Pharmaceutical composition for preventing or treating myocardial ischemia-reperfusion injury and application thereof

Abstract

The invention relates to a pharmaceutical composition for preventing or treating myocardial ischemia-reperfusion injury, the pharmaceutical composition contains luteolin and paeonol as active pharmaceutical ingredients, and the luteolin and the paeonol have a synergistic effect of preventing and treating myocardial ischemia-reperfusion injury.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a pharmaceutical composition for preventing or treating myocardial ischemia-reperfusion injury and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a serious cardiovascular disease caused by occlusion of the coronary artery supplying blood to the myocardium and is one of the main causes of death in humans. Currently, reperfusion therapy is the main treatment for AMI, but the restoration of blood perfusion to the ischemic myocardium may cause myocardial ischemia reperfusion injury (MIRI), which in turn induces myocardial cell apoptosis, myocardial damage, no reflow phenomenon, and microvascular endothelial damage, ultimately leading to arrhythmias, heart failure, cardiogenic shock, etc., which seriously affect the prognosis of AMI patients.

[0003] Studies have shown that the causes of MIRI are relatively complex. Oxidative stress, apoptosis, autophagy, pyroptosis, intracellular calcium overload, energy metabolism disorders, endoplasmic reticulum stress, ferroptosis and necroptosis may all be involved in its occurrence and development, involving multiple signaling pathways such as AMP-dependent protein kinase, Janus kinase / signal transducer and activator of transcription, nuclear transcription factor erythroid 2-related factor 2 / antioxidant response element / heme oxygenase 1, phosphatidylinositol-3-kinase / protein serine / threonine kinase, etc.

[0004] At present, the main drugs used in the treatment of MIRI by Western medicine include angiotensin receptor antagonists, adenosine receptor agonists, statins and magnesium preparations, but the effect is not ideal. Traditional Chinese medicine has received extensive clinical attention due to its advantages of multiple targets, multiple pathways and few adverse reactions. Studies have shown that many active monomers of traditional Chinese medicine have an improvement effect on MIRI, such as flavonoids (luteolin, ginkgo biloba, isoliquiritigenin, puerarin, dihydromyricetin, scutellariae A, calycosin isoflavones, curcumin, etc.), alkaloids (berberine, corydalis, evodiamine, anisodine, anisodine, scopolamine, etc.), phenols (eugenol, paeonol, etc.), saponins (citrinin, ginsenoside, gardenia glycoside, astragaloside A, baicalin, gypenoside, notoginseng saponin, salidroside, hesperidin, etc.), terpenes (atractylodes lactone, gardenia glycoside, saikosaponin, etc.), etc. However, the therapeutic effect of a single active monomer of traditional Chinese medicine is also insufficient. The development of a combination of active monomers of traditional Chinese medicine with synergistic effects is currently a research hotspot for anti-MIRI drugs. Summary of the invention

[0005] The purpose of the present invention is to provide a pharmaceutical composition which can effectively prevent and treat myocardial ischemia-reperfusion injury.

[0006] In one aspect of the present invention, the present invention provides a pharmaceutical composition comprising luteolin or a pharmaceutically acceptable salt thereof and paeonol or a pharmaceutically acceptable salt thereof, wherein the weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.1-10.

[0007] In one embodiment, the weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.5-2.

[0008] Preferably, the weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.5, 1:0.8, 1:1, 1:1.5, or 1:2.

[0009] More preferably, the weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.8-1.5.

[0010] In another aspect, the present invention also provides use of the pharmaceutical composition in preparing a drug for preventing or treating myocardial ischemia-reperfusion injury.

[0011] In the present invention, the pharmaceutically acceptable salt of luteolin includes a salt formed by luteolin and a pharmaceutically acceptable base.

[0012] In the present invention, the pharmaceutically acceptable salt of paeonol includes a salt formed by paeonol and a pharmaceutically acceptable base.

[0013] Preferably, the pharmaceutical composition or medicine of the present invention further comprises a pharmaceutically acceptable adjuvant.

[0014] Preferably, the luteolin or its pharmaceutically acceptable salt and paeonol or its pharmaceutically acceptable salt in the pharmaceutical composition or medicine of the present invention can be administered in the same or different pharmaceutical preparations. The pharmaceutical dosage forms of luteolin or its pharmaceutically acceptable salt and paeonol or its pharmaceutically acceptable salt can be the same or different. Luteolin or its pharmaceutically acceptable salt and paeonol or its pharmaceutically acceptable salt can be administered simultaneously or sequentially.

[0015] In the medical uses described above, the administration time, number of administrations and frequency of administration of luteolin or its pharmaceutically acceptable salt and paeonol or its pharmaceutically acceptable salt need to be determined according to the specific diagnosis results of the disease, which is within the technical scope mastered by those skilled in the art.

[0016] For example, when a treatment regimen for mice or rats is applied to humans, the effective doses of all drugs for humans can be converted by the effective doses of the drugs for mice or rats, which is also easy to achieve for ordinary technicians in this field.

[0017] As pharmaceutically acceptable adjuvants, any adjuvant known to be suitable for preparing a specific drug or pharmaceutical composition can be used. Examples include, but are not limited to, solvents, excipients, dispersants, emulsifiers, solubilizers, gel formers, ointment bases, antioxidants, preservatives, stabilizers, carriers, fillers, bonding agents, thickeners, complexing agents, disintegrants, buffers, penetration enhancers, polymerizers, lubricants, coating agents, propellants, tonicity regulators, surfactants, colorants, flavoring agents, sweeteners and dyes. Particularly adjuvants suitable for the type of desired formulation and desired mode of administration are used.

[0018] Furthermore, in the pharmaceutical composition, the pharmaceutically acceptable adjuvant is selected from, for example, water, starch, compressible starch, dextrin, sucrose, lactose, fructose, glucose, xylitol, mannitol, microcrystalline cellulose, calcium carbonate, magnesium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium sulfate, magnesium oxide, aluminum hydroxide, carboxymethyl cellulose calcium, carboxymethyl cellulose, sodium carboxymethyl starch, hydroxypropyl starch, cross-linked sodium carboxymethyl cellulose, cross-linked polyvinyl pyrrolidone, low-substituted hydroxypropyl methylcellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, Ketone, starch syrup, glucose syrup, sucrose syrup, fructose syrup, sorbitol, gelatin syrup, acacia syrup, tragacanth syrup, microcrystalline cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, calcium carboxymethylcellulose, polymethacrylate, alginic acid, sodium alginate, polyethylene glycol, colloidal magnesium aluminum silicate, stearic acid, calcium stearate, magnesium stearate, zinc stearate, talc, glyceryl monostearate, glyceryl palmitostearate, magnesium lauryl sulfate, polyethylene glycol, sodium stearyl fumarate.

[0019] The pharmaceutical composition is presented in a unit dosage form, such as tablets, pills, capsules, powders, granules, suspensions, oral liquids, aerosols or liquid sprays, drops; for oral parenteral, intranasal, sublingual or rectal administration or for inhalation or spray administration. Preferably, the pharmaceutical dosage form is an oral preparation, more preferably tablets, pills, capsules, granules, suspensions, powders, oral liquids.

[0020] The pharmaceutical composition of the present invention can be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, making sugar-coated pills, grinding, emulsifying, freeze-drying, etc. Pharmaceutically, the content of the active ingredient in the pharmaceutical composition of the present invention should be in the range of 0.05% to 90% by weight, preferably 0.1% to 50% by weight of the whole composition.

[0021] For adult patients, the compound of the present invention can be administered orally or parenterally in a dose of 0.001 to 500 mg once a day or several times a day. It should be noted that the dose can be appropriately increased or decreased depending on the type of disease to be treated, the patient's age, weight, symptoms, etc.

[0022] Beneficial effects:

[0023] The present invention provides a pharmaceutical composition for preventing or treating myocardial ischemia-reperfusion injury, wherein the pharmaceutical composition comprises luteolin and paeonol as active pharmaceutical ingredients, and the two traditional Chinese medicine active monomers themselves have the effect of preventing and treating myocardial ischemia-reperfusion injury, and the combination of the two achieves a synergistic effect, which can greatly improve the performance of preventing and treating myocardial ischemia-reperfusion injury, can reduce the dosage of the drug, has better prevention and treatment effects, and has better safety and application scenarios. DETAILED DESCRIPTION

[0024] The present invention is described in more detail hereinafter to facilitate understanding of the present invention.

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified. If no specific techniques or conditions are specified in the examples, they are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0026] Example 1: The therapeutic effect of the pharmaceutical composition of the present invention on MIRI

[0027] Main reagents: Luteolin group and paeonol were purchased from TCI (Shanghai) Company; nitro blue tetrazolium chloride (NBT) and propidium iodide (PI) were purchased from Amresco, USA; cysteine ​​protease protein caspase-3 and caspase-8 activity detection kit (fluorescence method) was purchased from Clontech, USA.

[0028] MIRI model establishment:

[0029] Fifty SD rats, half male and half female, weighing (200±20) g, were selected and kept in a shielded environment of the animal room at a temperature of 20-23°C, a relative humidity of 60-70%, and light and dark illumination for 12 hours respectively. After adaptive feeding, the rats were randomly divided into a sham operation group (equal volume of DMSO), a model group (equal volume of DMSO), a luteolin group (20 mg / kg), a paeonol group (20 mg / kg), and a luteolin+paeonol group (10 mg+10 mg / kg) (luteolin, paeonol, and "luteolin+paeonol" were prepared with DMSO to form a total concentration of 1wt% solution), 10 rats in each group, and intraperitoneal administration began 7 days before modeling, once a day, and once again 30 minutes before modeling.

[0030] The model was established by ligation of the left anterior descending coronary artery (Metformin alleviates left ventricular diastolic dysfunction in a rat myocardial ischemia reperfusion injury model., Woori Jo, K. Kang, et al., International Journal of Molecular Sciences, 2020, 21(4): 1489). After anesthesia, the rats were fixed on the operating table in a supine position, connected to an animal ventilator and monitored by electrocardiogram. The thoracotomy was performed at the 3rd and 4th intercostal spaces on the left side of the sternum, the pericardium was cut open, the heart was exposed, the needle was inserted 2 mm below the lower edge of the left atrial appendage, and the needle was removed from the pulmonary artery cone. The depth of the needle was 1 mm. The left anterior descending coronary artery was ligated with a 6-0 suture and an inflated balloon, and the chest was closed. After the balloon was inflated, the ST segment of the electrocardiogram showed obvious arch elevation as a sign of successful ischemia, which lasted for 45 minutes. The balloon was evacuated, and reperfusion was performed for 6 hours. The ST segment of the electrocardiogram decreased by more than 50% as a sign of successful reperfusion. After thoracotomy, the sham-operated rats only had their coronary arteries threaded but not ligated, and the rest of the procedures were the same as above.

[0031] 1. Determination of myocardial infarction area by NBT staining: After reperfusion, 5 rats were randomly selected from each group, and the hearts were cut out after being killed, washed, the atria and right ventricle were removed, and frozen at -20°C for 30 minutes, cut into 5 slices with a thickness of 2 mm along the long axis, immersed in 0.1% NBT solution, incubated at 37°C for 30 minutes, and fixed with 10% formaldehyde solution for 24 hours. Normal myocardium is blue, ischemic non-infarcted myocardium is light red, and infarcted myocardium is grayish white. Images were taken under a microscope and imported into the image analysis system to measure the infarct area (IA), area at risk (AAR) and total ventricular area (LVA), and IA / AAR and IA / LVA were calculated to evaluate the myocardial infarction.

[0032] 2. Determination of cell apoptosis rate by flow cytometry: The remaining 5 rats in each group were killed and their hearts were cut and washed. Part of the myocardial tissue was taken below the coronary artery ligature, cut into pieces, and fully ground with PBS buffer. The cell suspension was collected and centrifuged at 1000 r / min for 5 minutes. The supernatant was discarded, and the cells were washed several times with PBS buffer and resuspended to obtain relatively pure myocardial cells. The cells were counted under a light microscope and the concentration of myocardial cells was adjusted to about 1×10 6 / ml; the cardiomyocytes were resuspended in 70% pre-cooled ethanol and fixed overnight at low temperature. Before detection, they were centrifuged at a speed of 1000r / min at low temperature, and the ethanol was washed away with PBS buffer. The cell pellet was stained with PI at low temperature and in the dark for 30 minutes, and detected by flow cytometer.

[0033] 3. Fluorescence assay for cardiomyocyte caspase activity: Take the above concentration of 1×10 6 Cardiomyocytes at 10000 cells / ml were resuspended in pre-cooled lysis buffer, placed on ice for 5 minutes, centrifuged at 10000 r / min for 5 minutes, and the supernatant (cytoplasmic extract) was collected and placed in a 96-well plate. Reaction reagents and fluorescent substrates were added according to the instructions of the kit. Caspase inhibitors were added to the negative control wells. The plates were incubated at 37°C in the dark for 1 hour, and detected on a fluorescence microplate reader (excitation wavelength 400 nm, emission wavelength 505 nm). Fluorescence intensity (fluorescence units, FU) was used to represent the activity of caspase-3 and caspase-8.

[0034] 4. Results

[0035] 4.1 The effects of drugs on myocardial infarction area are shown in Table 1:

[0036] Table 1: Effects of drugs on myocardial infarction size

[0037] Group IA / AAR(%) IA / LVA(%) Sham operation group 0 0 Model Group 74.26±15.43 52.31±8.26 Luteolin group <![CDATA[57.63±9.84 a ]]> <![CDATA[41.26±6.21 a ]]> Paeonol group <![CDATA[59.22±10.23 a ]]> <![CDATA[43.59±6.58 a ]]> Luteolin+paeonol group <![CDATA[48.35±7.57 abc ]]> <![CDATA[31.26±5.74 abc ]]>

[0038] Note: Compared with the model group, a P<0.05; compared with the luteolin group, b P<0.05; compared with the paeonol group, c P<0.05

[0039] As shown in Table 1, the infarct area of ​​rats in the sham operation group was 0. Compared with the model group, the luteolin group, paeonol group and luteolin + paeonol group could significantly reduce IA / AAR and IA / LVA, and the differences were statistically significant; however, compared with the luteolin group and paeonol group, the effect of the luteolin + paeonol group was more prominent, and the difference was statistically significant.

[0040] 4.2 The effects of drugs on myocardial cell apoptosis rate are shown in Table 2:

[0041] Table 2: Effects of drugs on myocardial cell apoptosis rate

[0042] Group Myocardial cell apoptosis rate (%) Sham operation group 0.84±0.72 Model Group 50.42±6.33* Luteolin group <![CDATA[39.46±4.27 a ]]> Paeonol group <![CDATA[40.38±4.82 a ]]> Luteolin+paeonol group <![CDATA[30.59±3.94 abc ]]>

[0043] Note: Compared with the normal group, *P<0.05; compared with the model group, a P<0.05; compared with the luteolin group, b P<0.05; compared with the paeonol group, c P<0.05

[0044] As shown in Table 2, the apoptosis rate of cardiomyocytes in the sham operation group was low, and the apoptosis rate of cells in the model group was significantly increased. Compared with the model group, the luteolin group, paeonol group and luteolin + paeonol group could significantly reduce the apoptosis rate of cardiomyocytes, and the differences were statistically significant; however, compared with the luteolin group and paeonol group, the effect of the luteolin + paeonol group was more prominent, and the difference was statistically significant.

[0045] 4.3 The effects of drugs on the caspase activity of cardiomyocytes are shown in Table 3:

[0046] Table 3: Effects of drugs on caspase activity in cardiomyocytes

[0047] Group Caspase-3 activity (FU) Caspase-8 activity (FU) Sham operation group 94.42±10.24 121.98±13.25 Model Group 284.59±27.34* 201.45±21.78* Luteolin group <![CDATA[235.21±20.24 a ]]> <![CDATA[173.44±16.50 a ]]> Paeonol group <![CDATA[241.36±22.19 a ]]> <![CDATA[178.19±17.57 a ]]> Luteolin+paeonol group <![CDATA[198.45±20.33 abc ]]> <![CDATA[157.38±16.09 abc ]]>

[0048] Note: Compared with the normal group, *P<0.05; compared with the model group, a P<0.05; compared with the luteolin group, b P<0.05; compared with the paeonol group, c P<0.05

[0049] The cysteine ​​proteases caspase-3 and caspase-8 play an important role in the process of MIRI, and their increased activity leads to cardiomyocyte apoptosis. As shown in Table 3, compared with the sham operation group, the activities of caspase-3 and caspase-8 in cardiomyocytes in the model group were greatly increased. Compared with the model group, the luteolin group, paeonol group, and luteolin + paeonol group could inhibit the activities of caspase-3 and caspase-8 in cardiomyocytes, and the differences were statistically significant; however, compared with the luteolin group and paeonol group, the effect of the luteolin + paeonol group was more prominent, and the difference was statistically significant.

[0050] In summary, the experimental results show that the combination of luteolin + paeonol has significantly better activity in protecting myocardial cells from ischemia-reperfusion, and the luteolin and paeonol have achieved a synergistic effect, which can be used in the prevention and treatment of MIRI.

[0051] Example 2: Tablet composition

[0052] Take 2g each of luteolin and paeonol, add 20g of compressible starch and 10g of dextrin, mix well, granulate by 50% ethanol wet method, cool to below 60°C for drying, granulate, add 0.5g of talcum powder, mix well, and press into 100 tablets. Each tablet contains 20mg of luteolin and 20mg of paeonol on average.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition comprising luteolin or a pharmaceutically acceptable salt thereof and paeonol or a pharmaceutically acceptable salt thereof, wherein the weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.1-10.

2. The pharmaceutical composition according to claim 1, characterized in that The weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.5-2.

3. The pharmaceutical composition according to claim 1, characterized in that The weight ratio of luteolin or a pharmaceutically acceptable salt thereof to paeonol or a pharmaceutically acceptable salt thereof, calculated on the basis of luteolin and paeonol, is 1:0.8-1.

5.

4. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

5. The pharmaceutical composition according to claim 1, characterized in that The dosage form of the pharmaceutical composition is tablets, pills, capsules, powders, granules, suspensions, oral liquids, aerosols or liquid sprays, and drops.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for preventing or treating myocardial ischemia-reperfusion injury.

7. The use according to claim 6, characterized in that: The medicament further comprises a pharmaceutically acceptable adjuvant.