Traditional Chinese medicine composition for treating myocardial ischemia-reperfusion injury and preparation method thereof

Abstract

The invention relates to a traditional Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof, and belongs to the technical field of traditional Chinese medicine compositions. In order to solve the problem that a traditional Chinese medicine compound for treating myocardial ischemia-reperfusion injury is lacked in the prior art, the invention provides a traditional Chinese medicine composition for treating myocardial ischemia-reperfusion injury, and the traditional Chinese medicine composition comprises American ginseng, astragalus membranaceus, radix ophiopogonis, schisandra chinensis, radix rehmanniae recen, salvia miltiorrhiza, radix sophorae flavescentis, radix scrophulariae, honeysuckle, fructus forsythiae and liquorice. The traditional Chinese medicine composition provided by the invention has the effects of promoting blood circulation to remove blood stasis, reinforcing qi and nourishing yin, and activating meridians to stop pain, and is beneficial to improving the microcirculation state of cardiac muscle and relieving inflammatory response of cardiac muscle, so that damaged cardiac muscle tissues are protected. Animal experiments prove that the traditional Chinese medicine composition can effectively reduce the myocardial infarction area of MIRI model rats, remarkably improve the activity of myocardial cells subjected to hypoxia-reoxygenation injury and reduce the apoptosis rate of the myocardial cells subjected to hypoxia-reoxygenation injury. The invention provides a new way for clinical multi-target treatment of myocardial ischemia reperfusion injury.

Description

Technical Field

[0001] The invention belongs to the technical field of Chinese medicine compositions, and in particular relates to a Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof. Background Art

[0002] The World Health Organization reported that cardiovascular disease has become the leading cause of death and disability worldwide. It is estimated that by 2030, 23.6 million people will die from cardiovascular disease, and most of these deaths are caused by ischemic heart disease. Restoring blood perfusion in the coronary arteries can effectively reduce the damage caused by ischemic heart disease. At present, revascularization methods such as thrombolytic therapy, percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG) have effectively reduced the damage and necrosis caused by ischemia and reduced the mortality rate of cardiovascular disease. However, after myocardial ischemia, the restoration of blood and oxygen supply further leads to more serious damage and necrosis of myocardial cells, namely myocardial ischemia-reperfusion injury (MIRI). Clinically, it manifests as more serious conditions such as hypotension, arrhythmia and death. Therefore, how to reduce myocardial ischemia-reperfusion injury has become a hot issue in cardiovascular disease research. Modern research has found that increased production of oxygen free radicals, mitochondrial dysfunction, calcium overload, changes in pH, cell autophagy and apoptosis may be factors that induce myocardial ischemia-reperfusion injury. The use of various drugs for intervention treatment based on different mechanisms and targets has become the main means of Western medicine treatment. The drugs used clinically to treat myocardial ischemia mainly include statins, β-receptor blockers, calcium ion antagonists, nitrates, etc., which are expensive and often have unsatisfactory clinical efficacy due to the single treatment target.

[0003] Traditional Chinese medicine can significantly improve the degree of MIRI, and has the characteristics of multiple targets, multiple pathways, and few toxic side effects, and has attracted extensive attention from many researchers at home and abroad. Common Chinese herbal compound prescriptions include: Compound Danshen Dropping Pills, Suhexiang Pills, Suxiao Jiuxin Pills, etc., but there are relatively few studies on their application in myocardial ischemia-reperfusion injury. Summary of the invention

[0004] In order to solve the problem that the prior art lacks a Chinese medicine compound for treating myocardial ischemia-reperfusion injury, the present invention provides a Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof.

[0005] The technical solution of the present invention: A traditional Chinese medicine composition for treating myocardial ischemia-reperfusion injury comprises the following components in parts by weight: 1-20 parts of American ginseng, 20-40 parts of astragalus, 5-25 parts of ophiopogon, 1-20 parts of schisandra, 5-25 parts of raw rehmannia, 1-20 parts of salvia, 5-25 parts of sophora flavescens, 5-25 parts of figwort, 10-30 parts of biflorus, 10-30 parts of forsythia and 1-20 parts of liquorice.

[0006] Further, the invention comprises the following components in parts by weight: 5-15 parts of American ginseng, 25-35 parts of astragalus, 10-20 parts of ophiopogon, 5-15 parts of schisandra, 10-20 parts of raw rehmannia, 5-15 parts of salvia, 10-20 parts of sophora flavescens, 10-20 parts of figwort, 15-25 parts of bihua, 15-25 parts of forsythia and 5-15 parts of liquorice.

[0007] Further, the composition includes the following components in parts by weight: 10 parts of American ginseng, 30 parts of astragalus, 15 parts of ophiopogon, 10 parts of schisandra, 15 parts of raw rehmannia, 10 parts of salvia miltiorrhiza, 15 parts of sophora flavescens, 15 parts of figwort, 20 parts of bihua, 20 parts of forsythia and 10 parts of liquorice.

[0008] A method for preparing a Chinese medicine composition for treating myocardial ischemia-reperfusion injury comprises soaking all medicinal materials in 10 times the amount of water, decocting them twice and filtering and collecting the medicinal liquids respectively, mixing the medicinal liquids collected twice, and the obtained mixed medicinal liquid is the Chinese medicine composition for treating myocardial ischemia-reperfusion injury.

[0009] Furthermore, the soaking time is 90 minutes, the first decoction time of the two decoctions is 60 minutes, and the second decoction time is 30 minutes.

[0010] Furthermore, the method also includes a step of freeze-drying the obtained mixed drug solution.

[0011] Furthermore, the freeze-drying conditions are -80°C temperature and 0.002MPa pressure, freeze-drying for 48 hours.

[0012] Furthermore, the clinical adult dosage of the powdered Chinese medicine composition obtained by freeze-drying is 150-200 g / d.

[0013] Furthermore, the clinical adult dosage of the powdered Chinese medicine composition obtained by freeze-drying is 170 g / d.

[0014] Beneficial effects of the present invention: The Chinese medicine composition provided by the invention comprises American ginseng, astragalus, ophiopogon, schisandra, raw rehmannia, salvia miltiorrhiza, sophora flavescens, scrophularia ningpoensis, bihua, forsythia and liquorice. The whole prescription has the functions of invigorating qi and nourishing yin, promoting blood circulation and unblocking collaterals, and is used for treating chest pain and heart pain caused by deficiency of both qi and yin and obstruction of blood vessels.

[0015] In the Chinese medicine composition of the present invention, American ginseng and astragalus are the main medicines for replenishing qi and nourishing yin: American ginseng tastes sweet, slightly bitter and cool in nature, can replenish qi and nourish yin, clear away heat and promote the production of body fluid; astragalus is sweet and slightly warm, can replenish various deficiencies, especially good at replenishing the qi of the whole body, so as to treat various symptoms of asthenia.

[0016] Ophiopogon japonicus is sweet and slightly cold, and is a refreshing and moist product. It nourishes yin and clears the heart, and can be used to treat restlessness and palpitations caused by insufficient heart yin; Raw Rehmannia glutinosa is sweet, bitter and cold in nature, and nourishes yin and clears heat, cools blood and produces body fluid. It is used to treat febrile disease where evil heat enters the blood and damages yin; evil heat in febrile and hot diseases is easily accumulated and becomes toxic; therefore, Shuanghua and Forsythia suspensa have a fragrant smell, and can not only clear away heat and detoxify, but also eliminate filth and turbidity. Because of their light, clear and penetrating nature, they can make the heat evil in the blood flow outward from the qi and be resolved. The four are collectively called assistant drugs.

[0017] Sophora flavescens is bitter and cold and enters the heart meridian, which can clear the heat of the heart meridian to treat restlessness and palpitations. Salvia miltiorrhiza can both activate blood circulation and dissipate blood stasis and cool blood and clear the heart. Scrophularia ningpoensis nourishes yin, reduces fire and detoxifies, and cools blood. On the basis of the three ginsengs, Schisandra chinensis is added. Schisandra chinensis is sour and can produce body fluid and restrain yin. The four are adjuvants; Licorice is the adjuvant to coordinate the other drugs. The combination of the drugs has the functions of invigorating qi and nourishing yin, clearing away heat and detoxifying, and activating blood circulation and unblocking collaterals.

[0018] According to the clinical manifestations of myocardial ischemia-reperfusion injury, the basic principles of TCM diagnosis and treatment, and modern doctors' combination of clinical observations and experience summary and discussion, its basic pathogenesis is deficiency of the root and excess of the superficial symptoms. Because of the deficiency of qi, blood, yin and yang of the five internal organs, the circulation of qi, blood and body fluids is not smooth, forming blood stasis, qi stagnation, phlegm and water retention, which block the heart meridian, causing pain and resulting in illness.

[0019] The Chinese medicine composition provided by the present invention is used to treat myocardial ischemia-reperfusion injury, and is mainly used in the treatment and rehabilitation of cardiovascular diseases, especially for patients who have undergone heart surgery, coronary artery bypass grafting, recanalization after myocardial infarction, heart transplantation, and shock. The myocardial tissue of these patients is prone to obvious dysfunction during ischemia-reperfusion, and even irreversible myocardial damage occurs. The Chinese medicine composition provided by the present invention has the effects of promoting blood circulation and removing blood stasis, replenishing qi and nourishing yin, and dredging collaterals to relieve pain, which helps to improve the microcirculation state of the myocardium, reduce the inflammatory response of the myocardium, and thus protect the damaged myocardial tissue.

[0020] Animal experiments have shown that the Chinese medicine composition provided by the present invention can effectively reduce the myocardial infarction area of ​​MIRI model rats, significantly improve the viability of myocardial cells injured by hypoxia and reoxygenation, and reduce the apoptosis rate of myocardial cells injured by hypoxia and reoxygenation, and has a significant inhibitory effect on ROS generation and a certain protective effect on mitochondrial function. The present invention provides a new approach for the multi-target treatment of clinical myocardial ischemia-reperfusion injury, and provides a new method for preparing a Chinese medicine composition, which can improve its portability in clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1HE staining photos of myocardial tissues of three groups of rats in Example 4, A is the sham operation group, B is the model group, and C is the drug administration group; Figure 2 The TTC staining photos of the myocardial tissues of the three groups of rats in Example 4, A is the sham operation group, B is the model group, and C is the drug administration group; Figure 3 This is a comparison chart of the percentage of myocardial infarction area in the three groups of rats in Example 4, A is the sham operation group, B is the model group, and C is the drug administration group; Figure 4 The cell activity comparison diagram of each group of H9C2 cardiomyocytes in Example 5, A is the blank serum group, B is the model group, C is the low-dose drug-containing serum group, D is the medium-dose drug-containing serum group, and E is the high-dose drug-containing serum group; Figure 5 This is a comparison chart of the apoptosis rates of H9C2 cardiomyocytes in each group in Example 5, A is the blank serum group, B is the model group, C is the low-dose drug-containing serum group, D is the medium-dose drug-containing serum group, and E is the high-dose drug-containing serum group; Figure 6 The comparison chart of lactate dehydrogenase content of H9C2 cardiomyocytes in each group in Example 6, A is the blank group, B is the model group, C is the blank serum group, and D is the drug-containing serum group; Figure 7 The comparison chart of c-TnT content of H9C2 cardiomyocytes in each group in Example 6, A is the blank group, B is the model group, C is the blank serum group, and D is the drug-containing serum group; Figure 8 The fluorescence detection diagrams of ROS content and mitochondrial number of H9C2 cardiomyocytes in each group in Example 6, A is the blank group, B is the model group, C is the blank serum group, and D is the drug-containing serum group; Fig. 9 The mitochondrial content comparison diagram of each group of H9C2 cardiomyocytes stained with MitoTracker Green in Example 6, A is the blank group, B is the model group, C is the blank serum group, and D is the drug-containing serum group; Fig.10 Comparison of mitochondrial ROS content in H9C2 cardiomyocytes of each group under MitoSOX labeling in Example 6, A is the blank group, B is the model group, C is the blank serum group, and D is the drug-containing serum group. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the embodiments, but it is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the protection scope of the present invention. The process equipment or devices not specifically noted in the following embodiments are all conventional equipment or devices in the art. If not specifically specified, the raw materials used in the embodiments of the present invention can be obtained commercially; if not specifically specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0023] Example 1 This embodiment provides a Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof.

[0024] The Chinese medicine composition of this embodiment is made of the following components in parts by mass: 10 parts of American ginseng, 30 parts of astragalus, 15 parts of ophiopogon, 10 parts of schisandra, 15 parts of rehmannia, 10 parts of salvia miltiorrhiza, 15 parts of sophora flavescens, 15 parts of Scrophularia ningpoensis, 20 parts of bihua, 20 parts of forsythia and 10 parts of liquorice.

[0025] The preparation method of the Chinese medicine composition in this embodiment is: All medicinal materials are soaked in 10 times the amount of water for 90 minutes. The first decoction time is 60 minutes, and the medicinal liquid is collected by filtering. The second decoction time is 30 minutes, and the medicinal liquid is collected by filtering. The two medicinal liquids are mixed, cooled, and frozen in a -80°C refrigerator. Vacuum freeze-drying is carried out the next day. Freeze-drying is carried out at a temperature of -80°C and a pressure of 0.002MPa for 48 hours to obtain a powdered Chinese medicine composition for treating myocardial ischemia-reperfusion injury.

[0026] Example 2 This embodiment provides a Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof.

[0027] The Chinese medicine composition of this embodiment is made of the following components in parts by mass: 12 parts of American ginseng, 35 parts of astragalus, 16 parts of ophiopogon, 12 parts of schisandra, 16 parts of rehmannia, 12 parts of salvia, 17 parts of sophora flavescens, 17 parts of Scrophularia, 23 parts of bihua, 23 parts of forsythia and 12 parts of liquorice.

[0028] The preparation method of the Chinese medicine composition in this embodiment is: All medicinal materials are soaked in 10 times the amount of water for 90 minutes. The first decoction time is 60 minutes, and the medicinal liquid is filtered and collected. The second decoction time is 30 minutes, and the medicinal liquid is filtered and collected. The two medicinal liquids are mixed to obtain a Chinese medicine composition decoction for treating myocardial ischemia-reperfusion injury.

[0029] Example 3 This embodiment provides a Chinese medicine composition for treating myocardial ischemia-reperfusion injury and a preparation method thereof.

[0030] The Chinese medicine composition of this embodiment is made of the following components in parts by mass: 20 parts of American ginseng, 38 parts of astragalus, 25 parts of ophiopogon, 20 parts of schisandra, 25 parts of rehmannia, 20 parts of salvia miltiorrhiza, 25 parts of sophora flavescens, 25 parts of Scrophularia ningpoensis, 28 parts of bihua, 28 parts of forsythia and 20 parts of liquorice.

[0031] The preparation method of the Chinese medicine composition in this embodiment is: All medicinal materials are soaked in 10 times the amount of water for 90 minutes. The first decoction time is 60 minutes, and the medicinal liquid is filtered and collected. The second decoction time is 30 minutes, and the medicinal liquid is filtered and collected. The two medicinal liquids are mixed to obtain a Chinese medicine composition decoction for treating myocardial ischemia-reperfusion injury.

[0032] Example 4 This example investigates the protective effect of the Chinese medicine composition prepared in Example 1 on rat myocardial tissue through an animal model experiment.

[0033] 1. Animal model construction method 1. Experimental animals: clean grade male SD rats.

[0034] 2. Method for constructing myocardial ischemia-reperfusion animal model (MIRI model): The experimental animals were anesthetized by intraperitoneal injection and underwent coronary artery ligation to cause myocardial ischemia for 45 minutes. The sutures were then relaxed to restore coronary blood flow and establish an ischemia-reperfusion animal model.

[0035] 3. Grouping and administration method: Sham operation group: conventional feeding for 2 weeks, intragastric administration of normal saline for 7 days, 10 ml / kg, twice a day; Model group: conventional feeding for 2 weeks, oral administration of normal saline for 7 days, 10ml / kg, twice a day.

[0036] The drug administration group was conventionally fed for 2 weeks, and the drug dosage was converted to 30.6 g / kg·d according to the equivalent dose ratio of human and rat body surface area (1:0.018) according to the clinical adult dosage of 170 g / d. The lyophilized powder solution of the traditional Chinese medicine composition prepared in Example 1 was gavaged for 7 days, twice a day.

[0037] (II) Animal specimen collection method: After the last administration, the rats were fasted overnight but not water. They were weighed in the morning of the next day and anesthetized with 10% chloral hydrate (3.5 ml / kg) intraperitoneally. After the rats were fully anesthetized, the abdominal cavity was opened, the heart was removed and rinsed, the moisture in the heart was absorbed with filter paper, and the heart was fixed in 4% paraformaldehyde.

[0038] 3. HE staining to observe the pathological structural changes of myocardial tissue Hematoxylin-eosin (HE) staining was used for observation. Myocardial tissues of rats in each group were taken and dehydrated, paraffin-embedded, sliced ​​and dried, stained with hematoxylin-eosin, dehydrated and transparent again, and sealed. Photos were collected to observe the morphological changes of myocardial tissue ischemia. The results were as follows: Figure 1 shown.

[0039] Figure 1 HE staining photos of three groups of rat myocardial tissues, A is a sham operation group, B is a model group, and C is a drug administration group; It can be observed in the figure that the myocardial fiber structure of the sham operation group is intact without fracture, presenting a regular shape, the myocardial cells are arranged neatly, the nuclei are clearly visible, the cytoplasm color is uniform, and there is no abnormal morphology or color change; The myocardial fibers in the model group lose integrity, and there is a phenomenon of fracture loss, and vacuolar structures appear in the myocardial cells. The damaged myocardial tissue is replaced by fibrous tissue, and it presents a light pink in HE staining, which is different from the red color of normal myocardial tissue, showing that the myocardial tissue is damaged; The drug administration group can observe that the arrangement of myocardial fibers becomes neat, the vacuolar structure disappears, and the shape of myocardial cells also returns to normal. It is thus proved that the Chinese medicine composition provided by the present invention can effectively improve the myocardial damage of MIRI model rats.

[0040] 4. TTC staining to observe the changes in myocardial infarction area in rats Two rats were selected from each group. After myocardial ischemia and reperfusion were completed, the intact heart was removed as quickly as possible, and the blood on the myocardium was flushed with pre-cooled saline. The myocardial tissue was placed in a -20°C refrigerator and frozen for 20 minutes, then taken out. The myocardium was immediately cut into 5 slices with a thickness of about 2 mm using a scalpel. All the slices were immersed in 2% TTC (2,3,5-triphenyltetrazolium chloride) staining solution, and incubated in a 37°C water bath in the dark for 15-30 minutes. During the incubation process, the color changes of the myocardium should be observed while incubating, and the slices should be turned over from time to time to ensure uniform staining. After staining, the myocardium was rinsed with PBS buffer, fixed with 4% paraformaldehyde fixative, and photographed. The results are as follows. Figure 2 shown.

[0041] After saving the images, the image analysis software Image Pro Plus 6.0 was used to determine the size of the myocardial infarction area and the total area of ​​the myocardial tissue slices, and the myocardial infarction area was calculated as the percentage of myocardial infarction area / total myocardial slice area (IS%). The results were compared as follows: Figure 3 shown.

[0042] Figure 2 The TTC staining photos of myocardial tissues of three groups of rats, A is the sham operation group, B is the model group, and C is the drug-treated group; Figure 2It can be observed that in the normal myocardial tissue of the sham operation group, TTC staining showed a uniform red color, which indicated that the dehydrogenase activity in the myocardial cells was normal and the myocardial tissue was in a healthy state. At this time, the myocardial tissue was not damaged and there was no myocardial infarction area. After myocardial ischemia-reperfusion, TTC staining in the model group showed a white infarction area. TTC staining in the drug-treated group showed that the white infarction area was reduced, which indicated that the myocardial cells were recovering. Figure 3 The quantitatively calculated percentage of myocardial infarction area in rats also showed that the treatment with the Chinese medicine composition of the present invention can effectively reduce the myocardial infarction area in MIRI model rats.

[0043] Example 5 This example investigates the effect of the Chinese medicine composition prepared in Example 1 on the viability of myocardial cells through a cell model experiment.

[0044] 1.1 Cell model construction method 1. Experimental cells: rat myocardial H9C2 cell line.

[0045] 2. Method for constructing a cardiomyocyte model of hypoxia-reoxygenation injury (24h / 6h): Take H9C2 cardiomyocytes in the logarithmic growth phase, gently rinse twice with PBS, use a pipette to aspirate PBS, add trypsin to digest, and stop digestion after the cells slide off the wall of the culture flask. Centrifuge for 5 minutes and aspirate the supernatant, then add DMEM complete medium to resuspend the cells. Use a cell counting plate to count under an electron microscope and adjust the cell density to 5×10 4 The cells were inoculated into 6-well plates with 2 ml per well and grouped after culturing at 37°C and 5% CO2 for 24 hours.

[0046] The original culture medium was aspirated with a pipette, PBS was added for washing, and then the PBS was aspirated again, and replaced with sugar-free DMEM culture medium containing serum, and placed in an incubator ventilated with mixed gas (1% O2, 94% N2, 5% CO2) for 24 hours. After glucose and oxygen deprivation, the culture medium was replaced with DMEM complete culture medium containing 10% FBS and cultured under normoxic conditions for 6 hours to obtain a cardiomyocyte model of hypoxia-reoxygenation (24h / 6h) injury.

[0047] (II) The preparation method of drug-containing serum is as follows: Thirty healthy SPF male SD rats were randomly divided into two groups: a blank group of 10 rats and a Chinese medicine group of 20 rats.

[0048] The Chinese medicine group was given 15.3 g / kg·d of the lyophilized powder solution of the Chinese medicine composition prepared in Example 1 by intragastric administration, and the blank group was given an equal volume of normal saline. Each group was given intragastric administration for 14 days. One hour after the last intragastric administration, 10% chloral hydrate was used for anesthesia, and the anesthetic dose was 3 ml / kg according to the body weight of the rat. The abdomen of the rat was opened, the abdominal organs were manually separated, and blood was collected from the abdominal aorta to obtain blank serum of the blank group and drug-containing serum of the Chinese medicine group.

[0049] After the blood was allowed to stand for 1 hour, it was centrifuged at 4000r / min at 4℃ for 15 minutes to separate the serum, and the supernatant was extracted with a pipette. It was heated and inactivated in a 56℃ water bath for 30 minutes, and then filtered through a 0.22μm filter. The serum was collected in a sterile centrifuge tube, sealed with a sealing film, and placed in a -80℃ refrigerator for later use.

[0050] (III) Cell grouping and drug administration method: 1. Blank serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% blank serum for 24 h, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 h; 2. Model group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% blank serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 3. Low-dose drug-containing serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 5% drug-containing serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 4. Medium-dose group with drug-containing serum: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% drug-containing serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 5. High-dose drug-containing serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 20% ​​drug-containing serum under oxygen and glucose deprivation for 24 hours, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours.

[0051] (IV) CCK-8 detection of cell viability H9C2 cardiomyocytes in the logarithmic growth phase of each group were collected. When the cell density reached more than 80%, the cells were counted using a cell counting plate and the density was adjusted to 5×10 4 / mL, gently blow evenly, and place in the sample loading groove. Add 100uL of cell suspension to each well of a 96-well plate, set up 6 replicate wells in each group, and culture in a normoxic incubator for 24 hours to allow the cells to completely adhere to the wall. The modeling and administration methods are the same as above. According to the requirements of the kit, after the cell modeling of each group is completed, use a pipette to add 10uCCK-8 solution to each well under light-proof conditions, and continue to culture in a normoxic incubator for 2 hours. Use an enzyme reader to measure the absorbance at a wavelength of 450nM, collect the data and analyze them. The activity comparison of each group of cardiomyocytes is as follows. Figure 4 shown.

[0052] Figure 4 The cell activity comparison diagram of H9C2 cardiomyocytes in each group, A is the blank serum group, B is the model group, C is the low-dose drug-containing serum group, D is the medium-dose drug-containing serum group, and E is the high-dose drug-containing serum group; Figure 4 It can be seen that the drug-containing serum can significantly improve the viability of myocardial cells damaged by hypoxia and reoxygenation, which indicates that the traditional Chinese medicine composition provided by the present invention can improve the viability of myocardial cells damaged by hypoxia and reoxygenation.

[0053] 5. Flow cytometry detection of cell apoptosis According to the instructions of the kit, 10×Binding Buffer was diluted to 1×Binding Buffer with deionized water and stored in a refrigerator at 4°C. 5 / mL was inoculated in a 6-well plate, 1.5 mL per well. After being placed in a normoxic incubator for 12 hours, the model was established. The modeling and grouping and drug administration methods were the same as above. After the model was established, the culture medium on the well plate was aspirated into a new centrifuge tube and marked. It contained early apoptotic and necrotic cells. The cells on the well plate were rinsed twice with PBS, and the rinsing fluid was collected in a marked centrifuge tube. 1 mL of trypsin was added to each well, and 1 mL of complete medium was added after 1 minute to terminate the digestion. The cells were blown off by a pipette, and the liquid in each well was collected into the corresponding centrifuge tube, centrifuged at 2000rpm for 3 minutes, and the supernatant was discarded. Then 1 mL of PBS was added, blown evenly and centrifuged, and the supernatant was discarded. Repeat twice to complete the cell collection. 300uL of 1×Binding Buffer was added to each group, and the mixture was resuspended and mixed by a pipette, and then aspirated into a 1.5mL centrifuge tube, and 5uL of Annexin V-FITC was added and mixed, and incubated at room temperature in the dark for 15 minutes. Add 5uL PI dye solution and mix well, incubate at room temperature in the dark for 5min, and add 200uL 1×BindingBuffer 5min before loading. Detect by flow cytometry, AnnexinV-FITC is green fluorescence, PI is red fluorescence. Analysis to obtain the comparison of cardiomyocyte apoptosis rate in each group Figure 5 shown.

[0054] Figure 5 The figure is a comparison chart of the apoptosis rate of H9C2 cardiomyocytes in each group, A is the blank serum group, B is the model group, C is the low-dose drug-containing serum group, D is the medium-dose drug-containing serum group, and E is the high-dose drug-containing serum group; Figure 5 It can be seen that the drug-containing serum can significantly reduce the apoptosis rate of myocardial cells damaged by hypoxia and reoxygenation, which indicates that the Chinese medicine composition provided by the present invention can reduce the apoptosis of myocardial cells damaged by hypoxia and reoxygenation.

[0055] Example 6 This example investigates the effect of the Chinese medicine composition prepared in Example 1 on oxidative stress in myocardial cells by confocal microscopy.

[0056] 1.1 Cell model construction method 1. Experimental cells: rat myocardial H9C2 cell line.

[0057] 2. Method for constructing a cardiomyocyte model of hypoxia-reoxygenation injury (24h / 6h): Take H9C2 cardiomyocytes in the logarithmic growth phase, gently rinse twice with PBS, use a pipette to aspirate PBS, add trypsin to digest, and stop digestion after the cells slide off the wall of the culture flask. Centrifuge for 5 minutes and aspirate the supernatant, then add DMEM complete medium to resuspend the cells. Use a cell counting plate to count under an electron microscope and adjust the cell density to 5×10 4 The cells were inoculated into 6-well plates with 2 ml per well and grouped after culturing at 37°C and 5% CO2 for 24 hours.

[0058] The original culture medium was aspirated with a pipette, PBS was added for washing, and then the PBS was aspirated again, and replaced with sugar-free DMEM culture medium containing serum, and placed in an incubator ventilated with mixed gas (1% O2, 94% N2, 5% CO2) for 24 hours. After glucose and oxygen deprivation, the culture medium was replaced with DMEM complete culture medium containing 10% FBS and cultured under normoxic conditions for 6 hours to obtain a cardiomyocyte model of hypoxia-reoxygenation (24h / 6h) injury.

[0059] The method for preparing the drug-containing serum in this example is the same as that in Example 5.

[0060] (II) Grouping and dosing methods are as follows: 1. Blank serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% blank serum for 24 h, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 h; 2. Model group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% blank serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 3. Low-dose drug-containing serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 5% drug-containing serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 4. Medium-dose group with drug-containing serum: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 10% drug-containing serum for 24 hours under oxygen and glucose deprivation, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours; 5. High-dose drug-containing serum group: H9C2 cardiomyocytes were cultured in a sugar-free DMEM medium containing 20% ​​drug-containing serum under oxygen and glucose deprivation for 24 hours, and then replaced with DMEM complete medium and cultured under normoxic conditions for 6 hours.

[0061] (III) After 48 hours of drug administration, the levels of LDH (lactate dehydrogenase) and c-TnT (cardiac troponin T) in the myocardial cells of each group were measured. The results are as follows: Figure 6-Figure 7 shown.

[0062] from Figure 6-Figure 7 It can be seen that the drug-containing serum can reduce the levels of LDH and c-TnT.

[0063] (IV) Use basal culture medium to dilute MitoTracker Green and MitoSOX fluorescent probes to a working concentration of 100 nmol / L and pre-warm them in a 37°C incubator. Take out the cells 48 hours after administration, discard the culture medium, add the pre-warmed fluorescent dye solution, and incubate in a 37°C incubator. After 30 minutes, remove the dye solution, add Hoechst33342 for counterstaining, and detect the corresponding fluorescence intensity under a confocal microscope. Use ZEISS ZEN software to analyze the results. The results are as follows: Figure 8 As shown, the intensity of red fluorescence labeled with MitoSOX probe reflects the level of mitochondrial ROS content in cells, and the intensity of green fluorescence labeled with MitoTrackerGreen dye reflects the level of mitochondrial content in cells.

[0064] from Figure 9-10 It can be seen that the number of mitochondria in the drug-containing serum group was higher than that in the model group, and the mitochondrial ROS level was significantly reduced, which indicates that the Chinese medicine composition of the present invention has a certain protective effect on mitochondrial function.

Claims

1. A Chinese medicine composition for treating myocardial ischemia-reperfusion injury, characterized in that: The invention comprises the following components in parts by weight: 1-20 parts of American ginseng, 20-40 parts of astragalus, 5-25 parts of ophiopogon, 1-20 parts of schisandra, 5-25 parts of rehmannia, 1-20 parts of salvia miltiorrhiza, 5-25 parts of sophora flavescens, 5-25 parts of figwort, 10-30 parts of bihua, 10-30 parts of forsythia and 1-20 parts of liquorice.

2. A Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The invention comprises the following components in parts by weight: 5-15 parts of American ginseng, 25-35 parts of astragalus, 10-20 parts of ophiopogon, 5-15 parts of schisandra, 10-20 parts of raw rehmannia, 5-15 parts of salvia, 10-20 parts of sophora flavescens, 10-20 parts of figwort, 15-25 parts of bihua, 15-25 parts of forsythia and 5-15 parts of liquorice.

3. A Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 1, characterized in that: The invention comprises the following components in parts by weight: 10 parts of American ginseng, 30 parts of astragalus, 15 parts of ophiopogon, 10 parts of schisandra, 15 parts of raw rehmannia, 10 parts of salvia miltiorrhiza, 15 parts of sophora flavescens, 15 parts of figwort, 20 parts of bihua, 20 parts of forsythia and 10 parts of liquorice.

4. A method for preparing a Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to any one of claims 1 to 3, characterized in that: All the medicinal materials are soaked in 10 times the amount of water, decocted twice, filtered and collected respectively, and the collected medicinal liquids are mixed. The obtained mixed medicinal liquid is the traditional Chinese medicine composition for treating myocardial ischemia-reperfusion injury.

5. The method for preparing the Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 4, characterized in that: The soaking time is 90 minutes, the first decoction time of the two decoctions is 60 minutes, and the second decoction time is 30 minutes.

6. The method for preparing the Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 5, characterized in that: The method also includes a step of freeze-drying the obtained mixed drug solution.

7. The method for preparing the Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 6, characterized in that: The freeze-drying conditions are -80°C and 0.002MPa, and freeze-drying for 48h.

8. The method for preparing the Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 7, characterized in that: The clinical adult dosage of the powdered Chinese medicine composition obtained by freeze-drying is 150-200 g / d.

9. The method for preparing the Chinese medicine composition for treating myocardial ischemia-reperfusion injury according to claim 8, characterized in that: The clinical adult dosage of the powdered Chinese medicine composition obtained by freeze-drying is 170 g / d.

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