Application of Tibetan medicine composition extract in preparation of medicine for improving coronary heart disease

Abstract

The invention provides application of a Tibetan medicine composition extract in preparation of a medicine for improving coronary heart disease, and the Tibetan medicine composition extract is obtained by mixing rhododendron anthopogonoide, cardamom and galangal, performing water extraction and then performing alcohol precipitation. The invention provides the application of the Tibetan medicine composition extract, and the Tibetan medicine composition extract is applied to preparation of a medicine for improving coronary heart disease. The traditional Chinese medicine composition can effectively reduce the damage degree of myocardial tissues, improve the apoptosis condition of myocardial cells, reduce the level of myocardial damage markers in serum, improve the antioxidant index activity in the serum, reduce the blood fat index level in the serum and inhibit the expression of myocardial tissue inflammatory factors.

Description

Technical Field

[0001] The invention belongs to the technical field of drugs for improving coronary heart disease, and particularly relates to an application of a Tibetan medicine formula extract for improving coronary heart disease. Background Art

[0002] Coronary heart disease (CAD) is one of the most common and fatal cardiovascular diseases worldwide. In my country, due to the accelerated aging of the population, lifestyle changes (such as a high-salt, high-fat diet and sedentary lifestyles), and the high prevalence of metabolic diseases (such as hypertension and diabetes), the number of people with CAD has exceeded 120 million (according to 2025 epidemiological data), with a younger prevalence (over 40% of patients are aged 35 to 55). The disease is characterized by the formation of atherosclerotic plaques in the coronary arteries and vascular stenosis or occlusion. Clinical manifestations include angina pectoris and myocardial infarction, which severely impact patients' quality of life and place a heavy burden on medical resources.

[0003] While current treatments for coronary artery disease (CAD) can alleviate symptoms through medication and surgery, bottlenecks remain in key areas such as plaque stabilization, endothelial repair, and improved long-term prognosis. More precise and safe treatment options are urgently needed. Tibetan medicine, a key branch of traditional Chinese medicine, has accumulated extensive practical experience in the prevention and treatment of cardiovascular diseases. Tibetan medicine categorizes CAD as "cardiac wind syndrome" or "cardiac blood syndrome," and its treatment emphasizes holistic regulation and multi-target effects, aligning with modern medicine's understanding of atherosclerosis as a systemic inflammatory response. However, existing Tibetan medicines are mostly used in the form of compound preparations (such as Seventy-Ingredient Pearl Pills and Thirty-Five-Ingredient Agarwood Pills), whose complex composition limits their application in the treatment of CAD. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned existing technologies and provide an application of a Tibetan medicine formula extract in the preparation of a drug for improving coronary heart disease. The Tibetan medicine formula extract is used to prepare a drug for improving coronary heart disease, and can effectively reduce the degree of myocardial tissue damage, improve myocardial cell apoptosis, reduce the level of myocardial injury markers in serum, increase the activity of antioxidant indicators in serum, reduce the level of blood lipid indicators in serum and inhibit the expression of inflammatory factors in myocardial tissue.

[0005] To solve the above technical problems, the present invention adopts a technical solution: a use of a Tibetan medicine formula extract in the preparation of a drug for improving coronary heart disease, wherein the Tibetan medicine formula extract is obtained by mixing Rhododendron australis, Cardamom and Alpinia officinalis, extracting with water and then precipitating with alcohol; the Tibetan medicine formula extract is used to prepare a drug for improving coronary heart disease; Rhododendron australis is the main medicine, which has the effects of promoting diuresis and reducing swelling, clearing away heat and strengthening the heart, and improving bronchitis; cardamom is the auxiliary medicine, which has the effects of dispelling cold, warming the kidney, and astringing the meridians; galangal is the adjuvant medicine, which has the effects of warming the stomach and improving the spleen and stomach functions.

[0006] Preferably, the application includes one or more of the following applications: The Tibetan medicine formula extract is used to prepare a medicine for reducing the degree of myocardial tissue damage; The Tibetan medicine formula extract is used to prepare a drug for improving myocardial cell apoptosis; The Tibetan medicine formula extract is used to prepare a drug for reducing the level of myocardial injury markers in serum; The Tibetan medicine formula extract is used to prepare a drug for improving the activity of antioxidant indicators in serum; The Tibetan medicine formula extract is used to prepare a drug for lowering blood lipid index levels in serum; The Tibetan medicine formula extract is used to prepare a medicine for inhibiting the expression of myocardial inflammatory factors.

[0007] Preferably, when the Tibetan medicine formula extract is used to prepare a drug for reducing the level of myocardial injury markers in serum, the myocardial injury markers include one or more of CK-MB, CRP and LDH; When the Tibetan medicine formula extract is used to prepare a drug for improving the activity of antioxidant indicators in serum, the antioxidant indicators include one or more of SOD and GSH-Px; When the Tibetan medicine formula extract is used to prepare a drug for lowering the level of blood lipid indicators in serum, the blood lipid indicators include one or more of TC, TG and LDL-C; When the Tibetan medicine formula extract is used to prepare a drug for inhibiting the expression of myocardial inflammatory factors, the myocardial tissue inflammatory factors include one or more of IL-6, IL-1β and TNF-α.

[0008] Preferably, the drug further comprises a pharmaceutically acceptable carrier.

[0009] Preferably, the pharmaceutically acceptable carrier is suitable for oral liquid, tablet, pill, powder, capsule, patch or ointment.

[0010] Preferably, the mass ratio of the rhododendron serrata, cardamom and galangal is 2:1:1; the reasonable ratio of rhododendron serrata, cardamom and galangal has a good effect in treating coronary heart disease and improving hypoxic myocardial damage.

[0011] Preferably, the preparation method of the Tibetan medicine formula extract is: S1. Mixing Rhododendron australis, Cardamom and Alpinia officinalis to obtain a Tibetan medicine formula; S2. Add water to the Tibetan medicine formula obtained in S1, perform hot reflux extraction to obtain an extract a and a filter residue b; add water to the filter residue b, perform hot reflux extraction again to obtain an extract c and a filter residue d, combine the extracts a and c to obtain a combined extract, let the combined extract stand for 12 hours, filter the residue to obtain a filtered combined extract, add ethanol to the filtered combined extract, perform alcohol precipitation, filter, concentrate the filtrate under reduced pressure, and freeze-dry to obtain a Tibetan medicine formula extract.

[0012] Preferably, the mass ratio of water to Tibetan medicine formula is 10:1; the mass ratio of water to filter residue b is 8:1; the temperature of the hot reflux extraction is 100° C., and the extraction time for each time is 1 hour.

[0013] Preferably, the alcohol precipitation conditions are: the mass fraction of ethanol in the combined extract after filtration is 60%, and the time is 18 hours; the reduced pressure concentration method is: reduced pressure concentration until each 1 mL of filtrate contains 1.0 g of the Tibetan medicine formula; the freeze-drying conditions are: freeze-drying at -80°C for 48 hours.

[0014] Compared with the prior art, the present invention has the following advantages: The application of the Tibetan medicine formula extract provided by the present invention in the preparation of a drug for improving coronary heart disease can effectively reduce myocardial tissue damage, improve myocardial cell apoptosis, reduce the level of myocardial damage markers in serum, increase the activity of antioxidant indicators in serum, reduce the level of blood lipid indicators in serum and inhibit the expression of inflammatory factors in myocardial tissue, thereby achieving the purpose of improving coronary heart disease.

[0015] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a specific flow chart of establishing a rat coronary heart disease model in Example 2 of the present invention.

[0017] Figure 2 3 is a graph showing changes in the electrocardiogram of rats in the Con group and the CHD group in Example 2 of the present invention; wherein, “↓” indicates ST segment elevation.

[0018] Figure 3 It is the MA map of differential genes among the Con group, CHD group and SWDJ high-dose group in Example 2 of the present invention; wherein, Figure A is the MA map of differential genes between the CHD group and the Con group; Figure B is the MA map of differential genes between the SWDJ high-dose group and the CHD group; red represents upregulated genes, and blue represents downregulated genes.

[0019] Figure 4This is a heat map of differentially expressed genes in the Con group, CHD group, and SWDJ high-dose group in Example 2 of the present invention; green represents the Con group, pink represents the CHD group, and blue represents the SWDJ high-dose group.

[0020] Figure 5 These are the color Doppler ultrasound images of the hearts of rats in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention.

[0021] Figure 6 Figure 2 is a graph of cardiac function indices of rats in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention; Figure A is diastolic LVID; Figure B is systolic LVID; Figure C is FS; Figure D is HR; *P<0.05, **P<0.01 compared with the Con group; compared with the CHD group # P<0.05, ## P<0.01.

[0022] Figure 7 These are HE staining images of rat myocardial tissue in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention; wherein, the first row is HE staining images of rat myocardial tissue at 10× magnification; the second row is HE staining images of rat myocardial tissue at 100× magnification of the local area in the dotted box in the first row; “↓” indicates cellular inflammatory infiltration in HE staining.

[0023] Figure 8 3. These are TUNEL staining images of rat cardiomyocytes in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention.

[0024] Figure 9 Figure 2 is a graph showing the levels of myocardial injury markers in the serum of rats in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention; Figure A shows the CRP level; Figure B shows the CK-MB level; Figure C shows the LDH level; **P<0.01 compared with the Con group; **P<0.01 compared with the CHD group # P<0.05, ## P<0.01.

[0025] Figure 10 Figure 2 is a graph showing the levels of antioxidant and blood lipid indicators in the serum of rats in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention; wherein, Figure A shows GSH-Px activity; Figure B shows SOD activity; Figure C shows HDL-C level; Figure D shows LDL-C level; Figure E shows TC level; Figure F shows TG level; **P<0.01 compared with the Con group; compared with the CHD group # P<0.05, ## P<0.01.

[0026] Figure 11 These are the immunohistochemical images of inflammatory factors in the myocardial tissue of rats in the Con group, CHD group, At group and three different doses of SWDJ groups in Example 2 of the present invention; among them, the first row is the immunohistochemical image of the inflammatory factor IL-1β at a magnification of 10×, the second row is the immunohistochemical image of the inflammatory factor IL-1β at a local magnification of 40× in the dotted box in the first row; the third row is the immunohistochemical image of the inflammatory factor IL-6 at a magnification of 10×, the fourth row is the immunohistochemical image of the inflammatory factor IL-6 at a local magnification of 40× in the dotted box in the third row; the fifth row is the immunohistochemical image of the inflammatory factor TNF-α at a magnification of 10×, and the sixth row is the immunohistochemical image of the inflammatory factor TNF-α at a local magnification of 40× in the dotted box in the fifth row; "↓" indicates a positive result.

[0027] Figure 12 Graphs showing the relative mRNA expression of Beclin1, LC3, MFN2, P53, and P62 in myocardial tissue of rats in the Con group, CHD group, At group, and three different doses of SWDJ groups in Example 2 of the present invention; wherein, Graph A shows the relative expression of Beclin1; Graph B shows the relative expression of LC3; Graph C shows the relative expression of MFN2; Graph D shows the relative expression of P53; Graph E shows the relative expression of P62; **P<0.01 compared with the Con group; **P<0.01 compared with the CHD group # P<0.05, ## P<0.01. DETAILED DESCRIPTION

[0028] Example 1 This embodiment is a method for preparing a Tibetan medicine formula extract. The Tibetan medicine formula extract is prepared by mixing Rhododendron australis, Cardamom and Alpinia officinalis in a mass ratio of 2:1:1 to obtain a Tibetan medicine formula, adding water in a mass ratio of water to the Tibetan medicine formula of 10:1, and performing hot reflux extraction at a temperature of 100° C. for 1 hour to obtain an extract a and a filter residue b; adding water to the filter residue b in a mass ratio of water to the filter residue b of 8:1, and performing hot reflux extraction again at a temperature of 100° C. for 1 hour to obtain an extract c and a filter residue d. Combine extracts a and c to obtain a combined extract, let the combined extract stand for 12 hours, filter the residue to obtain a filtered combined extract, add ethanol to the filtered combined extract, wherein the mass fraction of ethanol in the filtered combined extract is 60%, perform alcohol precipitation for 18 hours, filter under reduced pressure until each 1 mL of filtrate contains 1.0 g of the Tibetan medicine formula, freeze-dry at -80°C for 48 hours, and obtain a Tibetan medicine formula extract, recorded as SWDJ; the Tibetan medicine formula extract is used to prepare a drug for improving coronary heart disease; Rhododendron australis is the main medicine, which has the effects of promoting diuresis and reducing swelling, clearing away heat and strengthening the heart, and improving bronchitis. Cardamom is the auxiliary medicine, which has the effects of dispelling cold, warming the kidneys, and astringing the meridians. Alpinia officinarum is the adjuvant medicine, which has the effects of warming the stomach and improving spleen and stomach functions. The reasonable ratio of Rhododendron australis, cardamom and Alpinia officinarum has a good effect in treating coronary heart disease and improving hypoxic myocardial damage.

[0029] Example 2 This embodiment is the use of the Tibetan medicine formula extract of Example 1 in the preparation of a drug for improving coronary heart disease. The Tibetan medicine formula extract is used to prepare a drug for improving coronary heart disease, which can effectively reduce the degree of damage to myocardial tissue, improve myocardial cell apoptosis, reduce the level of myocardial injury markers in serum, increase the activity of antioxidant indicators in serum, reduce the level of blood lipid indicators in serum and inhibit the expression of inflammatory factors in myocardial tissue.

[0030] 1. Experimental materials: (1) Experimental animals: 72 SPF male SD rats, weighing 250 g ± 20 g, were purchased from Hubei Beient Biotechnology Co., Ltd., license number: SCXK (E) 2021-0027; they were kept in the Experimental Animal Center of South-Central University for Nationalities, and the experimental institution's experimental animal use license number: SYXK (E) 2021-0089; the breeding conditions were a temperature of 20°C–26°C, a relative humidity of 40%–70%, an independent ventilation cage (IVC) with an air exchange rate of ≥15 times / h, and a light cycle of 12 h light / 12 h dark.

[0031] (2) Experimental drugs: Tibetan medicine formula extract was recorded as SWDJ.

[0032] (3) Experimental reagents: high-fat feed (Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.), conventional feed (Wuhan Wanqian Jiaxing Biotechnology Co., Ltd.), vitamin D3 (Beijing Solaibao Technology Co., Ltd.), isoproterenol hydrochloride (Aladdin Biochemical Technology Co., Ltd.), physiological saline (Sichuan Kelun Pharmaceutical Co., Ltd.), atorvastatin calcium tablets (Qilu Pharmaceutical Co., Ltd.), urethane (Sinopharm Chemical Reagent Co., Ltd.), TRNzol total RNA extract (Tiangen Biochemical Technology Co., Ltd.), chloroform (Sinopharm Chemical Reagent Co., Ltd.), isopropyl alcohol (Sinopharm Chemical Reagent Co., Ltd.), anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.), DEPC water (VETEC), total SOD activity detection kit (Shanghai Biyuntian Biotechnology Co., Ltd.), glutathione peroxidase detection kit (Shanghai Biyuntian Biotechnology Co., Ltd.), ABScript Ⅱ RT Master Mix for qPCR (Wuhan Aibotek Biotechnology Co., Ltd.), TBGreenTM Premix Ex TaqTM (Wuhan Aibotek Biotechnology Co., Ltd.).

[0033] 2. Experimental methods: (1) Experimental groups: After 7 days of adaptive feeding, 72 SD rats were randomly divided into a normal control group (Con), a model group (CHD), a positive control group (atorvastatin calcium tablets, At group), and three SWDJ groups with different doses (SWDJ low-dose group, SWDJ medium-dose group, and SWDJ high-dose group), with 12 rats in each group.

[0034] (2) Establishment of rat coronary heart disease model: Coronary heart disease models were established in rats in the model group, positive control group, SWDJ low-dose group, SWDJ medium-dose group, and SWDJ high-dose group. The specific process is as follows: Figure 1 As shown, including: feeding high-fat diet every day for 7 consecutive weeks, during which time 200,000 IU / kg of vitamin D3 was gavaged on the 7th day and 300,000 IU / kg of vitamin D3 was gavaged on the 14th day, and after 7 weeks, 5 mg / kg of isoproterenol hydrochloride was intraperitoneally injected at intervals of 24 hours once a day for 3 consecutive days; the normal control group was fed regular diet every day for 7 consecutive weeks, during which 10 mL / kg of normal saline was gavaged on the 7th day and 14th day once a day, and after 7 weeks, 10 mL / kg of normal saline was intraperitoneally injected at intervals of 24 hours once a day for 3 consecutive days; the BL-420F biofunctional laboratory system was used to detect the changes in the electrocardiogram of lead II of the rats, and the ST segment elevation of the electrocardiogram was used as the standard to determine that the rat coronary heart disease model was successfully established, as shown Figure 2 As shown in the figure, "↓" indicates ST segment elevation. Compared with the Con group, the ST segment elevation of the CHD group was ≥0.1 mV, indicating that the coronary heart disease models of all 60 rats in this study were successfully established.

[0035] (3) Experimental dosage design and administration: The clinical dosage of raw Tibetan medicine is 15g / d for adults, and the powder dosage for adults is 0.25g / kg / d, which is equivalent to 1.55g / kg / d for animals. The powder used in the experiment was extracted from the raw Tibetan medicine, with an extraction rate of 11.14%. The raw medicine dosage for animals was converted into the dosage of extracted powder, which was about 0.017g / kg. The commonly used dosage for animals was 1.55g / kg / d. Three dosing groups were designed, with the dosage of the three groups being 1 to 4 times the commonly used dosage and the ratio being 1:2:4. 72 SD rats were randomly divided into 6 groups. Figure 1 As shown in the figure, after the rat coronary heart disease model was successfully established, the drug was administered orally 7 days later. One atorvastatin calcium tablet contains 10 mg of active drug, that is, each atorvastatin calcium tablet is dissolved in 1.25 mL of normal saline, and each 17 mg of SWDJ powder is dissolved in 1 mL of normal saline; each rat in the normal control group was gavaged with 10 mL / kg of normal saline, each rat in the model group was gavaged with 10 mL / kg of normal saline, each rat in the positive control group was gavaged with 8 mg / kg of At, each rat in the low-dose SWDJ group was gavaged with 0.017 g / kg of SWDJ, each rat in the medium-dose SWDJ group was gavaged with 0.035 g / kg of SWDJ, and each rat in the high-dose SWDJ group was gavaged with 0.069 g / kg of SWDJ, once a day for 14 consecutive days.

[0036] (4) Sampling of drug-treated rats: Fourteen days after administration, each rat in the Con group, CHD group, At group and three different doses of SWDJ groups was anesthetized by intraperitoneal injection of urethane and underwent cardiac color Doppler ultrasound examination. After the color Doppler ultrasound, blood was collected from the rat abdominal aorta. The whole blood was centrifuged at 2500 rpm and 4°C for 15 min in a low-temperature high-speed centrifuge. The upper serum was aspirated, packaged, and stored in a -80°C refrigerator for subsequent testing. After blood was collected from the abdominal aorta, the rats were dissected and the rat hearts were collected and stored in a -80°C refrigerator and 4% formaldehyde fixative, respectively, for subsequent transcriptomic analysis, tissue hematoxylin-eosin (HE) staining, mRNA expression, immunohistochemistry detection, protein immunoblotting experiments, and related index detection.

[0037] 3. Transcriptomic Analysis (1) Extraction of total RNA from myocardial tissue: Myocardial tissue samples from rats in the Con, CHD, At, and SWDJ high-dose groups were obtained. Three samples (100 mg each) were randomly selected from each group and ground with liquid nitrogen. The samples were then placed in pre-cooled 1.5 mL centrifuge tubes and total RNA was extracted using TRNzol total RNA extraction solution. The specific procedures were as follows: 1 mL of TRIzol reagent was added to the ground product for thorough homogenization. After incubation at room temperature for 5 min, 200 μL of chloroform was added. After vigorous shaking for 15 s, the sample was allowed to stand for 2 min. The sample was centrifuged at 4°C and 12,000 rpm for 15 min. The upper aqueous phase was transferred to a new enzyme-free centrifuge tube and the volume of the upper aqueous phase was recorded. An equal volume of isopropanol was added to the centrifuge tube and the mixture was mixed. The sample was allowed to stand for 30 min at -20°C and then centrifuged at 4°C and 12,000 rpm for 30 min. The RNA precipitate was collected and 1 mL of 75% ethanol (11.25 mL of anhydrous ethanol was added to 3.75 mL of ethanol) was added. The RNA was washed with DEPC water (diluted to 15 mL) and centrifuged at 10,000 rpm for 5 min at 4°C, after which the supernatant was discarded. The RNA precipitate was allowed to dry naturally for 5 min and then dissolved in 30 μL of DEPC water. RNA integrity was assessed using an Agilent 2100 bioanalyzer, and RNA concentration and purity were determined using a Nano Drop spectrophotometer.

[0038] (2) Library construction and detection: A total of ≥1 μg of high-quality RNA sample was used for library construction. mRNA molecules with polyA tails were isolated by oligo (dT) magnetic bead enrichment. Ultrasonic shearing was used to fragment the mRNA to approximately 300 bp. First-strand cDNA was synthesized using random hexamer primers and reverse transcriptase using the fragmented mRNA as a template, followed by second-strand synthesis using DNA polymerase. cDNA fragments of approximately 450 bp were screened using AMPure XP magnetic beads, and the final sequencing library was obtained after PCR amplification and purification. The constructed library was sequenced by paired-end (PE) high-throughput sequencing using the Illumina sequencing platform.

[0039] (3) RNA sequencing data analysis: Raw sequencing data (Raw Data) were converted to FASTQ format files via base calling and quality assessment using FastQC software. Data preprocessing was completed using the Fastp tool: 3'-end adapter sequences were removed, reads with an average quality value below Q20 were filtered, sequences containing more than 10% N bases were removed, and valid reads ≥20 bp in length (Clean Data) were retained. Clean Data were aligned to the rat reference genome GRCm39 using HISAT2 software. The raw expression level of each gene was calculated using the HTSeq tool and normalized using the FPKM method. Differential gene expression was analyzed using the DESeq2 software, with the screening criteria of |log2FoldChange| ≥1 and P < 0.05. Cluster heat map analysis of all differentially expressed genes was performed using the Pheatmap package in R.

[0040] (4) Analysis of transcriptome sequencing results: like Figure 3 As shown in Figure A, there were 1663 differentially expressed genes between the CHD group and the Con group, of which 445 genes were upregulated and 1218 genes were downregulated; Figure 3 As shown in Figure B, there were 467 differentially expressed genes between the SWDJ group and the CHD group, of which 109 genes were upregulated and 358 genes were downregulated; 168 genes that were upregulated in the CHD group were reversed to downregulated by the high-dose SWDJ group, and 58 genes that were downregulated in the CHD group were reversed to upregulated by the high-dose SWDJ group.

[0041] Cluster heat map analysis was performed on all differentially expressed genes. The results are shown in the figure below. Figure 4 As shown, green represents the Con group, pink represents the CHD group, and blue represents the SWDJ high-dose group. The samples of the Con group, CHD group, and SWDJ high-dose group showed obvious clustering. The color gradient difference of the same gene between different groups (red represents high expression, blue represents low expression) intuitively reflects the relative change amplitude of gene expression. The gene expression pattern of the SWDJ high-dose group showed a significant separation trend from the CHD group.

[0042] 4. Effects of three different doses of SWDJ on cardiac function in rats with coronary heart disease: After 14 days of administration, if Figure 5 As shown, the anesthetized rats were subjected to cardiac ultrasound examination using a veterinary color Doppler ultrasound system, and the heart rate (HR), diastolic LVID, systolic LVID and left ventricular fractional shortening (FS) of the rats in the Con group, CHD group, At group and three different doses of SWDJ groups were measured.

[0043] like Figure 6As shown in Figure A, the three different doses of SWDJ groups all had a significant reversal effect on the diastolic LVID of rats with coronary heart disease; Figure 6 As shown in B, the high-dose SWDJ group had a significant reversal effect on the cardiac contraction LVID of rats with coronary heart disease; Figure 6 As shown in Figures C and D, the low-dose and high-dose SWDJ groups significantly improved FS and HR in rats with coronary heart disease, respectively.

[0044] 5. Effects of three different doses of SWDJ on the degree of myocardial tissue damage in rats with coronary heart disease model: HE staining was used to observe the pathological changes of myocardial tissue in rats with coronary heart disease. Figure 7 As shown in the figure, the cardiomyocytes of the rats in the Con group were arranged neatly without obvious abnormalities; the cardiomyocytes of the rats in the CHD group were hypertrophic and disordered, with widened intercellular spaces and increased connective tissue; some myocardial fibers were atrophied or degenerated, and a large number of inflammatory cells infiltrated; the cardiomyocyte necrosis and degeneration of the rats in the At group and three different doses of SWDJ groups were improved to varying degrees compared with those in the model group, and the degree of myocardial tissue damage gradually decreased with the increase of SWDJ concentration. "↓" indicates cellular inflammatory infiltration in HE staining.

[0045] 6. Effects of three different doses of SWDJ on myocardial cell apoptosis in rats with coronary heart disease model: Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining was used to observe cardiomyocyte apoptosis; Figure 8 As shown, a large number of cardiomyocytes in the rats in the CHD group underwent apoptosis (green indicates positive results), and the apoptosis of cardiomyocytes in the rats in the At group and three different doses of SWDJ groups was improved. The improvement of cardiomyocyte apoptosis in the rats was more obvious with the increase of SWDJ dose.

[0046] 7. Effects of three different doses of SWDJ on the levels of myocardial injury markers in the serum of rats with coronary heart disease: The Mindray BS-240VET automatic biochemical analyzer was used to detect the levels of three myocardial injury markers in serum: C-reactive protein (CRP), creatine kinase isoenzyme-MB (CK-MB), and lactate dehydrogenase (LDH); Figure 9 As shown in Figures A, B, and C, compared with those in the Con group, the levels of serum myocardial injury markers CK-MB, CRP, and LDH in the CHD group were significantly increased (P<0.01); after treatment with At and three different doses of SWDJ, the levels of the three serum myocardial injury markers were significantly downregulated compared with those in the CHD group (P<0.05).

[0047] 8. Effects of three different doses of SWDJ on serum antioxidant and blood lipid indicators in rats with coronary heart disease: The total SOD activity assay kit and glutathione peroxidase (GSH-Px) assay kit were used to detect the activities of SOD and GSH-Px in rat serum. The levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in serum were measured using a Mindray BS-240VET automatic biochemical analyzer.

[0048] like Figure 10 As shown in A and B, compared with the Con group, the activities of SOD and GSH-Px in the serum of rats in the CHD group were significantly decreased (all P < 0.05); compared with the CHD group, the levels of SOD and GSH-Px in the serum of rats in the At group and the three different doses of SWDJ groups were significantly increased; the results of serum lipid levels in rats are shown in Figure 10 As shown in Figures C, D, E, and F, compared with those in the Con group, the serum TC, TG, and LDL-C levels of the rats in the CHD group were significantly increased, and the HDL-C level was significantly decreased, and the differences were statistically significant (P<0.01). Compared with the CHD group, the serum TC, TG, and LDL-C levels of the rats in the At group and different doses of SWDJ were significantly decreased, and the HDL-C level was significantly increased.

[0049] 9. Effects of three different doses of SWDJ on the expression of inflammatory factors in myocardial tissue of rats with coronary heart disease model: Immunohistochemistry was used to detect the levels of inflammatory factors in the myocardial tissues of rats in the Con group, CHD group, At group and three different doses of SWDJ groups. The results were as follows: Figure 11 As shown in the figure, IL-6, IL-1β and TNF-α were positively expressed in the CHD group rats, with deeper tissue staining and obvious brown staining; in the Con group, At group and SWDJ low-, medium- and high-dose groups, the expressions of IL-6, IL-1β and TNF-α were decreased, indicating that the At and SWDJ low-, medium- and high-dose groups can inhibit the expression of inflammatory factors in myocardial tissue. "↓" indicates a positive result.

[0050] 10. Effects of three different doses of SWDJ on the mRNA expression levels of P62 (ubiquitin-binding protein P62), P53 (phosphoprotein 53), MFN2 (mitochondrial fusion protein Mitofusin 2), LC3 (autophagy microtubule-associated protein light chain 3), and Beclin1 (recombinant human autophagy effector protein Beclin 1) in myocardial tissue of rats with coronary heart disease model: Total RNA was extracted from myocardial tissue samples of rats in the Con, CHD, At, and three different doses of SWDJ groups using TRNzol total RNA extraction solution. The extracted total RNA was then reverse transcribed into cDNA using the ABScript Ⅱ RT Master Mix for qPCR reverse transcription kit. Real-time fluorescence quantitative PCR (qRT-PCR) was performed using the TB Green™ Premix Ex Taq™ kit. GAPDH gene was used as an internal reference gene to detect the mRNA expressions of P62, P53, MFN2, LC3, and Beclin1 in myocardial tissue. In the qRT-PCR experiment, the nucleotide sequence of the upstream primer P62-F of P62 is shown in SEQ ID NO.1, and the nucleotide sequence of the downstream primer P62-R is shown in SEQ ID NO.2; the nucleotide sequence of the upstream primer P53-F is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer P53-R is shown in SEQ ID NO.4; the nucleotide sequence of the upstream primer MFN2-F is shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer MFN2-R is shown in SEQ ID NO.6; the nucleotide sequence of the upstream primer LC3-F is shown in SEQ ID NO.7, and the nucleotide sequence of the downstream primer LC3-R is shown in SEQ ID NO.8; the nucleotide sequence of the upstream primer Beclin1-F is shown in SEQ ID NO.9, and the nucleotide sequence of the downstream primer Beclin1-R is shown in SEQ ID NO.10; the nucleotide sequence of the upstream primer GAPDH-F is shown in SEQ ID NO.11, and the nucleotide sequence of the downstream primer GAPDH-R is shown in SEQ ID NO.12.

[0051] like Figure 12 As shown in A, B, C, D and E, compared with those in the Con group, the mRNA levels of LC3, Beclin1 and P53 in the myocardial tissue of the rats in the CHD group were significantly increased (P<0.01), and the mRNA levels of MFN2 and P62 were significantly decreased (P<0.01); compared with the CHD group, the mRNA levels of LC3, Beclin1 and P53 in the myocardial tissue of the rats in the At group and different doses of SWDJ were significantly decreased (P<0.01), and the mRNA levels of P62 and MFN2 were significantly increased (P<0.01).

[0052] Therefore, the Tibetan medicine formula extract prepared by the present invention is used to prepare a medicine for improving coronary heart disease. When the Tibetan medicine formula extract is used to prepare a medicine for reducing the degree of myocardial tissue damage, the degree of myocardial tissue damage can be effectively reduced; when the Tibetan medicine formula extract is used to prepare a medicine for improving myocardial cell apoptosis, the apoptosis of myocardial cells can be improved; when the Tibetan medicine formula extract is used to prepare a medicine for reducing the content of myocardial injury markers in serum, the content of myocardial injury markers CK-MB, CRP and LDH can be effectively reduced; when the Tibetan medicine formula extract is used to prepare a medicine for improving the activity of antioxidant indicators in serum, the activity of SOD and GSH-Px in serum can be effectively increased; when the Tibetan medicine formula extract is used to prepare a medicine for improving the activity of antioxidant indicators in serum, the activity of SOD and GSH-Px in serum can be effectively increased. When the extract is used to prepare a drug for lowering the levels of blood lipid indicators in serum, it can effectively lower the levels of TC, TG and LDL-C indicators in serum; when the Tibetan medicine formula extract is used to prepare a drug for inhibiting the expression of inflammatory factors in myocardial tissue, it can effectively inhibit the expression of inflammatory factors IL-6, IL-1β and TNF-α in myocardial tissue; the above-mentioned drug also includes a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier is suitable for oral liquid, tablets, pills, powders, capsules, patches or ointments; through transcriptome analysis combined with rat coronary heart disease model verification, it was found that the Tibetan medicine formula extract has a significant alleviating effect on the rat coronary heart disease model, and its mechanism of action is related to protecting the heart tissue from oxidative stress damage after coronary heart disease damage.

[0053] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent variation made to the above embodiment based on the essence of the invention technology shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A use of a Tibetan medicine formula extract in preparing a drug for improving coronary heart disease, characterized in that: The Tibetan medicine formula extract is obtained by mixing Rhododendron australis, Cardamom and Alpinia officinalis, extracting with water and then precipitating with alcohol; the Tibetan medicine formula extract is used for preparing medicine for improving coronary heart disease.

2. The use of a Tibetan medicine formula extract according to claim 1 in preparing a drug for improving coronary heart disease, characterized in that: The application includes one or more of the following applications: The Tibetan medicine formula extract is used to prepare a medicine for reducing the degree of myocardial tissue damage; The Tibetan medicine formula extract is used to prepare a drug for improving myocardial cell apoptosis; The Tibetan medicine formula extract is used to prepare a drug for reducing the level of myocardial injury markers in serum; The Tibetan medicine formula extract is used to prepare a drug for improving the activity of antioxidant indicators in serum; The Tibetan medicine formula extract is used to prepare a drug for lowering blood lipid index levels in serum; The Tibetan medicine formula extract is used to prepare a medicine for inhibiting the expression of inflammatory factors in myocardial tissue.

3. The use of a Tibetan medicine formula extract according to claim 2 in preparing a drug for improving coronary heart disease, characterized in that: When the Tibetan medicine formula extract is used to prepare a drug for reducing the level of myocardial injury markers in serum, the myocardial injury markers include one or more of CK-MB, CRP and LDH; When the Tibetan medicine formula extract is used to prepare a drug for improving the activity of antioxidant indicators in serum, the antioxidant indicators include one or more of SOD and GSH-Px; When the Tibetan medicine formula extract is used to prepare a drug for lowering the level of blood lipid indicators in serum, the blood lipid indicators include one or more of TC, TG and LDL-C; When the Tibetan medicine formula extract is used to prepare a drug for inhibiting the expression of myocardial tissue inflammatory factors, the myocardial tissue inflammatory factors include one or more of IL-6, IL-1β and TNF-α.

4. The use of a Tibetan medicine formula extract according to claim 3 in preparing a drug for improving coronary heart disease, characterized in that: The drug further includes a pharmaceutically acceptable carrier.

5. The use of a Tibetan medicine formula extract according to claim 4 in preparing a drug for improving coronary heart disease, characterized in that: The pharmaceutically acceptable carrier is suitable for oral liquid, tablet, pill, powder, capsule, patch or ointment.

6. Use of a Tibetan medicine formula extract according to claim 1 in preparing a drug for improving coronary heart disease, characterized in that: The mass ratio of the rhododendron scabra, cardamom and galangal is 2:1:

1.

7. Use of a Tibetan medicine formula extract according to claim 1 in preparing a drug for improving coronary heart disease, characterized in that: The preparation method of the Tibetan medicine formula extract is: S1. Mixing Rhododendron australis, Cardamom and Alpinia officinalis to obtain a Tibetan medicine formula; S2. Add water to the Tibetan medicine formula obtained in S1, perform hot reflux extraction to obtain an extract a and a filter residue b, add water to the filter residue b, perform hot reflux extraction again to obtain an extract c and a filter residue d, combine the extracts a and c to obtain a combined extract, let the combined extract stand for 12 hours, filter the residue to obtain a filtered combined extract, add ethanol to the filtered combined extract, perform alcohol precipitation, filter, concentrate the filtrate under reduced pressure, and freeze-dry to obtain a Tibetan medicine formula extract.

8. Use of the Tibetan medicine formula extract according to claim 7 in preparing a drug for improving coronary heart disease, characterized in that: The mass ratio of water to the Tibetan medicine formula is 10:1; the mass ratio of water to filter residue b is 8:1; the temperature of the hot reflux extraction is 100° C., and the extraction time for each extraction is 1 hour.

9. Use of a Tibetan medicine formula extract according to claim 7 in preparing a drug for improving coronary heart disease, characterized in that: The conditions for the alcohol precipitation are: the mass fraction of ethanol in the combined extract after filtration is 60%, and the time is 18 hours; the method for the reduced pressure concentration is: reduced pressure concentration until each 1 mL of filtrate contains 1.0 g of the Tibetan medicine formula; the conditions for the freeze drying are: freeze drying at -80°C for 48 hours.

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