Use of myristicin in the preparation of a medicament for the treatment or prevention of cardiovascular disease

Abstract

The application provides application of myristic ether in preparation of drugs for treating or preventing cardiovascular diseases; it is found that myristic ether has multiple protective effects on myocardial ischemia-reperfusion injury: reducing myocardial infarction area; improving myocardial contraction function; reducing myocardial interstitial edema, myocardial cell morphological change and inflammatory cell infiltration; reducing AST, CK, CKMB and LDH levels in blood; reducing myocardial cell damage and death; reducing myocardial cell LDH leakage, enhancing intracellular SOD activity; reducing myocardial cell apoptosis.

Description

Technical Field

[0001] This application pertains to the field of cardiovascular disease treatment, and specifically, this application provides the use of myristole in the preparation of drugs for the treatment or prevention of cardiovascular diseases. Background Technology

[0002] Cardiovascular disease has become one of the leading causes of death worldwide. Clinical treatment for cardiovascular disease primarily involves surgery and medication, with surgery enabling rapid restoration of blood supply to ischemic myocardium. However, the sudden blood supply process increases collateral circulation volume, and revascularization can activate myocardial ischemia-reperfusion injury (MI / RI), which accounts for 40% of myocardial infarction areas. Myocardial infarction is a key factor affecting the treatment efficacy of coronary artery disease, and its pathogenesis is complex. MI / RI can induce an inflammatory response, damage the living tissue surrounding the infarct, and disrupt mitochondrial membrane potential, leading to the formation of reactive oxygen species (ROS). Clinically, various methods are used to treat myocardial infarction / RI, including anti-inflammatory, antioxidant, ischemic preconditioning, and pharmacological preconditioning. Post-coronary artery surgery administration of vitamin C and vitamin E can significantly reduce myocardial infarction by combating oxidative stress. However, many anti-inflammatory and antioxidant drugs are limited by pharmacokinetics and other factors. Finding suitable drugs to reduce MI / RI has become a critical issue that urgently needs to be addressed in the clinical treatment of myocardial ischemia.

[0003] Myristicin (abbreviated Myr), belonging to the allylbenzene family of compounds, is a natural active ingredient in many spices, medicinal plants, and some food flavorings (such as nutmeg, basil, cinnamon, cloves, parsley, carrots, and black pepper). It possesses a variety of biological effects, including antibacterial, antioxidant, anti-inflammatory, anti-proliferative, and anticancer properties. In traditional medicine, myristicin has been used to treat cholera, stomach cramps, nausea, diarrhea, and anxiety; no other pharmacological effects have been identified. Summary of the Invention

[0004] The applicant found that myristyl ether pre-administration has excellent cardioprotective effects: myristyl ether has significant protective effects against H9c2 cardiomyocyte H / R damage and against the heart of MIRI rats.

[0005] On the one hand, this application provides the use of myristole in the preparation of drugs for the treatment or prevention of cardiovascular diseases.

[0006] Furthermore, the cardiovascular disease is myocardial ischemia-reperfusion injury.

[0007] Furthermore, the drug provides one or more of the following effects:

[0008] (1) Reduce the area of ​​myocardial infarction;

[0009] (2) Improves myocardial contractile function;

[0010] (3) Reduces myocardial interstitial edema, myocardial cell morphological changes and inflammatory cell infiltration;

[0011] (4) Reduce the levels of AST, CK, CKMB, and LDH in the blood;

[0012] (5) Reduce myocardial cell damage and death;

[0013] (6) Reduce LDH leakage and enhance intracellular SOD activity;

[0014] (7) Reduce cardiomyocyte apoptosis.

[0015] Furthermore, myristole is the sole active ingredient in the drug.

[0016] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0017] Furthermore, the pharmaceutically acceptable excipients are selected from one or more of the following: fillers, binders, lubricants, disintegrants, coating agents, capsule shells, solvents, solubilizers, emulsifiers, pH adjusters, osmotic pressure adjusters, antioxidants, preservatives, flavoring agents, and coloring agents.

[0018] Furthermore, the drug is in the form of an oral or parenteral dosage form.

[0019] Furthermore, the drug is in the form of tablets, capsules, oral liquids, granules, drop pills, water injections, or powder injections.

[0020] Furthermore, the drug is in the form of an oral liquid.

[0021] Furthermore, the minimum dose unit of the drug contains an amount of myristole equivalent to 10 mg / kg of rat.

[0022] Those skilled in the art can calculate the dosage for different objects (e.g., people) based on algorithms known in the art (e.g., skin surface area conversion, weight conversion).

[0023] The smallest unit of dosage for a pharmaceutical composition refers to a tablet, a capsule, a sachet of granules, or an injection, etc.

[0024] On the other hand, this application provides non-therapeutic methods for protecting cardiomyocytes, including the application of myristole to cardiomyocytes.

[0025] The non-therapeutic methods can be used for non-therapeutic purposes such as scientific research and sample preservation, and are preferably performed in vitro.

[0026] Furthermore, the cardiomyocytes are H9c2 cardiomyocytes.

[0027] Furthermore, the method involves treating cardiomyocytes with 12.5-50 μM myristyl ether.

[0028] Furthermore, the cardiomyocytes undergo a hypoxia / reoxygenation process.

[0029] The dosage form of this invention can be produced using any method conventionally used in pharmaceutical manufacturing processes, without particular limitation. For example, the oral liquid of this invention can be extracted, purified, concentrated, dispensed, and sterilized using suitable methods known in the art. The oral liquid undergoes quality inspections, including visual inspection (clarity inspection), device variation inspection, hygiene inspection, qualitative identification, determination of active ingredient content, and determination of relative density, which effectively control the quality of the oral liquid.

[0030] The myristicin described in this application, CAS number 607-91-0, can be used interchangeably with myristicin, myr, myr and their different chemical names to represent the same meaning. Attached Figure Description

[0031] Figure 1 The chromatogram of the prepared myristole sample;

[0032] Figure 2 The results of echocardiographic examination of the effect of myristyl ether on cardiac function in MIRI rats;

[0033] Figure 3 The effect of myristyl ether on reducing the area of ​​myocardial infarction in MIRI rats (TTC staining);

[0034] Figure 4 To observe the effect of myristin on cardiac histological changes in MIRI rats by H&E staining (HE staining, magnification 800x);

[0035] Figure 5 The effect of myristyl ether on the activities of LDH (part A), AST (part B), CK (part C) and CKMB (part D) in the serum of MIRI rats;

[0036] Figure 6 The protective effects of myristyl ether against H / R damage in H9c2 cells: cytotoxicity (part A, CCK-8 assay), H / R (part B, CCK-8 assay), LDH (part C), and SOD (part D);

[0037] Figure 7 Results of myristyl ether reducing apoptosis in H9c2 cells after H / R damage (TUNEL staining, magnification 400x). Detailed Implementation

[0038] Related experimental instruments and materials

[0039]

[0040]

[0041] Example 1: Preparation of myristole:

[0042] Nutmeg was crushed, soaked overnight in 8 times its volume of 95% ethanol, and extracted twice by reflux, 2 hours each time. The residue was then extracted twice more by reflux in 6 times its volume of 70% ethanol, 2 hours each time. The ethanol was recovered under reduced pressure until no alcohol odor remained, yielding an extract. The extract was redissolved and adsorbed onto 1.5 times its volume of diatomaceous earth and completely dried. It was then extracted sequentially by reflux in a constant pressure funnel with petroleum ether, dichloromethane, ethyl acetate, and methanol. The extract was concentrated under reduced pressure to a thick paste, yielding the various fractions. The petroleum ether fraction was eluted using a gradient elution system of petroleum ether-ethyl acetate (100:1–0:1), and repeatedly separated by silica gel column chromatography and other methods to obtain the myristole sample (see...). Figure 1 ).

[0043] Purity: >98%

[0044] Instrument: Waters 2695;

[0045] Column: Agilent ZORBAX Eclipse Plus C18, 4.6*100mm, 3.5μm

[0046] Mobile phase: methanol-water 57:43;

[0047] Flow rate: 1.0 ml / min;

[0048] Column temperature: 25℃;

[0049] Detection wavelength: UV-276nm

[0050] Product chromatogram as follows: Figure 1 As shown.

[0051] Example 2: Study on the cardioprotective effect of myristyl ether on MIRI rats

[0052] Establishment of the rat MIRI model:

[0053] Sixty SPF-grade SD rats were randomly divided into six groups: Sham group (n=10), MIRI group (n=10), and MIRI + myristyl ether (10 ml·kg). -1 Group 1 (10 animals), MIRI + myristyl ether (20 ml / kg) -1 (10 pieces), MIRI + Myristyl Ether (40ml·kg) -1(10 pieces), MIRI+DZT (Diltiazem) (20mg·kg) -1 (10 rats). Adaptive feeding for 3-5 days. The low, medium, and high dose groups of myristole and the DZT group were administered the corresponding drugs by gavage (prepared with 3% Tween 80, Tween 80 was added first, mixed and dissolved, and then physiological saline was added), once a day for 7 consecutive days. The Sham group and the DCM group were administered the same amount of physiological saline. The weight of the rats was recorded daily for one week after administration.

[0054] One week after drug administration, the MIRI model was established by ligating the left anterior descending coronary artery (LAD) in rats to induce myocardial ischemia. In short, 60 SD rats were anesthetized with intraperitoneal injection of sodium pentobarbital (40 mg / kg), placed in a supine position, and cannulated. Electrocardiogram (ECG) was monitored dynamically. After hair removal, the chest was opened, and the LAD was ligated with 6-0 sutures for 30 minutes (ST segment elevation on ECG, and whitening of the area below the ligation site, indicating correct ligation). The ligation was then released, and the heart was reperfused for 24 hours. The sham-operated group did not undergo ligation. Postoperatively, when the rats were able to breathe spontaneously, the ventilator was removed. They were then placed on a heated blanket until they could walk, and then returned to their cages. 24 hours after reperfusion (day 1 post-modeling), the rats were anesthetized, and echocardiography was performed. On the second day post-modeling, blood was collected from the abdominal aorta, and the heart was removed and rinsed with physiological saline. Hearts were randomly selected from the experimental group and fixed in 4% paraformaldehyde. Another portion (≥5 hearts) was stained with TTC. The remaining hearts were stored at -80℃. Whole blood from the abdominal aorta was centrifuged (3500 rpm, 15 min), and the supernatant was collected and frozen at -80℃ for later use. Morphological and cardiac function examinations:

[0055] Twenty-four hours after reperfusion, echocardiography was performed using the Vevo 770 ultra-high resolution small animal ultrasound imaging system to evaluate the cardiac structure and function of rats. Each parameter was measured three times. The procedure involved anesthetizing the SD rats with 4% chloral hydrate, shaving the thoracic cavity, securing them to the operating table in a supine position with tape, applying coupling gel to the thoracic cavity, positioning the probe at the shaving location (left anterior chest), adjusting the probe, and evaluating the echocardiographic parameters in the long axis view parasternal to the left sternum. Figure 2 .

[0056] To observe the effect of myristole on the infarct area of ​​myocardial infarction in rats, TTC staining was performed on myocardial tissue. The results are shown below. Figure 3 After all treatments were completed, the heart was removed, rinsed with physiological saline, frozen in liquid nitrogen for 1 minute, and cut into 5 slices along the cardiac axis. The slices were then immersed in 2% TTC and incubated in the dark at 37°C for 15 minutes. Finally, they were fixed with 4% paraformaldehyde for 24 hours. Images of the heart were taken with a digital camera, and their proportions were determined. The infarct area was assessed based on the obtained images.

[0057] The remaining rat hearts were fixed with 4% formaldehyde. 5 mm tissue sections were stained with hematoxylin and eosin. Morphological examination was performed under a light microscope; results are shown below. Figure 4 .

[0058] like Figure 2 As shown, M-mode echocardiography was used to measure cardiac parameters to investigate the effect of myristole on cardiac function in MIRI rats. Compared with the Sham group, the MIRI group showed a significant decrease in left ventricular ejection fraction (EF) and left ventricular systolic fraction (FS), indicating myocardial contractile dysfunction. Compared with the MIRI group, the myristole (10ml / kg, 20ml / kg, 40ml / kg) administration groups and the DZT group showed significantly increased EF and FS, but still lower than the Sham group, indicating that cardiac contractile function was protected. This suggests that myristole can improve myocardial contractile function in rats after MIRI and has a protective effect on the heart of MIRI rats.

[0059] like Figure 3 As shown, the infarcted area is white, and the non-infarcted area is red. Observation showed that there was no myocardial infarction in the sham-operated group, while there was significant myocardial infarction in the MIRI group. Compared with the MIRI group, the myocardial infarction area was significantly reduced in the myocardial ether (10ml / kg, 20ml / kg, 40ml / kg) administration groups and the DZT group, indicating that myocardial infarction damage in rats was alleviated. This suggests that myocardial ether can reduce the myocardial infarction area in MIRI rats and has a protective effect on the heart of MIRI rats.

[0060] like Figure 4 As shown, H&E staining revealed that the myocardial fibers in the Sham heart tissue were regularly arranged and the myocardial cells had normal morphology. Compared with the Sham group, the MIRI group showed disordered myocardial tissue structure, interstitial edema, irregular cell nuclei, increased interstitial spaces, and inflammatory cell infiltration. Compared with the MIRI group, the myristole (10ml / kg, 20ml / kg, 40ml / kg) and DZT groups reduced myocardial interstitial edema, myocardial cell morphological changes, and inflammatory cell infiltration in MIRI rats. This indicates that myristole has a protective effect on the heart of MIRI rats.

[0061] Plasma collection and biochemical testing:

[0062] Blood samples were collected from the abdominal aorta of rats under chloral hydrate anesthesia using a 10 ml syringe. After the blood was allowed to stand at room temperature for 4 hours, it was incubated at 3500 rpm. -Centrifuge at 22℃ for 15 min, and store the supernatant at -20℃. Fix the rat on a cardboard and store the heart at -80℃. Subsequently, the activities of CKMB, LDH, CK, and AST in plasma were measured using an AU480 fully automated biochemical analyzer according to the instructions of the commercially available test kits purchased from Sinopharm Group. The results are shown in the table below. Figure 5 .

[0063] like Figure 5 As shown, compared with the Sham group, the MIRI group rats showed significantly increased levels of AST, CK, CKMB, and LDH. Compared with the MIRI group, the myristole (10ml / kg, 20ml / kg, 40ml / kg) and DZT groups showed significantly reduced levels of AST, CK, CKMB, and LDH, suggesting that myristole can reduce the activity of AST, CK, CKMB, and LDH enzymes and exert a cardioprotective effect.

[0064] Example 3: Study on the mechanism of myristyl ether's protective effect on H9c2 cardiomyocytes (H / R ratio). Cell viability was detected by CCK-8 assay.

[0065] This study investigated the protective effect of myristyl ether against H9c2 cell hydration / reoxygenation (H / R) damage. A 96-well cell culture plate was used, with a concentration of 1 × 10⁶ cells / well. 4 100 μL of cell suspension per mL was administered. Cells were seeded for 24 hours, reaching the logarithmic growth phase. 96-well plates were divided into: Control group, Model group (H / R group), H / R + Myr-12.5 μM group, H / R + Myr-25 μM group, and H / R + Myr-50 μM group. Twenty-four hours after pre-treatment, both the model and treatment groups underwent H / R treatment. High-glucose DMEM medium was removed from the culture plates, and then regular DMEM medium was added. The plates were then incubated in a CO2 anaerobic glove box at 37°C for 4 hours. The cell culture plates were then removed from the anaerobic glove box, the glucose-free DMEM medium was removed, and fresh, preheated (37°C) high-glucose DMEM medium was added. The plates were then incubated in a CO2 incubator at 37°C for 12 hours. During the incubation, the CO2 cell culture incubator was circulated with saturated humidity air containing 5% CO2.

[0066] H9c2 cardiomyocytes in the control group were cultured at 37°C in a carbon dioxide cell culture incubator with saturated humidity and 5% CO2 throughout the entire process, without any other treatment. Results are shown below. Figure 6 .

[0067] like Figure 6As shown, after treatment with myristole at concentrations of 1.5625 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM for 24 h, there was no significant difference in cell viability compared to the control group. After H / R treatment, the cell viability of the Model group was significantly lower than that of the control group. Compared with the Model group, the myristole-treated groups (12.5 μM, 25 μM, and 50 μM) significantly improved cell viability. These results suggest that myristole can alleviate H / R damage in H9c2 cells and exert a cardioprotective effect.

[0068] LDH and SOD detection:

[0069] After H / R treatment, the cell culture supernatant was collected, and the absorbance was measured and quantified using a lactate dehydrogenase assay kit (catalog number A020-2, Nanjing Jiancheng Bioengineering Institute) in accordance with the instructions.

[0070] Cell samples were collected and analyzed using a superoxide dismutase (SOD) assay kit (catalog number A001-3, Nanjing Jiancheng Biotechnology Institute), following the instructions. The absorbance was measured at 450 nm and quantified. Results are shown below. Figure 6 .

[0071] like Figure 6 As shown, compared with the control group, the Model group showed significantly increased LDH activity in the cell supernatant and significantly decreased intracellular SOD activity, indicating that the cardiomyocytes in the Model group suffered acute damage. Compared with the Model group, the myristole-treated groups (12.5uM, 25uM, 50uM) significantly reduced the LDH level in the supernatant of H9c2 cells and increased intracellular SOD enzyme activity. The results suggest that myristole can reduce LDH leakage from H9c2 cells after H / R and enhance intracellular SOD activity, thus exerting a cardiomyocyte protective effect.

[0072] TUNEL assay for cell apoptosis:

[0073] H9c2 cells were cultured in 24-well plates, treated with the drug, and subjected to hypoxia for 4 hours, followed by reoxygenation for 12 hours. Cells were washed once with PBS, fixed with 4% paraformaldehyde for 30 min, washed again with PBS, and incubated with PBS containing 0.3% Triton X-100 at room temperature for 5 min. TUNEL assay solution was prepared at 50 μL (5 μL TdT enzyme + 45 μL fluorescent labeling solution) per sample. After washing twice with PBS, 50 μL of TUNEL assay solution was added to the sample, and the sample was incubated at 37°C in the dark for 60 min. Cells were washed three times with PBS, mounted with anti-fluorescence quenching mounting medium, and observed under a fluorescence microscope. Three fields of view were selected for each sample, and the experiment was repeated three times. Results are shown below. Figure 7 .

[0074] like Figure 7 As shown, compared with the control group, the number of TUNEL-positive cells in the model group was significantly increased; compared with the model group, the number of TUNEL-positive cells in the myristole-treated groups (12.5uM, 25uM, 50uM) was significantly reduced. The results suggest that myristole can reduce apoptosis of H9c2 cells after H / R and exert a cardioprotective effect.

Claims

1. Use of myristicin in the preparation of a medicament for treating or preventing myocardial ischemia-reperfusion injury, wherein myristicin is the only active ingredient in the medicament.

2. The use according to claim 1, wherein the medicament further comprises a pharmaceutically acceptable excipient.

3. The use according to claim 2, wherein the pharmaceutically acceptable excipient is selected from one or more of a filler, a binder, a lubricant, a disintegrant, a coating agent, a capsule shell, a solvent, a cosolvent, an emulsifier, a pH adjuster, an osmotic pressure adjuster, an antioxidant, a preservative, a flavoring agent, a coloring agent.

4. The use according to claim 1, wherein the medicament is in an oral or parenteral administration dosage form.

5. The use according to claim 4, wherein the medicament is in the form of a tablet, a capsule, an oral solution, a granule, a dripping pill, an aqueous injection or a powder injection.

6. The use according to claim 5, wherein the medicament is in the form of an oral solution.

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