated by weighing the lyophilized myocardial peptides (n=3), and the statistical results are shown in Table 2. The yield is the mass ratio (%) of the lyophilized product obtained in production to the ventricular myocardium input in production. It can be seen that the myocardial peptide solutions prepared in Examples 1-3 have a higher peptide content and a higher proportion of low molecular weight peptides, resulting in a higher yield of the finished myocardial peptide product.

[0092] Table 2

[0093]

[0094] Note: In the same column of data, the same letter indicates that there is no significant difference between the data (P>0.05), and different letters indicate that there is a significant difference between the data (P<0.05).

[0095] Example 2: Treatment of ST-segment elevation myocardial infarction with cardiac peptides

[0096] 1. Research Subjects

[0097] We prospectively and consecutively enrolled 160 STEMI patients aged ≥18 years who were hospitalized in the Department of Cardiology at the General Hospital of the Chinese People's Liberation Army and underwent direct PCI treatment.

[0098] STEMI was defined as typical symptoms (chest pain or symptoms consistent with acute heart failure / unexplained syncope) lasting more than 30 minutes, with ischemic chest pain lasting more than 30 minutes and ST-segment elevation in two or more adjacent leads (≥0.1 mV in limb leads, ≥0.2 mV in chest leads), with or without elevated myocardial enzyme levels. This study complied with the Declaration of Helsinki and was approved by the Ethics Committee of the General Hospital of the People's Liberation Army. All enrolled patients or their families signed informed consent forms. Inclusion criteria: 1) Age ≥18 years; 2) Meeting the diagnostic criteria for STEMI; 3) STEMI patients had indications for direct coronary intervention; 4) Disease duration ≤12 hours; 5) Willingness to participate in this clinical trial and signing an informed consent form. Exclusion criteria: 1) Cardiogenic shock; 2) Papillary muscle rupture, ventricular septal rupture, or free wall rupture of the heart; 3) Ventricular fibrillation; 4) Recent severe infection or severe renal failure; 5) Recent history of bleeding or stroke (within six months); 6) Malignant tumor; 7) Allergy to the drugs used in this study.

[0099] 2. Methodology: Single-blind design and grouping

[0100] The cardiac peptide group and control group were designed in a 1:1 ratio. Patients were randomly assigned a number upon enrollment. Therefore, patients were unaware of their group assignment. After admission, patients were randomly assigned to receive either injectable cardiac peptide treatment or placebo treatment based on their random number. All patients received routine treatment according to the Chinese STEMI guidelines. The cardiac peptide group consisted of 80 patients who received intravenous infusion of the cardiac peptide prepared in Example 1 of this invention during PCI; the control group consisted of 80 patients who received a placebo of 0.9% sodium chloride injection in the same manner. The specific methods are as follows:

[0101] 1) Cardiac peptide group: In addition to routine treatment, cardiac peptide 3mg / kg was slowly infused intravenously before catheter insertion until the end of the procedure; cardiac peptide 3mg / kg / day was infused intravenously on the 1st, 2nd and 3rd days after the operation. Cardiac peptide was dissolved in 250ml of 0.9% sodium chloride injection before use. The fluid volume was halved for patients with severe heart failure.

[0102] 2) Control group: In addition to routine diagnosis and treatment, the myocardial peptide solution was replaced with 0.9% sodium chloride injection, and the rest was the same as the myocardial peptide group.

[0103] 3. Observation indicators

[0104] The results of myocardial MRI with contrast enhancement, cardiac function indicators (left ventricular ejection fraction), and brain natriuretic peptide (BNP) levels were compared between the two groups of patients.

[0105] 1) Seven days after surgery, myocardial MRI with contrast was performed to assess the infarct area (IS). The specific method was to present the infarct site through delayed contrast-enhanced CMR, and to use image post-processing tools to delineate the infarct area of ​​each scan layer. Based on the number of layers, the percentage of the infarct area in the left ventricle was calculated, i.e., IS.

[0106] 2) Instrumental monitoring of cardiac function parameters: LVEF was measured using color Doppler ultrasound, and echocardiography was performed 7 days after direct PCI. Echocardiograms were recorded and analyzed by three cardiologists (radiologists) in randomized groups. All echocardiographic studies were performed using an echocardiography system (GEVingmed; GE Healthcare) equipped with a 2.5 MHz multi-frequency phased array transducer. To determine the reproducibility of echocardiographic parameters, two independent observers analyzed 40 randomly selected patients. The correlation coefficients for inter-observer variability in LVEF were 0.91 and 0.90, respectively.

[0107] 3) Brain natriuretic peptide (BNP) detection: BNP levels were monitored in all enrolled patients before PCI treatment and 7 days after PCI.

[0108] 4. Sample size calculation

[0109] Based on the sample size calculation formula for the t-test between the two groups, π1 represents the myocardial infarction area in the cardiac peptide group, π2 represents the myocardial infarction area in the control group, and Zα and Zβ represent the corresponding standard normality differences. Review of authoritative literature and preliminary experimental results suggest that the cardiac peptide intervention group can reduce the myocardial infarction area by up to 12%. Furthermore, this study was designed as a one-sided test; when α was 0.05, Zα was 1.64 (one-sided); when β was 0.10 (power 80%), Zβ was 1.28. Substituting the formula, each group required 70 patients; however, for safety, 80 patients were included in each group based on a 10% loss to follow-up rate.

[0110] 5. Statistical processing

[0111] SPSS 19.0 software was used to perform normality and homogeneity of variance tests on continuous data. Data are expressed as mean ± standard deviation (s) or the median interquartile range (Md) (IQR), and independent samples t-tests or independent samples rank tests were used. Comparisons between categorical data were performed using χ² tests or Fisher's tests. All statistical tests were two-tailed, and p < 0.05 was considered statistically significant.

[0112] 6. Results Statistics and Analysis

[0113] Before PCI, there were no statistically significant differences in age, sex, clinical history, heart rate, blood pressure, cardiac function, creatinine, and other biochemical indicators between the cardiac peptide group and the control group, making them comparable. After PCI, as shown in Table 3, the cardiac peptide prepared in Example 1 can significantly reduce the myocardial infarction area, increase LVEF during the STEMI recovery period after PCI, and reduce serum BNP levels after emergency PCI.

[0114] Table 3

[0115]

[0116] Note: Compared with the control group, * represents P<0.05, ** represents P<0.01, *** represents P<0.001, and no indication means there is no significant difference.

[0117] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing cardiac peptides, characterized in that, Includes the following steps: S1. Take the ventricular muscle of a piglet, mix it with water, and homogenize it to obtain a homogenate. S2. The homogenate is subjected to acoustic treatment at a frequency of 30-40Hz for 20-30 minutes. S3. Apply alternating current and process for 5-10 minutes. Then, disconnect the power supply and apply constant voltage reverse current for 15-25 minutes. Finally, apply constant voltage forward current for 12-18 minutes to obtain a mixture. The frequency of the alternating current is 38-46Hz and the voltage is 300-360V. The voltage of the constant voltage reverse current and constant voltage forward current is 220-300V. S4. The mixture is filtered through gauze to obtain coarse filtrate. The coarse filtrate is then passed through a hollow fiber column to obtain fine filtrate. S5. Heat the filtrate to inactivate it, and then filter it through a filter membrane to obtain the inactivated solution; S6. Concentrate the inactivation solution to obtain a myocardial peptide solution; S7. The myocardial peptide solution is freeze-dried to obtain the finished myocardial peptide product.

2. The preparation method according to claim 1, characterized in that, Step S1 includes the following steps: The materials were added according to the mass ratio of ventricular myocardium to water for injection of 1:1-2, and homogenized 3-4 times using a colloid mill to obtain a homogenate.

3. The preparation method according to claim 1, characterized in that, Step S4 includes the following steps: The coarse filtrate was filtered twice under positive pressure through a three-column hollow fiber column connected in series to obtain a fine filtrate with a molecular weight cutoff of ≤12KD.

4. The preparation method according to claim 1, characterized in that, Step S5 includes the following steps: Heat the filtrate to 75-85℃ and stir in a water bath for 1-2 hours, then filter it through a microporous membrane for sterilization.

5. The preparation method according to claim 1, characterized in that, In step S6, a reverse osmosis concentration column is used for concentration.

6. The myocardial peptide prepared by the preparation method according to any one of claims 1-5.

7. The preparation method according to any one of claims 1-5 or the application of the myocardial peptide according to claim 6, characterized in that, The application is to prepare drugs for ischemia-reperfusion injury after interventional myocardial infarction or to prepare drugs for treating ST-segment elevation myocardial infarction.

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