Application of herba lycopi flavone or pharmaceutically acceptable salt or solvate thereof in preparation of medicine for preventing, delaying and / or treating heart aging and / or myocardial injury

Abstract

The invention discloses application of herba lycopi flavone or pharmaceutically acceptable salts or solvates thereof in preparation of medicines for preventing, delaying and / or treating heart aging and / or myocardial injury, and particularly discloses application of herba lycopi flavone or pharmaceutically acceptable salts or solvates thereof in preparation of medicines for preventing, delaying and / or treating heart aging and / or myocardial injury. The herba lycopi flavone disclosed by the invention has a structure as shown in a formula (I). The herba lycopi flavone disclosed by the invention can be used for improving heart function indexes of senescent heart, reducing the expression level of gamma-H2AX in senescent heart tissues and effectively improving myocardial fiber breakage and arrangement disorder of organisms. (I).

Description

Technical Field

[0001] This invention relates to the use of a flavonoid from Zephyranthes or a pharmaceutically acceptable salt or solvate thereof in the preparation of medicaments for the prevention, delay and / or treatment of cardiac aging and / or myocardial injury. Background Technology

[0002] As the population ages, the burden of age-related diseases has become a major health and social problem. Age-related degenerative changes in the structure and function of the heart are a major risk factor for cardiovascular disease, which in turn accelerates cardiac aging. Therefore, early detection of cardiac degenerative changes and timely warning, prevention, and treatment of cardiovascular diseases are crucial for achieving healthy aging.

[0003] Currently, strategies for delaying cardiac aging mainly include lifestyle interventions and drug therapy. Lifestyle interventions, including calorie restriction and regular exercise, have some effect, but strict adherence to these diet and exercise programs is often poor. Drug therapy, such as metformin, rapamycin, NAD+ precursors (e.g., NMN / NR), and senescent cell scavengers (e.g., dasatinib + quercetin), has shown potential in research, but significant limitations and challenges remain. Long-term use of rapamycin may cause serious adverse reactions such as immunosuppression and metabolic disorders; the long-term safety of metformin and senescent cell scavengers in off-label populations still requires large-scale clinical validation.

[0004] Natural product monomers are monomeric components extracted and isolated from natural products. They are characterized by their singular composition and minimal side effects, making them a valuable resource for new drug development. They also possess a variety of pharmacological activities, such as antioxidant, antitumor, antibacterial, and anti-inflammatory effects. Current research indicates that *Eupatorium fortunei* flavonoids exhibit multiple activities, including antioxidant, antitumor, and anti-inflammatory effects. However, there are no reports of *Eupatorium fortunei* flavonoids' activity in delaying cardiac aging and / or treating myocardial injury. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the use of Zephyranthes flavonoids in the preparation of drugs for the prevention, delay, and / or treatment of cardiac aging and / or myocardial injury. It can reduce the senescence positivity rate of senescent cells, improve weight loss and diastolic dysfunction caused by cardiac aging, shorten isovolumetric relaxation time, reduce the expression level of γ-H2AX in cardiac tissue, and also improve myocardial injury.

[0006] The present invention achieves the above objectives using the following technical solutions.

[0007] This invention provides the use of Lycopus lucidus flavonoids or pharmaceutically acceptable salts or solvates thereof in the preparation of medicaments for the prevention, delay, and / or treatment of cardiac aging and / or myocardial injury, wherein the Lycopus lucidus flavonoids have a structure as shown in formula (I): (I).

[0008] In this invention, the molecular formula of Lycopus lucidus flavonoids is C2. 16 H 12 O7, with a molecular weight of 316.26 and CAS No.: 520-11-6, can be extracted and isolated from plants containing this active ingredient (such as Eupatorium fortunei and ginseng) using conventional natural product extraction methods in the field, or it can be purchased from commercially available products.

[0009] Pharmaceutically acceptable salts of Lycopus lucidus flavonoids include, but are not limited to, phenoxy anion salts (i.e., the limited phenolic hydroxyl groups in Lycopus lucidus flavonoids that lose protons to form anoxy anions), and sodium salts (Na... + ), potassium salt (K) + ), magnesium salt (Mg 2+ ), calcium salts (Ca 2+ ), meglumine salt, sulfate, succinate, maleate, fumarate, etc.

[0010] Solvates of Lycopus lucidus include, but are not limited to, Lycopus lucidus hydrates (e.g., monohydrates, dihydrates), Lycopus lucidus ethanolates, Lycopus lucidus acetoneates, Lycopus lucidus ethyl acetateates, Lycopus lucidus N,N-dimethylformamide (DMF) compounds, or Lycopus lucidus dimethyl sulfoxide (DMSO) compounds.

[0011] In existing research, Lycopus lucidus flavonoids are mainly used to study their effects on esophageal cancer, eye inflammation, pneumonia, allergy, macrophage inflammation, epilepsy, acute kidney injury, and pollution-resistant cosmetics. The inventors have conducted in-depth research on Lycopus lucidus flavonoids and, through numerous experiments, have for the first time discovered that it possesses pharmacological activities that delay cardiac aging and improve myocardial damage.

[0012] According to the intended use of the present invention, preferably, the myocardial injury is caused by one or more of the following: coronary heart disease, myocardial hypertrophy, heart failure, angina pectoris, arrhythmia, pulmonary heart disease, myocardial infarction, myocardial ischemia, myocardial ischemia-reperfusion, chemotherapy drugs, bacteria, and viruses.

[0013] Cardiac aging, as a driving factor in myocardial injury, lowers the heart's damage threshold and reduces its functional reserve, making the heart more vulnerable. When subjected to damaging stimuli, an aging heart can trigger severe and irreversible myocardial damage. Simultaneously, myocardial damage further accelerates cardiac aging.

[0014] Current treatment strategies for myocardial injury primarily intervene by reducing cardiac load (e.g., diuretics, vasodilators), improving myocardial metabolism (e.g., beta-blockers), or revascularization (e.g., stent surgery). However, these methods cannot fundamentally reverse or prevent the progressive loss and functional decline of cardiomyocytes. The flavonoids of this invention, or pharmaceutically acceptable salts or solvates thereof, can address the upstream drivers of myocardial injury, fundamentally preventing its occurrence and development by delaying cardiac aging. Furthermore, the flavonoids of this invention, or pharmaceutically acceptable salts or solvates thereof, improve myocardial injury in organisms.

[0015] According to the intended use of the present invention, preferably, the medicament comprises zeylan flavonoids or a pharmaceutically acceptable salt or solvate thereof and pharmaceutically acceptable excipients.

[0016] In this invention, pharmaceutically acceptable excipients refer to pharmaceutical excipients conventional in the field of pharmaceutical formulations, which may be selected from one or more of fillers, binders, disintegrants, lubricants, suspending agents, pigments, flavoring agents, solvents, and surfactants.

[0017] In this invention, examples of fillers include, but are not limited to, starch, microcrystalline cellulose, sucrose, dextrin, lactose, powdered sugar, glucose, etc.; examples of binders include, but are not limited to, water, ethanol, starch paste, syrup, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium alginate, polyvinylpyrrolidone, etc.; examples of disintegrants include, but are not limited to, starch effervescent mixtures (i.e., sodium bicarbonate and citric acid), tartaric acid, low-substituted hydroxypropyl cellulose, etc.; examples of lubricants include, but are not limited to, magnesium stearate, stearic acid, sodium chloride, sodium oleate, sodium lauryl sulfate, etc.; suspending agents... Examples of agents include, but are not limited to, polysaccharides (e.g., farnesian gum), agar, sodium alginate, cellulose ether, carboxymethyl chitosan, etc.; examples of pigments include, but are not limited to, iron oxide, chlorophyll, β-carotene, tartrazine, indigo, etc.; examples of flavoring agents include, but are not limited to, sucrose, fructose, sorbitol, mannitol, aspartame, sodium saccharin, fruit flavoring, peppermint flavoring, etc.; examples of solvents include, but are not limited to, water, balanced salt solutions, etc.; examples of surfactants include, but are not limited to, polysorbates (e.g., Tween-80), poloxamer, sodium lauryl sulfate, etc.

[0018] According to the intended use of the present invention, preferably, the drug has Lycopus lucidus flavonoids as the sole active ingredient; the dosage form of the drug is an oral preparation.

[0019] The oral formulations of the present invention can be oral solid dosage forms, such as tablets, dispersible tablets, enteric-coated tablets, granules, capsules, pellets, powders, etc.; or oral liquid dosage forms, including but not limited to drug solutions, suspensions, and emulsions. In the present invention, the oral formulations are preferably capsules, tablets, granules, or oral liquid dosage forms; more preferably capsules or tablets.

[0020] Experimental results show that the flavonoids of *Zephyranthes bidentata* or its pharmaceutically acceptable salts or solvates can reduce the expression level of β-galactosidase in cardiomyocytes, improve weight loss caused by cardiac aging, improve cardiac function indicators of aging hearts, and reduce the expression level of the DNA damage marker γ-H2AX in aging cardiac tissue, and can also improve myocardial damage caused by cardiac aging. According to certain embodiments of the present invention, the active ingredient in the pharmaceutical preparation contains only flavonoids of *Zephyranthes bidentata*.

[0021] According to the intended use of the present invention, preferably, the amount of *Zephyranthes bidentata* flavonoids added in a unit formulation is 2.4–3.9 mg / kg. More preferably, the amount added is 2.45–3.83 mg / kg.

[0022] According to the intended use of the present invention, preferably, the flavonoids of *Zephyranthes bidentata* or its pharmaceutically acceptable salts or solvates are capable of reducing the expression level of β-galactosidase in cardiomyocytes.

[0023] According to a specific embodiment of the present invention, in the SA-β-Gal staining experiment, senescent H9c2 cardiomyocytes were treated with Lycopus lucidus flavonoids. When the concentration of Lycopus lucidus flavonoids was 20 μM, the expression level of β-galactosidase in senescent H9c2 cardiomyocytes was significantly reduced. The positive rate of senescent cardiomyocytes decreased from 57.5 ± 9.7% to 23.5 ± 5.5%. Cardiac senescence at the cellular level can be induced by doxorubicin.

[0024] According to the intended use of the invention, preferably, the *Eupatorium fortunei* flavonoids or their pharmaceutically acceptable salts or solvates are able to improve weight loss due to cardiac aging. Cardiac aging at the animal level can be induced by D-galactose.

[0025] The flavonoids of Zephyranthes described in this invention, or their pharmaceutically acceptable salts or solvates, can significantly improve the physical performance of organisms with aging hearts.

[0026] According to specific embodiments of the present invention, Lycopus lucidus flavonoids or pharmaceutically acceptable salts or solvates thereof can significantly improve the physical performance of cardiac aging mice. In mouse physical performance testing experiments (specifically grip strength test and rotarod test), cardiac aging mice treated with Lycopus lucidus flavonoids showed significant improvement in grip strength and a significant reduction in the number of drops during the rotarod test.

[0027] Furthermore, the flavonoids of *Zephyranthes* or their pharmaceutically acceptable salts or solvates described in this invention can significantly improve cardiac function in organisms with aging hearts.

[0028] According to the intended use of the present invention, preferably, the flavonoids of *Zephyranthes bidentata* or its pharmaceutically acceptable salts or solvates can improve cardiac diastolic function and shorten isovolumetric relaxation time.

[0029] According to a specific embodiment of the present invention, by observing echocardiographic images of mice, the inventors found that after treatment with Lycopus lucidus flavonoids, the diastolic function of the heart in cardiac aging mice was significantly improved. Specifically, the maximum flow rate ratio (E / A) of the cardiac aging mice was significantly reduced, and the isovolumetric relaxation time was significantly shortened. This indicates that Lycopus lucidus flavonoids or their pharmaceutically acceptable salts or solvates described in this invention can effectively delay cardiac aging.

[0030] According to the intended use of the invention, preferably, the flavonoids of *Zephyranthes bidentata* or its pharmaceutically acceptable salts or solvates are capable of reducing the expression level of γ-H2AX in aging cardiac tissue.

[0031] According to a specific embodiment of the present invention, by detecting DNA damage markers in cardiac aging mice, the expression level of γ-H2AX in the heart tissue of cardiac aging mice was significantly higher than that in cardiac healthy mice. The expression level of γ-H2AX in the heart tissue of cardiac aging mice treated with Lysimachia christinae was significantly reduced.

[0032] Cellular functional decline makes the aging heart extremely vulnerable, lowering the cardiac damage threshold and making it prone to myocardial injury. Furthermore, myocardial relaxation depends on the regulation of intracellular calcium ions. Due to the decreased function of calcium ion processing proteins in the aging heart, myocardial relaxation is slowed, failing to achieve effective relaxation and adequate blood filling. This reduces the heart's functional reserve, hindering effective compensation and exacerbating the damage burden. Therefore, the aging heart is more susceptible to myocardial injury.

[0033] According to the use described in this invention, preferably, the flavonoids of Lycopus lucidus or its pharmaceutically acceptable salts or solvates are able to improve myocardial fiber rupture and disordered arrangement.

[0034] According to a specific embodiment of the present invention, in an HE staining experiment of mouse heart tissue, the inventors found significant myocardial fiber rupture in the hearts of mice with aging hearts. After treatment with Lycopus lucidus flavonoids, the myocardial cells of the mice became more tightly packed, and the myocardial fiber rupture was significantly improved. This further demonstrates that Lycopus lucidus flavonoids or its pharmaceutically acceptable salts or solvates can effectively improve myocardial damage in organisms.

[0035] The flavonoids of the present invention, or their pharmaceutically acceptable salts or solvates, can significantly reduce the senescence positivity rate of senescent cells, improve cardiac function indicators of senescent hearts, improve weight loss caused by cardiac aging, reduce the expression level of γ-H2AX in cardiac tissue of senescent hearts, and improve myocardial fiber breakage and disordered arrangement, thereby delaying cardiac aging and / or improving myocardial damage. Attached Figure Description

[0036] Figure 1 The expression level of β-galactosidase in H9c2 cells of each group.

[0037] Figure 2 The senescence positivity rate of H9c2 cells.

[0038] Figure 3 The results of the grip strength test for each group of mice are shown.

[0039] Figure 4 The results of the rotarod experiment for each group of mice are shown.

[0040] Figure 5 Echocardiographic images of cardiac contractile function in mice of each group.

[0041] Figure 6 Echocardiographic images of diastolic function in mice from each group.

[0042] Figure 7 The results of the detection of cardiac diastolic function indicators in mice of each group.

[0043] Figure 8 HE staining results of heart tissue sections from each group of mice.

[0044] Figure 9 The expression level of γ-H2AX in the heart tissue of mice in each group is shown. Detailed Implementation

[0046] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are commercially available products.

[0048] Example 1: In vitro efficacy study of Lycopus lucidus flavonoids 1.1 Experimental Materials and Modeling 1.1.1 Experimental Materials Rat cardiomyocytes (H9c2) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai). SA-β-Gal reagent kit, model G1580, was purchased from Beijing Solarbio Technology Co., Ltd. Zeylan flavonoids, verbascoside isoflavones, kaempferol, gentianin, and daidzein were all purchased from Chengdu Pusi Biotechnology Co., Ltd. Among them, the structure of verbascoflavonoid is shown in formula (II); the structure of kaempferol is shown in formula (III); the structure of gentianin is shown in formula (IV); and the structure of daidzein is shown in formula (V).

[0049] (II); (III); (IV); (V).

[0050] 1.1.2 Establishment of an doxorubicin-induced H9c2 cardiomyocyte senescence model H9c2 cells were cultured in complete medium for 12 hours at 37°C, 95% humidity, and 5% CO2 concentration to obtain cultured H9c2 cells. The complete medium was DMEM high-glucose medium containing 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin mixed solution.

[0051] After culture, H9c2 cells were planted at 3×10⁻⁶. 3 / well cells were seeded in 96-well plates, and control groups (senescent H9c2 cardiomyocytes), experimental groups (treated with *Eupatorium fortunei* flavonoids), and control groups (treated with *Vernicia fordii*, *Kaempferol*, *Aristolochia*, and *Glycine*, respectively, and labeled as *Vernicia fordii*, *Kaempferol*, *Aristolochia*, and *Glycine*, respectively) were set up. After 24 hours of complete cell adhesion, the complete culture medium in the experimental groups was replaced with complete culture medium containing 0.2 μM doxorubicin to induce senescence, and 20 μM *Eupatorium fortunei* flavonoids were added. The control group was treated with an equal volume of complete culture medium containing 0.2 μM doxorubicin. The complete culture medium in the control groups was replaced with complete culture medium containing 0.2 μM doxorubicin to induce senescence, and 20 μM *Vernicia fordii*, *Kaempferol*, *Aristolochia*, and *Glycine*, respectively, were added. After 24 hours of intervention, the culture medium of the experimental group was replaced with a complete culture medium containing 20 μM of Lycopus lucidus flavonoids, the culture medium of the control group was replaced with a complete culture medium of the same volume as that of the experimental group, and the culture medium of the control group was replaced with a complete culture medium containing the same amount of Lycopus lucidus flavonoids, kaempferol, gentianin and daidzein as that of the experimental group. After 24 hours of intervention, the cells were obtained after the experiment.

[0052] Experimental methods 1.2.1 SA-β-Gal staining experiment Cells from the control, experimental, and comparative groups were washed three times with PBS after the experiment to remove any residual culture medium. A staining working solution with a pH of 6.0 was prepared according to the SA-β-Gal kit instructions. The staining working solution was added to each group of cells and incubated at 37°C in the dark for 24 h. During incubation, the SA-β-Gal enzyme decomposes the staining substrate, producing a blue staining product, thus marking senescent cells. After incubation, the staining working solution was aspirated, and the cells were washed with PBS for 3 min. Then, the PBS was aspirated, and 0.5 μg / mL DAPI (4',6-diamidinyl-2-phenylindole) solution was added to the wells, and the cells were incubated at 25°C in the dark for 15 min to stain the cell nuclei. DAPI specifically binds to DNA in the cell nucleus, causing the nucleus to emit blue fluorescence. After incubation, the DAPI solution was aspirated, and PBS was added, and the cells were washed three times for 10 min each time on a shaker in the dark to remove unbound DAPI. After thorough washing, the cells were observed and images were acquired under a fluorescence microscope. Under bright-field fluorescence microscopy, the cytoplasm of senescent cells appears blue-green. The nuclei of all cells appear blue; therefore, the number of nuclei observed represents the total number of cells in the field of view. The expression levels of β-galactosidase in H9c2 cells of each group are as follows: Figure 1 As shown in the figure, the more blue-green cells appearing in the cytoplasm of the SA-β-Gal group, the greater the number of senescent cells; the stronger the blue fluorescence in the DAPI group, the greater the number of cells. Image merging was used to reduce background error. Three fields of view were randomly selected from each well for imaging, and cell counts were performed using ImageJ software. The β-gal positivity rate was calculated using the following formula: β-gal positivity rate = (number of senescent cells / total number of cells) × 100%. The senescence positivity rate of H9c2 cells in each group is shown below. Figure 2 As shown.

[0053] Results and Discussion Depend on Figure 1 It can be seen that the number of senescent cells in the control group was significantly greater than that in the experimental group. Furthermore, from... Figure 2 It was found that the senescence positivity rate of H9c2 cells in the experimental group was significantly lower than that in the control group. Lycopus lucidus flavonoids effectively reduced the senescence positivity rate in the doxorubicin-induced H9c2 cardiomyocyte senescence model. Compared with isoflavones, kaempferol, gentianin, and genistein, Lycopus lucidus flavonoids exhibited more significant biological activity in delaying cardiomyocyte senescence. This suggests that the good biological activity of Lycopus lucidus flavonoids in delaying cardiomyocyte senescence is not easily expected.

[0054] Example 2: In vivo efficacy study of Lycopus lucidus flavonoids 2.1 Experimental Materials and Modeling 2.1.1 Experimental Materials C57BL / 6N mice, 3 months old, weighing 18-25g, were purchased from the Department of Laboratory Animal Science, Peking University School of Medicine. The DNA damage detection kit, model C2037S, was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0055] 2.1.2 Establishment of a D-galactose-induced mouse aging model C57BL / 6N mice were acclimatized for 3 days under conditions of 20±2℃ and 60±5% humidity, following a 12h / 12h light-dark cycle, and fed a maintenance diet (provided by the Department of Laboratory Animal Science, Peking University School of Medicine, in accordance with national standard GB 14924.3-2010). They were also allowed free access to water before the experiment was conducted.

[0056] Forty mice were randomly divided into four groups: control group, model group (D-gal), drug treatment group (D-gal+N), and positive control group (D-gal+R). Drug treatment was administered for 12 weeks, with the following administration methods: Control group: 0.1 mL of normal saline was injected subcutaneously into the back, and 0.1 mL of 0.5 wt% sodium carboxymethyl cellulose (CMC-Na) solution was administered by gavage.

[0057] Model group (D-gal): D-gal (120 mg / kg / d) was injected subcutaneously into the back, and 0.1 mL of 0.5 wt% sodium carboxymethyl cellulose (CMC-Na) solution was administered by gavage.

[0058] The treatment group (D-gal+N) received a subcutaneous injection of D-gal (120 mg / kg / d) in the back and gavage administration of Lycopus lucidus flavonoids (25 mg / kg / d).

[0059] Positive drug group (D-gal+R): D-gal (120 mg / kg / d) was injected subcutaneously into the back, and rapamycin (10 mg / kg / d) was administered by gavage.

[0060] Experimental methods 2.2.1 Experimental Principle By injecting excessive amounts of D-galactose (D-gal) into mice, the mice were induced to produce excessive reactive oxygen species (ROS) through metabolism, which triggered severe oxidative stress and led to damage to proteins, lipids, and DNA. This process simulated and accelerated cell and tissue damage during the natural aging process, causing the mice to exhibit physiological and behavioral characteristics similar to those of natural aging.

[0061] 2.2.2 Mouse grip strength test The experimental mice were placed on a grip strength meter, and their tails were gently pulled to encourage them to grip the probe with their forelimbs. The readings on the grip strength meter were recorded when maximum force was applied to the mice. Each mouse was measured three times, with at least 30 minutes between each measurement. The grip strength test results for each group of mice are as follows: Figure 3 As shown.

[0062] 2.2.3 Mouse rotarod experiment Mice were placed on the rotating bar of a rotarod apparatus and subjected to acclimatization training in an environment with a temperature of 22±1℃ and in the absence of strong light. The rotation speed of the bar was fixed at 12 rpm, and training lasted for 5 minutes each day for three days. During the formal test, the rotation speed was set to 30 rpm, and the number of times the mouse fell onto the infrared sensor plate below within 3 minutes was recorded. Each mouse underwent three rounds of testing, with a rest interval of at least 30 minutes between each round. The results of the rotarod experiment for each group of mice are as follows: Figure 4 As shown.

[0063] 2.2.4 Cardiac Function Evaluation Cardiac function was assessed using a photoacoustic / ultrasound multimodal small animal imaging system (Vevo 3100, MX550D probe). Mice induced anesthesia with 2% isoflurane (mixed with oxygen) were transferred from the induction chamber to a 37°C constant-temperature platform in a prone position. 0.5% isoflurane (mixed with oxygen) was continuously administered via a nasal cone to maintain anesthesia. Hair was removed from the anterior chest region of the mice, and preheated ultrasound coupling agent was applied. M-mode ultrasound images (depth 15 mm, frame rate >200 fps) were acquired at the short-axis level of the left ventricular papillary muscles, using the long-axis view of the left ventricle as a reference, and at least three stable cardiac cycles were continuously recorded. Echocardiograms of cardiac systolic function in each group of mice are shown below. Figure 5 As shown.

[0064] Blood flow spectra at the mitral valve orifice were acquired in pulsed Doppler mode, and isovolumetric relaxation time (IVRT) was measured. Simultaneously, peak early diastolic (E-wave) and late diastolic (A-wave) velocities were measured, and the maximum velocity ratio E / A was calculated. Echocardiographic images of diastolic function in each group of mice are shown below. Figure 6 As shown in the figure, the detection results of cardiac diastolic function indicators in each group of mice are as follows: Figure 7 As shown.

[0065] 2.2.5 HE staining experiment Mice were euthanized, their thoracic cavities were opened, and their intact hearts were harvested. The hearts were fixed in 4% paraformaldehyde, rinsed thoroughly, dehydrated and cleared, and then embedded in paraffin to form paraffin blocks. The paraffin blocks were then sliced ​​into 5 μm thin sections using a microtome. The sections were laid flat on glass slides and baked at 37°C for 2 hours to ensure close adhesion. The slides were then dewaxed until hydrated, and the cell nuclei were stained with hematoxylin and the cytoplasm with eosin. After dehydration and clearing, the slides were mounted with coverslips. HE staining of mouse heart tissue is shown below. Figure 8 As shown.

[0066] 2.2.6 Detection of DNA damage markers Mouse heart tissue, frozen in liquid nitrogen, was placed in a mold containing OCT embedding medium at -20°C for equilibration for 30 minutes. Then, serial sections of 8 μm thickness were prepared using a cryostat and mounted on detachable glass slides. Staining was performed according to the instructions of the DNA damage detection kit. γ-H2AX expression was observed under a fluorescence microscope. Results are shown below. Figure 9 As shown in the figure, the stronger the blue fluorescence of the DAPI group, the greater the number of cells; the stronger the green fluorescence of the γ-H2AX group, the greater the DNA damage; the merged images are used to reduce background error.

[0067] Results and Discussion 2.3.1 Changes in mouse body weight Mice were weighed after 12 weeks of feeding, and the changes in body weight are shown in Table 1.

[0068] Table 1 Grouping Initial body weight / g Body weight after experiment / g control group 20.3±1.0 26.7±2.4 Model group 20.2±0.8 24.5±1.5# Drug administration group 20.5±1.1 27.3±2.9** Positive drug group 20.2±0.8 25.3±2.0 Note: Compared with the control group, # indicates P<0.05; compared with the model group, ** indicates P<0.01.

[0069] As shown in Table 1, the weight gain of mice in the model group was significantly lower than that in the control group (P<0.05, compared with the control group); the weight gain of mice in the treatment group was significantly higher than that in the control group, and extremely significantly higher than that in the model group (P<0.01, compared with the model group). Therefore, Lycopus lucidus flavonoids can improve the weight loss in mice with cardiac aging.

[0070] 2.3.2 Changes in mouse physical condition Depend on Figure 3 and Figure 4 It was found that in the mouse grip strength test, the grip strength of the model group mice was significantly lower than that of the control group mice. After treatment, the grip strength of the mice improved, and the grip strength of the treated group mice was significantly higher than that of the model group mice, and also higher than that of the positive drug group mice. In the mouse rotarod test, the number of falls in the model group mice was significantly increased compared with the control group. After treatment, the number of falls in the treated group mice was significantly reduced. This indicates that Lycopus lucidus flavonoids can significantly improve the physical performance of cardiac aging mice.

[0071] 2.3.3 Results of cardiac function testing in mice Depend on Figure 5 It can be seen that there is no significant difference in the echocardiogram of cardiac contractile function among the mice in each group, that is, the cardiac contractile function of the mice in the drug-treated group is basically unchanged compared with that of naturally aged mice.

[0072] Depend on Figure 6It was found that echocardiograms of diastolic function in the control group mice showed clear ventricular wall motion and valvular activity, while ventricular wall motion in the model group mice was weakened. Echocardiograms of diastolic function in the drug-treated and positive control groups mice showed significant improvement in ventricular wall motion and valvular activity.

[0073] Depend on Figure 7 It was found that, compared with the control group mice, the model group mice had a significantly increased maximum flow rate ratio and a significantly prolonged isovolumetric relaxation time, indicating that the diastolic function of the heart was impaired in the model group mice. Compared with the model group mice, the drug-treated group mice had a significantly decreased maximum flow rate ratio and a significantly shortened isovolumetric relaxation time. This indicates that Lycopus lucidus flavonoids significantly improved the diastolic dysfunction of the heart in aging mice and shortened the isovolumetric relaxation time.

[0074] 2.3.4 Results of HE staining of mouse heart tissue Depend on Figure 8 It was found that myocardial fibers in the heart tissue of mice in the model group were ruptured. After treatment with Lycopus lucidus flavonoids, compared with the model group, the rupture of myocardial fibers in the heart tissue of mice in the treatment group was improved, and the myocardial cells were more tightly arranged. This indicates that Lycopus lucidus flavonoids have the effect of improving myocardial injury.

[0075] 2.3.5 Results of DNA damage marker detection in mouse heart tissue Depend on Figure 9 It was found that the expression level of the DNA damage marker γ-H2AX in the heart tissue of the model group mice was significantly higher than that in the control group. After treatment with Ze Lan flavonoids, the expression level of γ-H2AX in the treatment group mice was significantly reduced compared with the model group.

[0076] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. Use of a Vitis quinquangulalia flavone or a pharmaceutically acceptable salt or solvate thereof for the manufacture of a medicament for preventing, delaying and / or treating cardiac aging and / or myocardial injury, characterized in that, The flavonoids from *Ze Lan* have the structure shown in formula (I): （I）。 2. The use according to claim 1, characterized in that, The myocardial injury is caused by one or more of the following: coronary artery disease, myocardial hypertrophy, heart failure, angina pectoris, arrhythmia, pulmonary heart disease, myocardial infarction, myocardial ischemia, myocardial ischemia-reperfusion, chemotherapy drugs, bacteria, and viruses.

3. The use according to claim 1, characterized in that, The drug comprises zeylan flavonoids or their pharmaceutically acceptable salts or solvates and pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that, The drug has Lycopus lucidus flavonoids as its sole active ingredient; the drug is an oral preparation.

5. The use according to claim 4, characterized in that, In a unit formulation, the amount of Zeylan flavonoids added is 2.4–3.9 mg / kg.

6. The use according to claim 1, characterized in that, The flavonoids from *Zephyranthes* or their pharmaceutically acceptable salts or solvates can reduce the expression level of β-galactosidase in cardiomyocytes.

7. The use according to claim 1, characterized in that, The flavonoids of *Zephyranthes* or their pharmaceutically acceptable salts or solvates can improve weight loss caused by cardiac aging.

8. The use according to claim 1, characterized in that, The flavonoids of *Zephyranthes* or their pharmaceutically acceptable salts or solvates can improve cardiac diastolic function and shorten isovolumetric relaxation time.

9. The use according to claim 1, characterized in that, The flavonoids of Zephyranthes or their pharmaceutically acceptable salts or solvates can improve myocardial fiber breakage and disordered arrangement.

10. The use according to claim 1, characterized in that, The flavonoids of Zephyranthes or their pharmaceutically acceptable salts or solvates can reduce the expression level of γ-H2AX in cardiac tissue.

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