Application of sedum spectabile total flavonoids in preparation of medicine for treating myocardial infarction

Abstract

The invention provides application of sedum spectabile total flavonoids in preparation of a medicine for treating myocardial infarction. The sedum spectabile total flavonoids can relieve myocardial injury after myocardial infarction, reduce the myocardial infarction area and scar tissue, improve the heart function, inhibit myocardial remodeling after myocardial infarction and relieve mitochondrial injury in myocardial tissue after myocardial infarction.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating myocardial infarction. Background Technology

[0002] Myocardial infarction (MI) is a common acute and critical cardiovascular disease, primarily caused by myocardial necrosis due to acute or chronic ischemia and hypoxia of the coronary arteries. Clinically, it is characterized by sudden onset of severe and persistent oppressive pain in the retrosternal or precordial region, which cannot be completely relieved by rest or nitroglycerin, accompanied by elevated serum myocardial enzyme activity and progressive electrocardiographic changes. It can induce arrhythmias, shock, or heart failure. The onset is sudden and often life-threatening. Current treatments for myocardial infarction mainly include medication, interventional procedures, and surgery, but these cannot fundamentally and effectively repair the ischemic and necrotic myocardium. After myocardial infarction, a large number of functional cardiomyocytes undergo apoptosis and necrosis. The subsequent cardiac remodeling leads to the loss of the integrity of the original cardiac structure and function, which is replaced by fibrous scar tissue, ultimately inevitably leading to heart failure.

[0003] Mitochondria are the main organelles for energy supply in cardiomyocytes, and mitochondrial oxidative phosphorylation is a crucial process for generating ATP, the direct energy source. Mitochondrial integrity is a vital structural basis for ATP production, and damage to the mitochondrial cristae is a major cause of impaired myocardial energy metabolism. Energy deficiency is the root cause of myocardial remodeling after myocardial infarction; therefore, addressing energy metabolism issues and improving mitochondrial damage can further prevent myocardial injury after myocardial infarction. Thus, treatments targeting mitochondrial energy metabolism show great potential in myocardial infarction. Currently, the main measures for treating myocardial injury and remodeling after myocardial infarction include reperfusion therapy, RAAS inhibitors (including beta-blockers, ACE inhibitors, ARBs, and aldosterone receptor antagonists), statins, anti-inflammatory therapy, and stem cell transplantation. However, due to the complex pathogenesis and drug side effects, the treatment effect of myocardial infarction remains unsatisfactory, and the mortality rate remains high. Therefore, finding new treatment methods to reduce mitochondrial cristae damage and myocardial energy metabolism may be an important strategy for improving myocardial infarction.

[0004] Total flavonoids (TFP) from Sedum spectabile are flavonoids extracted from the plant and possess antioxidant and antibacterial properties. Current research reveals that TFP can significantly reduce fasting blood glucose in alloxan-induced diabetic rats. However, there are no reports on the therapeutic effects of TFP on myocardial infarction. Therefore, clarifying the regulatory role of TFP in mitochondrial function and energy metabolism, and accelerating its translational application in cardiovascular medicine, is urgent and important for the prevention and treatment of myocardial infarction. Summary of the Invention

[0005] To address the problems existing in the background art, this invention provides the application of total flavonoids from Sedum spectabile in the preparation of drugs for treating myocardial infarction. Total flavonoids from Sedum spectabile can alleviate myocardial damage after myocardial infarction, reduce the infarct area and scar tissue, improve cardiac function, inhibit myocardial remodeling, and reduce mitochondrial damage to myocardial tissue after myocardial infarction.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: In a first aspect, the present invention provides the application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating myocardial infarction.

[0007] According to the above plan, the myocardial infarction is an acute myocardial infarction.

[0008] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs for treating myocardial infarction caused by obstruction of the left anterior descending coronary artery.

[0009] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs to reduce myocardial damage after myocardial infarction, reduce the area of ​​myocardial infarction and scar tissue, improve cardiac function, and inhibit myocardial remodeling after myocardial infarction.

[0010] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs for treating myocardial infarction mediated by mitochondrial structural and functional damage.

[0011] According to the above scheme, the total flavonoids of Rhodiola rosea are used in the preparation of drugs to alleviate mitochondrial damage and metabolic disorders in myocardial tissue after myocardial infarction.

[0012] According to the above scheme, the total flavonoids from Rhodiola rosea can reduce mitochondrial damage by improving the mitochondrial oxidative phosphorylation function of myocardial tissue after myocardial infarction.

[0013] In a second aspect, the present invention provides a medicament for treating myocardial infarction, comprising total flavonoids from Rhodiola rosea and / or pharmaceutically acceptable salts thereof.

[0014] According to the above scheme, it also includes pharmaceutical components that have a positive effect on the treatment of myocardial infarction when used simultaneously with total flavonoids of Rhodiola rosea and / or pharmaceutically acceptable components that improve the stability of total flavonoids of Rhodiola rosea and / or pharmaceutically acceptable auxiliary components.

[0015] According to the above plan, the dosage forms of the drug are powder, granules, tablets, pills, capsules, oral liquid, gel, cream, spray or injection.

[0016] The beneficial effects of this invention are: 1. This invention is the first to apply total flavonoids from Rhodiola rosea to the preparation of a drug for treating myocardial infarction. Total flavonoids from Rhodiola rosea can significantly improve myocardial damage after myocardial infarction, providing a theoretical basis for clinical treatment of myocardial infarction. 2. This invention provides the application of total flavonoids from Rhodiola rosea in the treatment of mitochondrial damage and metabolic disorders. Total flavonoids from Rhodiola rosea can improve mitochondrial damage in cardiomyocytes after myocardial infarction and increase the level of oxidative phosphorylation in cardiomyocytes, thus having positive clinical application value in the treatment of myocardial mitochondrial dysfunction after myocardial injury. Attached Figure Description

[0017] Figure 1 In this paper, A represents the survival rate analysis of mice treated with solvent or total flavonoids from Sedum aizoon within 2 weeks after myocardial infarction; B and C represent the infarct area and quantitative analysis results of mice treated with solvent or total flavonoids from Sedum aizoon 7 days after myocardial infarction, as shown by TTC (2,3,5-triphenyltetrazolium chloride) staining. Figure 2 The levels of serum troponin (cTnT) and creatine kinase isoenzyme (CK-MB) in mice treated with solvent or total flavonoids from Sedum truncatum 2 weeks after sham surgery or myocardial infarction were measured. Figure 3 Masson staining images (1X) of heart sections from mice treated with solvent or total flavonoids from Sedum truncatum 2 weeks after sham surgery or myocardial infarction. Figure 4 In this invention, the echocardiographic results of mice in the sham-operated group and the MI group after administration of solvent or total flavonoids from Sedum truncatum are shown in Figure A, which is a representative M-mode echocardiogram of mice in the sham-operated group and the MI group after administration of solvent or total flavonoids from Sedum truncatum; Figure B is a two-dimensional speckle tracking imaging (VevoStrain) analysis of left ventricular strain during the cardiac cycle of mice in each group (parasternal long axis view) to assess myocardial strain of the heart in each group of mice; Figures C, D and E are the results of cardiac ejection fraction (EF), fractional shortening (FS), and global longitudinal strain rate (GLS) analysis of mice in each group of mice, respectively. Figure 5 In the image, A represents the mitochondrial morphology of myocardial tissue from mice treated with solvent or total flavonoids from Sedum spectabile two weeks after sham surgery or myocardial infarction, as shown by transmission electron microscopy; B represents the mitochondrial morphology of mice treated with solvent or total flavonoids from Sedum spectabile. Figure 5 A. Quantitative analysis results of the percentage of intact mitochondrial cristae in myocardial tissue; Figure 6The present invention describes the quantitative analysis results of mitochondrial oxygen consumption curve (OCR) and basal respiration, maximum respiratory rate and ATP production of neonatal rat cardiomyocytes treated with solvent or total flavonoids from Sedum spectrophotometry after culturing for 24 hours under normal oxygen and hypoxia (1.0% O2) conditions. A is the mitochondrial oxygen consumption curve (OCR) of cardiomyocytes, B is the quantitative analysis result of basal respiration, C is the quantitative analysis result of maximum respiratory rate and D is the quantitative analysis result of ATP production. Detailed Implementation

[0018] The principles and features of the present invention are described below with reference to the accompanying drawings and specific embodiments. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0019] The inventors discovered that total flavonoids from *Sedum spectabile* can alleviate myocardial damage after myocardial infarction, thereby delaying the progression of heart failure. Through establishing hypoxic models of neonatal rat cardiomyocytes and a mouse model of myocardial infarction induced by ligation of the left anterior descending coronary artery, they found that total flavonoids from *Sedum spectabile* significantly improved mitochondrial oxidative phosphorylation function in cardiomyocytes under hypoxic conditions. Animal experiments showed that after myocardial infarction, mice treated with total flavonoids from *Sedum spectabile* had reduced mortality, significantly reduced infarct area and scar tissue, and significantly improved cardiac function. Simultaneously, total flavonoids from *Sedum spectabile* can alleviate mitochondrial damage in myocardial tissue of mice after myocardial infarction. Therefore, this study provides the application of total flavonoids from *Sedum spectabile* in the preparation of drugs for treating myocardial infarction. Total flavonoids from *Sedum spectabile* have a good therapeutic effect on myocardial damage after myocardial infarction and can also prevent and treat mitochondrial damage after myocardial infarction.

[0020] In some specific embodiments of the present invention, the dosage of total flavonoids from Sedum spectabile in animal models can be 5-20 mg / kg / day, preferably 10 mg / kg / day. Total flavonoids from Sedum spectabile (at a dose of 0.1 mg / ml) can significantly improve hypoxia-induced mitochondrial oxidative phosphorylation dysfunction in cardiomyocytes.

[0021] In vivo model: This invention used 8-10 week old C57BL / 6J mice weighing 23.5g-27.5g as experimental subjects. A myocardial infarction (MI) animal model was induced by ligation of the left anterior descending coronary artery. Small animal ultrasound, 2,3,5-triphenyltetrazolium chloride (TTC) staining, Masson staining, and transmission electron microscopy were used to evaluate changes in cardiac function, infarct area, scar tissue, and mitochondrial morphology after myocardial infarction. Results showed that after establishing the myocardial infarction model, mice treated with total flavonoids from *Sedum spectabile* (at a dose of 10mg / kg / d) had reduced mortality, significantly reduced infarct area, significantly reduced scar tissue, and significantly improved cardiac function. Simultaneously, total flavonoids from *Sedum spectabile* could alleviate mitochondrial damage in the myocardial tissue of mice after myocardial infarction.

[0022] In vitro model: This invention establishes a cardiomyocyte hypoxia model by extracting cardiomyocytes from newborn rats. The mitochondrial oxidative phosphorylation function of cardiomyocytes after hypoxia treatment was evaluated using biological techniques such as the Seahorse Cell Energy Metabolism Analyzer. Experimental results showed that, compared with the control group (normal oxygen treatment group), total flavonoids from *Sedum spectabile* (dose 0.1 mg / ml) significantly improved the mitochondrial oxidative phosphorylation function of cardiomyocytes under hypoxic conditions.

[0023] This invention provides a new target and strategy for the clinical treatment of myocardial injury after myocardial infarction using total flavonoids from Rhodiola rosea.

[0024] Laboratory animals and their feeding The experimental subjects were wild-type C57BL / 6J mice aged 8-10 weeks and weighing 23.5-27.5g. All experimental mice were housed in the Specific Pathogen Free (SPF) Laboratory Animal Center of the Institute of Cardiovascular Diseases, Wuhan University. The housing conditions were as follows: room temperature between 22-24℃, humidity between 50-70%, 12-hour alternating light and dark lighting, and ample water and food.

[0025] Example 1: Obtaining and Proportioning Total Flavonoids from Rhodiola Rosea 1. Acquisition: Total flavonoids (TFP) from Rhodiola rosea were purchased from Shanghai Ronghe Pharmaceutical Technology Development Co., Ltd., batch number: 250903, product number: RH1126-5.5g, in the form of brown powder, with a purity ≥95%.

[0026] The extraction method for total flavonoids from *Sedum spectabile* is as follows: *Sedum spectabile* herb is pulverized and soaked twice in 75% ethanol (material-to-liquid ratio 1:20, soaking for at least 10 hours each time). After filtration, the ethanol extracts are combined and collected. The extracts are then adsorbed using DM30 macroporous adsorption resin, followed by gradient elution with 20%, 40%, 60%, 80%, and 90% ethanol, and the fractions are collected. Analyzing is performed using high-performance liquid chromatography (HPLC). The flavonoid-containing fractions are combined and concentrated to remove ethanol. The concentrate is loaded onto a C18 column and eluted with pure methanol. The methanol solution is concentrated to remove methanol, then concentrated to dryness and placed in a vacuum drying oven at 50℃ for 12 hours to obtain the total flavonoids from *Sedum spectabile*.

[0027] 2. Preparation: Take an appropriate amount of total flavonoid powder from *Sedum spectabile* and dissolve it in dimethyl sulfoxide (DMSO, purchased from Wuhan Sewell Biotechnology Co., Ltd., catalog number GC203006) to prepare a stock solution, making the concentration of total flavonoids from *Sedum spectabile* in the stock solution 25 mg / ml. Then, dilute the stock solution with physiological saline at a ratio of 1:50 to prepare a working solution, making the concentration of total flavonoids from *Sedum spectabile* in the working solution 0.5 mg / ml. Administer 0.5 ml / mouse (25 g) / day to the corresponding groups of mice to achieve a dosage of 10 mg / kg / day. A similar procedure was followed when treating isolated myocardial cells of the corresponding groups to achieve a drug concentration of 0.1 mg / ml.

[0028] Example 2: Construction of a mouse model of myocardial infarction (MI) The mouse model of myocardial infarction was established by ligation of the left anterior descending coronary artery (LAD). The model operation procedure is as follows: 1. Preoperative preparation (1) Anesthesia: First, weigh the mice and calculate the required amount of anesthetic (3% sodium pentobarbital) based on 90 mg / kg body weight. Administer the anesthetic via intraperitoneal injection and record the injection time. Successful anesthesia is defined as no obvious reaction to tail or toe clamping and the mouse being in good condition (generally, there is no obvious reaction about 10 minutes after injection; the optimal time for surgery is about 30 minutes after anesthesia, with the mouse showing a toe clamping reaction approximately 50 minutes after anesthesia).

[0029] (2) Surgical area preparation: Remove hair from the skin of the left chest, left lateral chest, and left forelimb axilla of the mouse. After shaving, wipe the surgical area with a damp gauze to remove the mouse hair, so as not to affect the surgical field.

[0030] (3) Endotracheal intubation: Secure the mouse's upper incisors to the inclined surface of the V-shaped plate with a rubber band, and quickly and accurately insert the endotracheal tube into the trachea through the glottis. Then, place the mouse in a right lateral decubitus position on a heating pad (the heating pad needs to be preheated). Connect the endotracheal tube to the ventilator and fix the mouse in place. If the rise and fall of the mouse's chest is consistent with the ventilator frequency, it indicates that the endotracheal intubation was successful.

[0031] 2. Left anterior descending coronary artery ligation (LAD) Mice were placed in a supine position. The muscles between the third and fourth ribs on the left side were bluntly separated. Hemostatic forceps were used to open the third and fourth ribs, and the heart was quickly squeezed out. The left anterior descending coronary artery was ligated with a 6-0 suture needle, 1 mm below the left atrial appendage, with a needle depth of 0.5 mm. During the operation, the myocardium in the infarct area below the ligation site turned pale to determine whether the ligation was successful. An electrocardiogram was connected to further determine whether the model was successful. Postoperatively, each mouse was routinely given intraperitoneal injection of penicillin at 100,000 IU per day for 3 consecutive days. The incision skin was also disinfected to prevent infection.

[0032] 3. Postoperative care After ligation of the left anterior descending coronary artery, once the mice exhibited spontaneous breathing and a strong toe-clamping response, the endotracheal tube was removed, and the mice were placed in a rearing cage containing autoclaved bedding, feed, and drinking water for continued rearing and observation in the rearing room.

[0033] 4. Group processing Mice in the sham-operated group (Sham group, with only thoracotomy to expose the heart and no other treatment) and the MI group (a mouse model of myocardial infarction obtained by thoracotomy followed by ligation of the left anterior descending coronary artery) were randomly assigned to two groups. On postoperative day 3, mice were intraperitoneally injected daily for 10 consecutive days with total flavonoids from *Sedum spectabile* (using physiological saline containing 2% DMSO as the solvent, at a dose of 10 mg / kg / day). The control group received only the same dose of solvent. Based on the treatment method, mice were divided into four groups: sham-operated group (Sham + solvent group), sham-operated group + total flavonoids from *Sedum spectabile* (Sham + TFP group), surgical group (MI + solvent group), and surgical group + total flavonoids from *Sedum spectabile* (MI + TFP group), with 15 mice in each group. Mice mortality was recorded daily in each group.

[0034] Postoperative survival rate of mice as follows Figure 1 As shown in Figure A, the survival rates of the surgical group (MI + solvent group) and the surgical group + total flavonoids of Rhodiola rosea group (MI + TFP group) were 40% and 80% respectively 7-15 days after surgery. Treatment with total flavonoids of Rhodiola rosea after myocardial infarction can significantly improve the survival rate of mice with myocardial infarction.

[0035] One week after surgery, tissue samples were taken for TTC staining; two weeks after surgery, various experiments were performed, including cardiac ultrasound and gross tissue sampling, and data such as heart weight, lung weight, and body weight of the mice were recorded.

[0036] Example 3: Pathological examination of a mouse model of myocardial infarction 1. Get materials (1) Preliminary work: Prepare a urine cup containing 20 mL of 10% formaldehyde and label it (mouse number, group, surgery type, and collection date). Place a petri dish filled with 10% KCl solution at the collection point. Turn on the analytical balance and zero it for later use. Weigh and euthanize the mice.

[0037] (2) Sample collection: The vascular pedicle below the atrial appendage was grasped with ophthalmic curved forceps, the heart was cut off, and quickly placed in a 10% KCl solution. After the heart stopped beating in diastole, it was placed on sterile gauze, the fluid in the heart chamber was gently squeezed out, the surface fluid was dried, the weight was weighed and recorded, the heart was placed in the corresponding urine cup, fixed for 48 hours, and then used for pathological examination.

[0038] (3) Related measurements and calculations: Remove the mouse heart and lungs, trim them, blot them dry with filter paper, weigh and record the weight. Cut open the skin at the tibia of the mouse's hind limb, measure and record the tibia length. Calculate the ratio of lung weight to body weight (LW / BW) and the ratio of heart weight to tibia length (HW / TL).

[0039] 2. Pathological examination 2.1 TTC (2,3,5-triphenyltetrazolium chloride) staining: After removing the fresh heart, place it in a -20℃ freezer for 20 minutes to harden the heart → use a blade to cut the heart into five equal parts perpendicular to the longitudinal axis of the heart → place the cut heart tissue in 2% TTC staining solution → then place it in a 37℃ constant temperature water bath for 20-30 minutes → remove the heart slices, arrange them neatly and take pictures. TTC staining results are as follows Figure 1 As shown in B and C, the results showed that the myocardial infarction area in the surgery group (MI + solvent group) reached 56.88%, while the myocardial infarction area in the surgery group + Rhodiola rosea total flavonoids group (MI + TFP group) was 25.36%, indicating that treatment with Rhodiola rosea total flavonoids after myocardial infarction can significantly reduce the infarction area.

[0040] 2.2 Preparation of paraffin-embedded specimen sections The main procedures include: trimming heart or lung tissue → embedding frame preparation → rinsing with running water → dehydration → clearing → wax infiltration → embedding → sectioning → spreading → air drying or baking for later use.

[0041] 2.3 Specific steps of Masson's trichrome staining Baking at 55℃ for 30 min → Xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → Rinse with running water for 10 min → Double distilled water for 1 min → Weigert's ferrohematoxylin staining for 5 min → Rinse with tap water for 5 min → Remove residual liquid → Differentiate with 1% hydrochloric acid alcohol for 4 s → Rinse with tap water for 5 min to regain blue color → Stain with Ponceau S and acid fuchsin solution for 10 min → Rinse with distilled water for 5 min → Treat with phosphomolybdic acid aqueous solution for about 5 min → Aniline blue solution Counterstain for 5 min → treat with 1% glacial acetic acid for 1 min → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → xylene for 2 min, 3 times → immediately cover with a slide while the xylene is still wet and take a picture under a microscope.

[0042] Masson staining results are as follows: Figure 3 As shown, the results showed that treatment with total flavonoids from Rhodiola rosea after myocardial infarction could significantly reduce scar tissue.

[0043] Example 4: Transmission electron microscopy of myocardial tissue After euthanizing mice by cervical dislocation, the heart was removed within 3 minutes. Before sampling, a culture dish containing electron microscopy fixative was prepared in advance. After removing small pieces of myocardial tissue from the body, it was immediately placed in the culture dish and cut into small pieces in the fixative of the culture dish with a scalpel. The sampled tissue volume was controlled to be 1.0 mm × 1.0 mm × 1.0 mm cube. Care was taken to avoid mechanical damage such as squeezing with tweezers during sampling. The blade should be sharp to avoid bruising the tissue. After the myocardial tissue was removed, it was immediately placed in electron microscopy fixative and fixed at room temperature in the dark for 2 hours, and then transferred to 4°C for storage. Subsequently, an electron microscopy sample was prepared, and the morphology of mitochondria in the myocardial tissue was observed by transmission electron microscopy.

[0044] Transmission electron micrograph of mouse myocardial tissue as shown below Figure 5 As shown, the results indicate that mitochondria in the myocardial tissue of mice after MI surgery were severely damaged, with a large number of mitochondrial cristae destroyed; however, after intraperitoneal injection of total flavonoids from Rhodiola rosea for 2 weeks, the proportion of intact mitochondrial cristae in the myocardial tissue of myocardial infarction model mice increased from 3.7% to 9.3%, an increase of 5.6%, proving that total flavonoids from Rhodiola rosea can significantly improve mitochondrial damage in myocardial infarction model mice (MI mice).

[0045] Example 5: Echocardiography to detect cardiac function in mice 1. Preliminary preparations (1) Preparation of the anesthesia machine: First connect the oxygen cylinder to the air inlet on the anesthesia machine, then unscrew the sealing cap of the drug delivery port on the anesthesia machine, quickly add isoflurane to the safe mark and then tighten the sealing cap. Unscrew the main valve on the oxygen cylinder, adjust the knob of the flow control valve, and maintain the outlet pressure at 0.2-0.3 MPa.

[0046] (2) Preparation of test mice: After the test mice are rapidly anesthetized with isoflurane, the hair in the chest area is shaved, and the head of the prepared mouse is inserted into the anesthetic catheter. The mouse is kept under stable anesthesia with 1.5-2.0% isoflurane.

[0047] 2. Echocardiography Anesthetized mice were placed on an ultrasound platform, their limbs secured with adhesive tape. A suitable amount of ultrasound coupling gel was evenly applied to the skin of the precordial region and the ultrasound probe. The ultrasound probe was aimed at the point of strongest cardiac contraction, parallel to the long axis of the mouse's heart. The long axis section of the left ventricle was first determined using ultrasound. The probe was then adjusted, and after each mouse's heart rate stabilized, the M-mode ultrasound was switched to record and measure video and imaging data over multiple cardiac cycles. The following parameters were analyzed for at least three consecutive cardiac cycles: heart rate, ejection fraction, and fractional shortening. All parameters were measured at least three times, and the average values ​​are provided.

[0048] 3. Two-dimensional speckle tracking analysis Two-dimensional speckle tracking was performed on dynamic images of the long-axis section of the heart acquired by ultrasound using the Vevo3100's Vevoostrain software to trace the trajectory of myocardial motion. At least six independent cardiac cycles were analyzed for each heart. Myocardial strain parameters such as global longitudinal strain rate (GLS) were obtained through analysis.

[0049] The cardiac function test results of mice after sham surgery or left anterior descending coronary artery ligation modeling are as follows: Figure 4 As shown in the figure, compared with the sham-operated group, mice in the MI group showed weakened cardiac contractility and reduced local ventricular wall motion 2 weeks after surgery, mainly manifested as a decrease in left ventricular ejection fraction and overall longitudinal strain; while the treatment with total flavonoids from Rhodiola rosea showed an increase in left ventricular ejection fraction and overall longitudinal strain, suggesting that total flavonoids from Rhodiola rosea can significantly improve cardiac dysfunction in mice after myocardial infarction.

[0050] Example 6: Detection of myocardial injury biomarkers in a mouse model of myocardial infarction Before gross sampling, the whiskers of the mice were trimmed. Under inhalation anesthesia, blood was collected from the orbital veins. The skin of the mouse's nape and periauricular region was lifted with the thumb and forefinger of the left hand, and pressure was applied firmly above the eye socket to induce congestion of the posterior orbital venous plexus. The blood collection device was inserted into the eyeball from the inner corner of the eye at a 45° angle to the plane of the eye, penetrating 3 mm. The device was then rotated to collect blood from the posterior orbital border. After blood collection from each mouse, the eyeball was pressed with sterile gauze to stop bleeding. The blood was allowed to stand for 10 minutes, then centrifuged at 3000 rpm for 3 minutes. The supernatant serum was aliquoted into labeled Eppendorf tubes, and the expression levels of myocardial injury markers cTnT and CK-MB were detected using an ELISA kit.

[0051] Results of myocardial injury marker testing, such as Figure 2 As shown, this indicates that severe myocardial injury in mice after MI surgery can be improved by intraperitoneal injection of total flavonoids (TFP) from Rhodiola rosea for 2 weeks.

[0052] Example 7: Effects of total flavonoids from Rhodiola rosea on mitochondrial function in hypoxic-stimulated primary cardiomyocytes 1. Primary neonatal SD rat cardiomyocyte culture (1) Ten Sprague-Dawley infant rats aged 1-3 days were disinfected below the neck with 75% alcohol. The heart was removed with ophthalmic scissors and microforceps and placed in a glass petri dish containing 10 ml of LDM / F12 solution. The above process was repeated for another rat.

[0053] (2) Wash the heart with DMEM / F12 medium and cut the heart into 1-2 mm pieces. 3The fragments were transferred to a serum bottle containing a rotor, DMEM / F12 was removed, and trypsin digestion solution was added. The rotor speed was 120 r / min, and digestion was carried out for 15 min. After standing for a few seconds, the supernatant was discarded.

[0054] (3) Add trypsin digestion solution, rotate at 120 r / min, and digest for 15 min. Let stand for a few seconds, aspirate the supernatant, terminate digestion with DMEM / F12 medium containing 20% ​​fetal bovine serum, and store at 4°C. Repeat this step several times. When collecting the supernatant, try to collect as much as possible. When the tissue block turns white and becomes significantly smaller, stop digestion.

[0055] (4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min and discard the supernatant. Add an appropriate amount of culture medium to the centrifuge tube, gently pipette to resuspend the cells, and concentrate them into a 50 mL centrifuge tube. Filter the cell suspension through a 40 μm cell filter.

[0056] (5) Seed the cells in a 100 mm culture dish and allow them to adhere for 90 min. Then, filter the unadhered cell suspension. Add BrdU (final concentration 0.1 mM) to the total amount of cell suspension, mix well, and then add the mixture to a dish coated with 0.1% gelatin.

[0057] (6) Gently shake to disperse the cells, do not vortex. Incubate at 37°C and 5% CO2 for 48 hours, wash once with PBS, and change the culture medium.

[0058] (7) Cell grouping and treatment: Primary cardiomyocytes were seeded in 24-well culture plates and the experiments were conducted when the cell confluence reached 70-80%. After different treatments, the cells were divided into the following four groups: normal oxygen + solvent group; normal oxygen + TFP group; hypoxia + solvent group; and hypoxia + TFP group. The specific treatments were as follows: TFP was added to the normal oxygen + TFP group and the hypoxia + TFP group at a dose of 0.1 mg / ml. Then, the cells were treated in a hypoxic environment (1.0% O2, tri-gas incubator) for 24 hours to construct an in vitro cardiomyocyte hypoxia model. The control group was cultured under normal oxygen. Mitochondrial function was then measured in each group of cells.

[0059] 2. Effects of total flavonoids from Rhodiola rosea on mitochondrial function in primary cardiomyocytes under hypoxia stimulation Mitochondrial oxidative respiration rate (OCR) in neonatal rat cardiomyocytes was measured using a SeahorseXFe24 cell energy analyzer. Working concentrations of the required mitochondrial stress assay drugs (oligomycin A and FCCP) for primary cardiomyocytes were determined by dostotation. Primary cardiomyocytes were cultured in V-7 hippocampal plates at a density of ≥10⁵ cells per well. Before assay, primary cardiomyocytes were cultured for 1 hour at 37°C in a CO₂-free incubator (1 mM pyruvate + glucose-free Seahorse assay medium). A syringe was then loaded with 1.0 μM oligomycin, 1.0 μM FCCP, and 2.0 μM antimycin A. Mitochondrial oxidative respiration parameters, such as basal respiration, ATP production, maximum oxygen consumption, and respiratory potential, were recorded as mitochondrial oxygen consumption rate (OCR).

[0060] The Seahorse Cell Energy Metabolism Analyzer was used to detect the mitochondrial oxygen consumption curves (OCR) of cardiomyocytes in each group, as follows: Figure 6 As shown. Basal respiration represents the rate at which cells consume oxygen through mitochondrial oxidative phosphorylation under basal conditions, reflecting the energy consumption level of cells to maintain basic physiological functions. ATP production: The portion of the OCR that decreases after the addition of oligomycin (which inhibits ATP synthase) represents ATP production-related respiration, indicating the amount of oxygen consumed by mitochondria for ATP synthesis. Maximum respiration refers to the maximum value of the OCR reached after the addition of the uncoupling agent FCCP, representing the mitochondrial respiratory capacity of cells under maximum energy demand. Finally, the OCR that remains after the addition of rotenone and antimycin A (which inhibits the mitochondrial electron transport chain) represents oxygen consumption from non-mitochondrial sources. After hypoxia stimulation, basal respiration, ATP synthesis, and maximum respiration of cardiomyocytes were significantly reduced, indicating mitochondrial dysfunction; while under hypoxia conditions, basal respiration, ATP synthesis, and maximum respiration of cardiomyocytes treated with total flavonoids from Sedum sarmentosum were significantly improved, indicating that total flavonoids from Sedum sarmentosum significantly improve mitochondrial function of cardiomyocytes under hypoxia conditions.

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of total flavonoids from Rhodiola rosea in the preparation of drugs for treating myocardial infarction.

2. The application according to claim 1, characterized in that, The myocardial infarction mentioned refers to acute myocardial infarction.

3. The application according to claim 1, characterized in that, The application of the total flavonoids from Rhodiola rosea in the preparation of drugs for treating myocardial infarction caused by obstruction of the left anterior descending coronary artery.

4. The application according to claim 1, characterized in that, The total flavonoids from Rhodiola rosea are used in the preparation of drugs to reduce myocardial damage after myocardial infarction, reduce the infarct area and scar tissue, improve cardiac function, and inhibit myocardial remodeling after myocardial infarction.

5. The application according to claim 1, characterized in that, The application of the total flavonoids from Rhodiola rosea in the preparation of drugs for treating myocardial infarction mediated by mitochondrial structural and functional damage.

6. The application according to claim 5, characterized in that, The application of the total flavonoids from Rhodiola rosea in the preparation of drugs to alleviate mitochondrial damage and metabolic disorders in myocardial tissue after myocardial infarction.

7. The application according to claim 6, characterized in that, The total flavonoids from Rhodiola rosea can reduce mitochondrial damage by improving the mitochondrial oxidative phosphorylation function of myocardial tissue after myocardial infarction.

8. A drug for treating myocardial infarction, characterized in that, Including total flavonoids from Sedum spectabile and / or its pharmaceutically acceptable salts.

9. The medicament according to claim 8, characterized in that, It also includes pharmaceutical components that have a positive effect on the treatment of myocardial infarction when used concurrently with total flavonoids of Rhodiola rosea and / or pharmaceutically acceptable components that improve the stability of total flavonoids of Rhodiola rosea and / or pharmaceutically acceptable adjuvant components.

10. The medicament according to claim 9, characterized in that, The dosage forms of the drug are powder, granules, tablets, pills, capsules, oral liquid, gel, cream, spray or injection.

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