Traditional Chinese medicine composition for treating ischemic heart disease as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition for treating ischemic heart disease and a preparation method and application thereof. The traditional Chinese medicine composition is formed by compatibility of salvia miltiorrhiza and hawthorn, active functional components for protecting cardiac muscle in the salvia miltiorrhiza comprise water-soluble phenolic acids and fat-soluble tanshinone, and active functional components for protecting cardiac muscle in the hawthorn comprise procyanidins, flavonoids and triterpenic acids. Through systematic evaluation and research on the traditional Chinese medicine composition, the functional components for treating the IHD are screened out, the action mechanism of the functional components is clarified, and a new thought can be provided for precise treatment of the IHD.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a traditional Chinese medicine composition for treating ischemic heart disease, and a preparation method and application thereof. Background Art

[0002] Ischemic heart disease (IHD) is a condition characterized by impaired myocardial blood flow caused by coronary atherosclerosis. As one of the most fatal cardiovascular diseases worldwide, its high morbidity, disability, and recurrence rates pose a significant challenge to public health systems. While modern medicine can alleviate clinical symptoms through percutaneous coronary intervention and drug therapy, widespread technical bottlenecks remain, such as postoperative restenosis, drug resistance, and poor long-term prognosis. New treatment strategies with greater safety and sustained efficacy are urgently needed.

[0003] In traditional Chinese medicine, IHD is classified under the categories of "chest pain" and "heart pain." Its core pathogenesis, characterized by "blood stasis leading to paralysis," and its treatment principles and methods of "activating blood circulation and removing stasis," have formed a comprehensive system of syndrome differentiation and treatment. Existing clinical evidence demonstrates that Traditional Chinese Medicine (TCM) exhibits unique advantages in improving myocardial microcirculation, alleviating ischemic damage, and enhancing long-term quality of life in IHD patients. Leveraging the synergistic properties of TCM across multiple components, targets, and pathways, the systematic development of TCM compositions with a clear material basis for efficacy will provide a new path for the precision treatment of IHD. Summary of the Invention

[0004] The present invention aims to provide a traditional Chinese medicine composition for treating ischemic heart disease, specifically a Danshen-Hawthorn combination (DSHP), which comprises Danshen (Salviae Miltiorrhizae Radix et Rhizoma) and Hawthorn (Crataegi Fructus) in a specific ratio. Danshen is bitter and slightly cold, promoting blood circulation, removing blood stasis and promoting new blood circulation; Hawthorn is sour, sweet, and slightly warm, effectively dispersing qi and blood stasis, and reducing turbidity and lipids. The combination of the two adheres to the traditional Chinese medicine compatibility principle of "mutually complementary" and, through synergistic synergy, significantly enhances the therapeutic effects of promoting qi and blood circulation, removing blood stasis, and alleviating pain. The DSHP is particularly suitable for treating chest pain and heart pain caused by blood stasis.

[0005] It should be noted that existing systematic research on Danshen-Hawthorn combinations has significant shortcomings: first, a holistic quality evaluation system is lacking; second, the key active ingredients in the treatment of IHD remain unidentified; and third, research on the relevant pharmacological mechanisms remains elusive. To address these technical deficiencies, the present invention further provides a preparation method and application scheme for this combination. By combining modern separation and analysis techniques with pharmacodynamic evaluation, a chemometrically-based "spectrum-effect" correlation network is established, thereby clarifying the material basis and mechanism of action for its treatment of IHD.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a traditional Chinese medicine composition for treating ischemic heart disease, wherein the traditional Chinese medicine composition is composed of salvia miltiorrhiza and hawthorn, wherein the active functional ingredients in salvia miltiorrhiza for protecting the myocardium include water-soluble phenolic acids and fat-soluble tanshinones, and the active functional ingredients in hawthorn for protecting the myocardium include proanthocyanidins, flavonoids and triterpenoid acids.

[0008] Preferably, the water-soluble phenolic acids include rosmarinic acid, lithospermic acid, danshensu, salvianolic acid A, salvianolic acid B and protocatechuic aldehyde; the fat-soluble tanshinones include dihydrotanshinone I, tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone and dihydrotanshinone; the proanthocyanidins include proanthocyanidin B2; the flavonoids include vitexin, rutin, quercetin, naringin, hyperoside, isoquercetin and epicatechin; the triterpene acids include citric acid, ursolic acid, malic acid, gallic acid, fumaric acid, caffeoylshikimic acid, caffeic acid and chlorogenic acid.

[0009] Preferably, the mass ratio of the salvia miltiorrhiza to the hawthorn is 1-2:1-5.

[0010] More preferably, the mass ratio of the Salvia miltiorrhiza and the Crataegus chinensis is 1:3.

[0011] Preferably, the myocardial protective active functional ingredients in the traditional Chinese medicine composition include tanshinone IIA, isoquercetin and tanshinone I.

[0012] In a second aspect, a medicine for treating ischemic heart disease is provided, wherein the medicine comprises the Chinese medicine composition of the present invention.

[0013] In a third aspect, the present invention provides a use of the Chinese medicine composition of the present invention in the preparation of a drug for treating ischemic heart disease.

[0014] In a fourth aspect, the present invention provides the use of the traditional Chinese medicine composition of the present invention in improving left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS).

[0015] A fifth aspect provides the use of the traditional Chinese medicine composition of the present invention in alleviating myocardial pathological damage and myocardial fibrosis.

[0016] In a sixth aspect, the present invention provides the use of the traditional Chinese medicine composition of the present invention in reducing the release of lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB).

[0017] In the present invention, MI specifically refers to acute myocardial infarction; DSHP specifically refers to the Danshen-Hawthorn combination, which is composed of Danshen (Salviae Miltiorrhizae Radix et Rhizoma) and Hawthorn (Crataegi Fructus); LDH specifically refers to lactate dehydrogenase; CK-MB specifically refers to creatine kinase isoenzyme; OPLS specifically refers to orthogonal partial least squares; and GRA specifically refers to grey relational analysis.

[0018] In addition, a preparation method of the traditional Chinese medicine composition of the present invention is also provided, wherein the preparation method comprises the following steps: preparing the traditional Chinese medicine composition from salvia miltiorrhiza and hawthorn.

[0019] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0020] (1) Innovatively established characteristic fingerprints of six batches of Danshen-Hawthorn combinations (DSHP) with different ratios. Twenty-six stable common peaks were successfully calibrated by UHPLC, and 17 characteristic chemical components were accurately identified, including active ingredients such as tanshinones and flavonoids, providing a scientific basis for the quality control of the combination.

[0021] (2) The therapeutic effect of DSHP was confirmed for the first time in a mouse model of MI induced by coronary artery ligation. After intervention with different ratios of DSHP, the cardiac function parameters (including LVEF, LVFS, etc.), myocardial enzyme spectrum (LDH, CK-MB), myocardial histopathological morphology, and myocardial fibrosis degree of mice were significantly improved compared with those of model mice. This confirmed that DSHP can effectively reduce myocardial ischemic damage and has a significant myocardial protective effect.

[0022] (3) For the first time, two chemometric methods, orthogonal partial least squares (OPLS) and grey relational analysis (GRA), were combined to establish a "spectrum-effect" correlation model of "chemical components-efficacy indexes". The optimal compatibility ratio of DSHP was determined to be salvia miltiorrhiza:crataegus pinnatifida = 1:3 (w / w), and six core active ingredients were screened out: F9, F10, F23 (tanshinone IIA), F12 (isoquercetin), F20 (tanshinone I) and F22, providing an accurate ratio basis for the clinical application of the composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The overlay of fingerprints of DSHP (N3-N8) with different compatibility ratios and their control fingerprints (R).

[0024] Figure 2 This is a comparison chart of retention times of reference substances.

[0025] Figure 3 For the identification of common peaks in the reference spectrum.

[0026] Figure 4 Electrocardiograms of mice after myocardial infarction surgery; A represents the electrocardiogram of normal mice; B represents the electrocardiogram of a mouse model with acute myocardial infarction.

[0027] Figure 5 Figure 3 M-mode ultrasound images and statistical results of the parasternal short-axis section of the left ventricle of mice in each group; A represents a representative M-mode ultrasound image in the short-axis view; B represents the left ventricular ejection fraction (%); C represents the left ventricular fractional shortening (%).

[0028] Figure 6 This is the H&E staining image of heart tissue.

[0029] Figure 7 This is a Masson staining image of heart tissue.

[0030] Figure 8 are the levels of serum myocardial injury markers in MI mice; A represents the LDH level in serum samples; B represents the CK-MB level in serum samples.

[0031] Figure 9A Biplot diagram of ingredients, pharmacodynamic indicators and samples.

[0032] Figure 9B VIP diagram of the OPLS model.

[0033] Figure 9C is the OPLS regression coefficient of each common peak and LDH.

[0034] Figure 9D is the OPLS regression coefficient of each common peak and CK-MB. DETAILED DESCRIPTION

[0035] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0036] Example 1 Experimental Example

[0037] 1. Materials

[0038] 1.1 Experimental animals and medicinal materials

[0039] C57BL / 6J mice, weighing 18.0–22.0 g and aged 6–8 weeks, were purchased from the Experimental Animal Center of the Air Force Medical University. Experiments were conducted after 48 hours of acclimatization. All animal experiments were conducted in accordance with the requirements of the Animal Ethics Committee of the Air Force Medical University (ethics approval number: IACUC-20241152). Salvia miltiorrhiza (Batch number: 20210402, from Shaanxi) and Crataegus pinnatifida (Batch number: 20201001, from Shandong) were purchased from Shaanxi Duoyao Chinese Medicine Pieces Co., Ltd.

[0040] 1.2 Experimental instruments

[0041] As shown in Table 1.

[0042] Table 1 List of experimental instruments

[0043]

[0044] 1.3 Experimental Reagents

[0045] As shown in Table 2.

[0046] Table 2 Experimental reagent list

[0047]

[0048]

[0049] 2 Methods

[0050] 2.1DSHP sample preparation and yield

[0051] Salvia miltiorrhiza and hawthorn were dried in an oven at 50°C. The dried Salvia miltiorrhiza and hawthorn were chopped and divided into the following groups: Salvia miltiorrhiza (1:0) group, hawthorn (0:1) group, DSHP (2:1) group, DSHP (1:1) group, DSHP (1:2) group, DSHP (1:3) group, DSHP (1:4) group, and DSHP (1:5) group, with a total weight of 60 g of medicinal materials in each group. Place the weighed medicinal materials in batches into a 2000mL round-bottom flask. Add 15 times the volume of 70% ethanol and soak for 30 minutes. Then, extract under reflux twice, each time for 40 minutes. Combine the two filtrates, recover the ethanol, and concentrate to a thick paste. Dry in a freeze dryer for 48 hours. Collect the dry powder, seal it, and store it in a desiccator until ready for use. Weigh accurately and calculate the dry extract yield. [Dry extract yield (%) = dry extract mass / total mass of extracted medicinal materials × 100%).

[0052] 2.2 Construction of chemical fingerprints of DSHP with different ratios

[0053] 2.2.1 Chromatographic conditions

[0054] Chromatograph: Thermo Vanquish Flex UHPLC liquid chromatograph; chromatographic column: Waters ACQUITY UPLC BEH C18 (2.1 mm × 150 mm, 1.7 μm); flow rate: 0.2 mL / min; column temperature: 40°C; injection volume: 2 μL; detection wavelength: 210 nm; mobile phase: acetonitrile (A)-0.1% phosphoric acid (B), gradient elution as shown in Table 3.

[0055] Table 3 Elution gradient

[0056]

[0057]

[0058] 2.2.2 Preparation of test solution

[0059] Accurately weigh 100 mg of each sample batch into a 10 mL volumetric flask. Two replicates were added to each sample and ultrasonically extracted (40 kHz) for 1 hour. After cooling, the volume was adjusted to the mark with 70% methanol. The solution was filtered through a 0.22 μm filter membrane, and the filtrate was collected to obtain the test solution. The details are shown in Table 4.

[0060] Table 4 Sample weight (mg)

[0061] batch number Medicinal material ratio Sample 1 (mg) Sample 2 (mg) N1 Salvia miltiorrhiza (1:0) 99.29 99.31 N2 Hawthorn (0:1) 98.38 99.02 N3 Salvia miltiorrhiza: Crataegus pinnatifida (2:1) 96.59 96.62 N4 Salvia miltiorrhiza:Hawthorn (1:1) 98.75 98.47 N5 Salvia miltiorrhiza: Crataegus pinnatifida (1:2) 100.41 100.23 N6 Salvia miltiorrhiza:Hawthorn (1:3) 99.72 99.55 N7 Salvia miltiorrhiza: Crataegus pinnatifida (1:4) 97.86 97.93 N8 Salvia miltiorrhiza:Hawthorn (1:5) 99.12 99.03

[0062] 2.2.3 Preparation of reference solution

[0063] Accurately weigh 35.56 mg of 5-hydroxymethylfurfural, 5.80 mg of danshensu, 5.45 mg of chlorogenic acid, 26.98 mg of procyanidin B, and 6.88 mg of epicatechin into a 50 mL volumetric flask, dissolve in 10% methanol, and dose to volume. Accurately weigh 4.23 mg of isoquercetin, 5.35 mg of hyperoside, 5.71 mg of dihydrotanshinone I, 11.01 mg of tanshinone I, 6.17 mg of cryptotanshinone, and 5.80 mg of tanshinone IIA into a 50 mL volumetric flask; 6.53 mg of oleanolic acid, 6.68 mg of ursolic acid, 5.54 mg of rosmarinic acid, 5.40 mg of lithospermic acid, and 4.16 mg of salvianolic acid A into a 25 mL volumetric flask; and 5.82 mg of salvianolic acid B into a 10 mL volumetric flask, dissolve in methanol, and dose to volume. Then dilute salvianolic acid B to 10 times, and dilute the other reference substances to 20 times, with the concentrations shown in Table 5.

[0064] Table 5 Concentration of mixed reference substances (μg / mL)

[0065]

[0066]

[0067] 2.2.4 Methodological Investigation

[0068] Precision test: Prepare a test solution of DSHP sample N6 according to the method in "2.2.2." Repeat the injection six times using the chromatographic conditions in "2.2.1." Using salvianolic acid B (peak 16, S), which has the highest content and best resolution, as the reference peak, calculate the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks. The results showed that the RSDs were all less than 3%, indicating good precision of the instrument.

[0069] Stability test: Prepare a test solution of DSHP sample N6 according to the method in "2.2.2". After standing at room temperature for 0, 1, 2, 4, 8, 12, and 24 hours, sample injection and analysis were performed according to the chromatographic conditions in "2.2.1". Using salvianolic acid B (peak 16, S), which has a large response and good resolution, as the reference peak, calculate the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks. The results showed that the RSDs were all less than 3%, indicating that the prepared test solution was stable within 24 hours.

[0070] Repeatability test: Prepare six test solutions of DSHP sample N6 according to the method in "2.2.2." Each solution was injected and analyzed using the chromatographic conditions in "2.2.1." Using salvianolic acid B (peak 16, S), which has a high response and good resolution, as the reference peak, the relative standard deviations (RSDs) of the relative retention times and relative peak areas of the common peaks were calculated. The results showed that the RSDs were all less than 3%, indicating that the selected chromatographic method had good repeatability.

[0071] 2.2.5 Establishment of chemical fingerprint

[0072] Chromatograms of eight DSHP samples with different ratios were obtained by injection and measurement according to the test solution preparation method and chromatographic conditions. The chromatograms were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 edition) software for analysis. The reference chromatogram was generated using the median method, with N1 as the reference chromatogram, a time window width of 0.2 minutes, and full peak matching. Superimposed fingerprints of the eight DSHP samples (N1-N8) were generated, and the similarity of samples with different DSHP compatibility ratios N3-N8 was calculated.

[0073] 2.3 Myocardial protective effects of different ratios of DSHP on MI mice

[0074] 2.3.1 Experimental groups and dosage

[0075] One hundred and twenty C57 BL / 6J mice were randomly divided into 10 groups: sham group (MI), MI group, MI + Danshen-Hawthorn 1:0 group (MI+DSHP-1:0), MI + Danshen-Hawthorn 0:1 group (MI+DSHP-0:1), MI + Danshen-Hawthorn 1:1 group (MI+DSHP-1:1), MI + Danshen-Hawthorn 2:1 group (MI+DSHP-2:1), MI + Danshen-Hawthorn 1:2 group (MI+DSHP-1:2), MI + Danshen-Hawthorn 1:3 group (MI+DSHP-1:3), MI + Danshen-Hawthorn 1:4 group (MI+DSHP-1:4), and MI + Danshen-Hawthorn 1:5 group (MI+DSHP-1:5). Except for the sham group, MI models were established in mice by ligating the left anterior descending coronary artery. In the sham group, mice underwent the same surgical procedure without coronary artery ligation. The drug was administered by gavage on the day of modeling for four consecutive weeks.

[0076] It is recorded in the prior art that the commonly used dosage of Salvia miltiorrhiza and Crataegus pinnatifida is 15-30 g of Salvia miltiorrhiza and 15-30 g of Crataegus pinnatifida; calculated based on the commonly used dosage of 30 g of medicinal materials, the raw drug dosage of DSHP extracts with different ratios is 60 g. After conversion according to the equivalent dose ratio table (Table 6) calculated based on the body surface area of ​​humans and animals (the conversion coefficient for humans and mice is 9.1), the raw drug dosage of different DSHP extracts for mice is determined to be 7.80 g / kg. The specific calculation is: 60 g / 70 kg × 9.1 ≈ 7.80 g / kg.

[0077] Table 6 Equivalent dose ratios based on body surface area for humans and animals

[0078]

[0079] 2.3.2 Construction of MI Mouse Model

[0080] An MI mouse model was established by ligating the left anterior descending coronary artery. Mice were anesthetized with 1% isoflurane, and 80 mg / kg ketamine and 7 mg / kg xylazine were administered intraperitoneally. The mice were intubated with a 16-gauge intravenous catheter for mechanical ventilation. A left thoracotomy was performed to expose the heart, and the left anterior descending coronary artery was ligated with 6-0 silk. Limb lead II electrocardiograms were performed using a BL-420 biofunctional experimental system, and ST segment changes were observed and recorded. The acute myocardial ischemia model was considered successful if abnormal ST segment elevation was observed on the lead II electrocardiogram compared with the control group. The chest cavity was closed, and the mice were placed on a heating plate until they recovered. Postoperative anti-infection treatment was administered for 7 days (penicillin 200,000 units / kg intraperitoneally).

[0081] 2.3.3 Echocardiography

[0082] Four weeks after administration, the mice were depilated of their thoracic hair and anesthetized with a small animal anesthesia machine using a mixture of isoflurane and oxygen. A small amount of ultrasound coupling agent was applied, and M-mode images of the left ventricular short-axis and long-axis sections were acquired using a Vevo3100 LT ultra-high-resolution small animal ultrasound imaging system. All images were saved in the original format (DICOM). Ultrasound data were acquired using VevoLAB 3.0, the accompanying software from FUJIFILM VisualSonics, to calculate LVEF and LVFS and perform statistical analysis.

[0083] 2.3.4 HE staining

[0084] Cardiac tissue was fixed with 4% paraformaldehyde and then embedded in paraffin. 4 μm serial sections were prepared. Pretreatment was performed using a gradient dewaxing and hydration system (xylene I / II for 20 min each, followed by ethanol to 70% alcohol for 5 min each). Hematoxylin staining parameters were as follows: Harris staining for 5 min, 1% hydrochloric acid-ethanol differentiation for 10 s, and 0.6% ammonia bluing. Eosin counterstaining was controlled for 2 min. Subsequently, the sections were dehydrated using a reversed gradient alcohol system (70% to anhydrous ethanol for 5 min each), cleared with xylene, and mounted with neutral resin. Myocardial tissue images were acquired using a digital pathology scanning system (×200), focusing on the morphological changes of cardiomyocytes and inflammatory infiltration.

[0085] 2.3.5 Masson staining

[0086] After standard dewaxing and hydration, paraffin sections were stained with a multi-color staining system for collagen-specific labeling: nuclear staining with Weigert iron hematoxylin for 5 minutes, staining with acid fuchsin-ponceau complex for 8 minutes, mordanting with phosphomolybdic acid solution for 3 minutes, and extracellular matrix-specific staining with aniline blue for 5 minutes. Key quality control steps included color separation with 1% glacial acetic acid for 1 minute to remove nonspecific staining and dehydration with graded alcohols to ensure staining stability. Myocardial fibrosis area percentage was quantified using an automated image analysis system, and the collagen volume fraction (CVF) was calculated as an indicator of fibrosis severity.

[0087] 2.3.6 Serum biochemical index detection

[0088] Four weeks after administration, blood samples were collected from the abdominal aorta of each group. The samples were centrifuged at room temperature for 60 minutes, followed by 10 minutes at 3500 rpm. The supernatant was collected and stored at -80°C for later use. Serum levels of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB), markers of myocardial injury, were measured using an automated biochemical analyzer.

[0089] 2.3.7 Analysis of spectrum-effect relationship of DSHP myocardial protection in different compatibility ratios

[0090] Spectrum-effect relationship analysis was performed using SIMCA 14.1 software and SPSS PRO online software (https: / / www.spsspro.com / ). Serum levels of myocardial injury marker enzymes in mice treated with different DSHP ratios were correlated with the peak areas of the common peaks in the corresponding samples. Spectrum-effect relationship analysis was performed using orthogonal partial least squares (OPLS) and grey relation analysis (GRA).

[0091] 2.3.8 Statistical analysis

[0092] All data are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 10.0, and differences between groups were compared using one-way analysis of variance followed by Tukey's test. All experiments were randomized and blinded and repeated at least three times to confirm their reproducibility. Statistical significance was defined as P < 0.05.

[0093] Example 2 Results

[0094] 3 Results

[0095] 3.1 Dry extract extraction rate of DSHP extracts with different ratios

[0096] As shown in Table 7, the dry extract extraction yields of the combined groups were generally higher than those of the single-herb groups (N1: 35.08%; N2: 23.3%), indicating a synergistic effect between the combination of Danshen and Crataegus pinnatifida. The 2:1 ratio (N4) achieved the highest extraction yield (51.22%), followed by the 1:3 ratio (N6) (50.33%), with extraction efficiencies exceeding 50%. The 1:3 ratio (N6) achieved a high extraction yield of 50.33% with a lower Danshen dosage (15g), conserving Danshen resources more than the 2:1 ratio (N4), suggesting potential advantages for industrial production.

[0097] Table 7 Dry extract extraction rate of DSHP extracts with different ratios

[0098] serial number Combination ratio Weight of medicinal materials Freeze-dried powder weight Dry extract extraction rate N1 Salvia miltiorrhiza (1:0) Salvia miltiorrhiza 60g 21.05 35.08％ N2 Hawthorn (0:1) 60g hawthorn 14.00 23.3％ N3 Salvia miltiorrhiza:Hawthorn (1:1) 30g of Salvia miltiorrhiza and 30g of Hawthorn 23.66 39.43％ N4 Salvia miltiorrhiza: Crataegus pinnatifida (2:1) 40g of Salvia miltiorrhiza and 20g of Hawthorn 30.73 51.22％ N5 Salvia miltiorrhiza: Crataegus pinnatifida (1:2) 20g of Salvia miltiorrhiza and 40g of Hawthorn 19.05 31.75％ N6 Salvia miltiorrhiza:Hawthorn (1:3) Salvia miltiorrhiza 15g, Hawthorn 45g 30.20 50.33％ N7 Salvia miltiorrhiza: Crataegus pinnatifida (1:4) Salvia miltiorrhiza 12g, Hawthorn 48g 18.68 31.13％ N8 Salvia miltiorrhiza:Hawthorn (1:5) Salvia miltiorrhiza 10g, Hawthorn 50g 20.19 33.65％

[0099] 3.2 Establishment of feature maps and similarity evaluation

[0100] Take DSHP extracts of different proportions from N3 to N8, prepare the test solution in sequence according to the method in item "2.2.2", and inject the sample for determination according to the chromatographic conditions in item "2.2.1". The determination results are exported in CDF format and imported into the "Similarity Evaluation System of Chromatographic Fingerprints of Traditional Chinese Medicine" (2012 edition) software in sequence to obtain the superimposed spectrum of 6 batches of samples. The median method is used with a time width of 0.2min and N4 as the reference spectrum. After full spectrum matching, a total of 26 common peaks are determined to generate a reference spectrum (R), see Figure 1 .

[0101] Taking the control fingerprint (R) as a reference, the similarity of the fingerprints of the six batches of DSHP extracts with different compatibility ratios is shown in Table 8. As can be seen from Table 8, the similarity of the fingerprints of the different DSHP ratios is greater than 0.99, which meets the requirements of the fingerprint, indicating that their chemical composition is similar, the homogeneity is good, and the quality is stable.

[0102] Table 8 Similarity results

[0103] / N3 N4 N5 N6 N7 N8 R N3 1 1 0.999 0.999 0.992 0.997 0.999 N4 1 1 0.999 0.999 0.992 0.997 0.999 N5 0.999 0.999 1 1 0.997 0.999 1 N6 0.999 0.999 1 1 0.996 0.999 1 N7 0.992 0.992 0.997 0.996 1 0.999 0.996 N8 0.997 0.997 0.999 0.999 0.999 1 0.999 R 0.999 0.999 1 1 0.996 0.999 1

[0104] 3.3 Common peak identification

[0105] By comparing the retention time of the reference spectrum with that of the reference substances in Table 5, 17 common peaks in the reference spectrum can be identified, namely 5-hydroxymethylfurfural (F1), tanshinone (F2), chlorogenic acid (F4), proanthocyanidin B2 (F6), epicatechin (F7), hyperoside (F11), isoquercetin (F12), rosmarinic acid (F14), lithospermic acid (F15), salvianolic acid B (F16, S), salvianolic acid A (F18), dihydrotanshinone I (F19), tanshinone I (F20), cryptotanshinone (F21), tanshinone IIA (F23), oleanolic acid (F25), and ursolic acid (F26). See Table 5 for details. Figure 2 and Figure 3 .

[0106] 3.4 Successful establishment of MI mouse model

[0107] The present invention establishes an MI mouse model by ligating the left anterior descending coronary artery, and uses a high-precision electrocardiogram (ECG) instrument to monitor ECG changes during the operation. Figure 4 As shown in A, the electrocardiogram of mice in the sham group was normal, while the electrocardiogram of mice in the MI group showed ST segment elevation ( Figure 4 Middle B) shows that the acute myocardial infarction mouse model was successfully established.

[0108] 3.5 DSHP in different ratios can improve cardiac function in MI mice to varying degrees

[0109] After modeling, the general condition of the mice deteriorated, including poor appetite, poor mental state, lethargy, decreased activity intensity, and gradual death of animals. After 14 days, the condition of the mice stabilized. As shown in Table 9, after 28 days, only 4 animals remained in the MI group. The number of surviving animals in each drug-treated group was higher than that in the MI group, and all animals in the 1:3 group survived. The overall condition of the animals was good. Echocardiography was used to observe the effect of DSHP on the cardiac function of MI mice. Figure 5 As shown (when compared with the Sham group, * P<0.05, **** P < 0.0001; when compared with MI, # P<0.05, ## P<0.01, #### P < 0.0001). Figure 5 It can be seen that compared with the sham group, the LVEF and LVFS of the MI group rats were significantly reduced (P<0.0001, P<0.05). In contrast, different DSHP ratios can effectively improve LVEF and LVFS values, among which the DSHP-1:3 group was the most significant (P<0.0001, P<0.05), suggesting that the DSHP-1:3 group can significantly improve cardiac function after myocardial infarction.

[0110] Table 9 Record of mouse survival status (number)

[0111]

[0112] 3.6 DSHP in different ratios can alleviate myocardial pathological morphology in MI mice to varying degrees

[0113] HE staining ( Figure 6 ) showed that the myocardial tissue structure of mice in the sham group was intact and clear, with no inflammatory cell infiltration. However, the heart tissue of mice in the MI group showed irregular arrangement of cardiomyocytes, nuclear fragmentation, accompanied by edema and even necrosis, proliferation of fibrous connective tissue, and infiltration of inflammatory factors. Compared with the MI group, different DSHP-administered groups improved myocardial tissue lesions caused by MI to varying degrees and delayed the pathological progression of MI. The DSHP-1:3 group had the most significant effect in improving myocardial damage.

[0114] 3.7 DSHP in various proportions can alleviate myocardial fibrosis in MI mice to varying degrees

[0115] In Masson staining, muscle fibers appear red and collagen fibers appear blue ( Figure 7In the sham group, the myocardial cells were arranged in an orderly pattern, and no significant collagen fiber proliferation was observed in the myocardial interstitium. However, in the MI group, myocardial tissue showed a large amount of irregularly shaped, disordered, and prominent fibrosis. Compared with the MI group, the different DSHP-administered groups improved MI-induced myocardial fibrosis to varying degrees, with the DSHP-1:3 group showing the most significant effect.

[0116] 3.8 DSHP in different ratios can reduce the levels of serum myocardial injury markers in MI mice to varying degrees

[0117] like Figure 8 As shown (when compared with the Sham group, *** P<0.001, **** P < 0.0001; when compared with MI, # P<0.05, ### P<0.001, #### Compared with the Sham group, the serum LDH and CK-MB levels of the MI group mice were increased (P<0.001, P<0.0001); compared with the MI group, the DSHP-1:3 group could significantly reduce the serum LDH level ( Figure 8 A, P < 0.05), while the DSHP treatment groups of each ratio could reduce the serum CK-MB level to varying degrees, and the differences were statistically significant ( Figure 8 (B, P < 0.001).

[0118] 3.9 Spectrum-effect relationship analysis

[0119] 3.9.1 Orthogonal Partial Least Squares Method

[0120] OPLS is a regression modeling method for multiple dependent variables to multiple independent variables. It can remove data variations in the independent variable X that are irrelevant to the categorical variable Y, so that the categorical information is mainly concentrated in one principal component. The model becomes simple and easy to interpret. It is an effective method to construct the spectrum-effect relationship of traditional Chinese medicine. Using SIMCA14.1 software, the peak area of ​​the common peak of 8 batches of DSHP with different proportions was standardized. The results were used as independent variables (X), and the mouse myocardial enzyme spectrum LDH and CK-MB were used as dependent variables (Y) for OPLS analysis (see Table 10). In general, independent variables with a variable projection importance (VIP) value greater than 0.5 are considered to be meaningful in explaining the dependent variable, and independent variables with a VIP value greater than 1 are considered to have greater significance in explaining the dependent variable. At the same time, when the standardized regression coefficient is negative, it means that the component content is negatively correlated with the secretion of myocardial injury markers, and vice versa. Figures 9A-9D As shown. Figure 9AIt can be seen that sample N6 (Danshen: Crataegus 1:3) has the best effect in terms of component content and inhibition of mouse myocardial enzymes LDH and CK-MB. Figure 9B It can be seen that there are 9 peaks with VIP values ​​greater than 1.0 in DSHP, namely F9, F10, F23 (tanshinone IIA), F6 (proanthocyanidin B2), F12 (isoquercetin), F8, F20 (tanshinone I), F22 and F7 (epicatechin). Among them, the standardized regression coefficients of F9, F10, F23 (tanshinone IIA), F20 (tanshinone I) and F22 are negative, which are negatively correlated with LDH secretion ( Figure 9C The standardized regression coefficients of F9, F10, F23 (tanshinone IIA), F20 (tanshinone I), F22, and F7 (epicatechin) were negative, and were negatively correlated with CK-MB secretion ( Figure 9D In conclusion, F9, F10, F23 (tanshinone IIA), F20 (tanshinone I), F22, and F7 (epicatechin) may be the active components of DSHP that exert myocardial protection, and the 1:3 group has the best overall effect.

[0121] Table 10. Original measurement data of common peak areas and efficacy indices in spectrum-effect analysis

[0122]

[0123]

[0124] 3.9.2 Grey correlation analysis

[0125] SPSSPRO online software (https: / / www.spsspro.com / ) was used. The anti-myocardial injury efficacy of Danshen-Hawthorn combination in different proportions, LDH and CK-MB (efficacy), were used as parent sequences, and the common peak area (spectrum) of the combination was used as subsequence. The data were normalized and the "spectrum-efficacy" correlation was calculated. When the common peak area correlation was >0.6, the common peak was correlated with the efficacy; when the correlation was >0.8, the two were highly correlated. The results are shown in Table 11. As can be seen from Table 11, the top 10 contributions of the common peaks to the indicator LDH are: F12 (isoquercetin) > F9 > F10 > F23 (tanshinone IIA) > F1 (5-hydroxymethylfurfural) > F4 (chlorogenic acid) > F22 > F20 (tanshinone I) > F21 (cryptotanshinone) > F11 (hyperoside); the top 10 contributions of the common peaks to the indicator CK-MB are: F12 (isoquercetin) > F9 > F10 > F1 (5-hydroxymethylfurfural) > F23 (tanshinone IIA) > F20 (tanshinone I) > F22 > F21 (cryptotanshinone) > F4 (chlorogenic acid) > F16 (salvianolic acid B). In summary, the top 10 common peaks had a high correlation with the efficacy indicators, and the higher the correlation, the stronger the correlation, indicating that multiple components in DSHP synergistically exerted anti-myocardial injury effects.

[0126] Table 11 Correlation between DSHP anti-myocardial injury activity and common peak area

[0127]

[0128]

[0129] Because each statistical method has its own limitations and adaptability, no single mathematical model can fully reflect the relationship between chemical fingerprints and pharmacological effects. Therefore, we integrated the results of the two mathematical statistical methods, OPLS and GRA, and took their intersection to ensure the completeness and accuracy of the experimental results. Ultimately, we identified six major active ingredients in DSHP for the treatment of ischemic heart disease: F9, F10, F23, F12, F20, and F22. Among them, F23 (tanshinone IIA), F12 (isoquercetin), and F20 (tanshinone I) have been identified.

[0130] As described above, the basic principles, main features and advantages of the present invention are well described. The above embodiments and descriptions are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the scope of protection determined by the present invention.

[0131] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but rather represents selected embodiments of the present invention. All other embodiments obtained without creative effort and through deduction and substitution by a person of ordinary skill in the art based on the concepts of the present invention are within the scope of protection of the present invention.

Claims

1. A Chinese medicine composition for treating ischemic heart disease, characterized in that: The traditional Chinese medicine composition is prepared by combining salvia miltiorrhiza and hawthorn. The active functional components of salvia miltiorrhiza for protecting the myocardium include water-soluble phenolic acids and fat-soluble tanshinones, and the active functional components of hawthorn for protecting the myocardium include proanthocyanidins, flavonoids and triterpenoid acids.

2. The Chinese medicine composition according to claim 1, characterized in that The water-soluble phenolic acids include rosmarinic acid, lithospermic acid, danshensu, salvianolic acid A, salvianolic acid B and protocatechuic aldehyde; the fat-soluble tanshinones include dihydrotanshinone I, tanshinone I, tanshinone IIA, tanshinone IIB, cryptotanshinone and dihydrotanshinone; the proanthocyanidins include proanthocyanidin B2; the flavonoids include vitexin, rutin, quercetin, naringin, hyperoside, isoquercetin and epicatechin; the triterpene acids include citric acid, ursolic acid, malic acid, gallic acid, fumaric acid, caffeoylshikimic acid, caffeic acid and chlorogenic acid.

3. The Chinese medicine composition according to claim 1, characterized in that The mass ratio of the salvia miltiorrhiza and the hawthorn is 1-2:1-5.

4. The Chinese medicine composition according to claim 3, characterized in that The mass ratio of the salvia miltiorrhiza and the hawthorn is 1:

3.

5. The Chinese medicine composition according to claim 1, characterized in that The myocardial protective active functional components in the traditional Chinese medicine composition include tanshinone IIA, isoquercetin and tanshinone I.

6. The method for preparing the Chinese medicine composition according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: preparing the drug from salvia miltiorrhiza and hawthorn.

7. Use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in the preparation of a medicament for treating ischemic heart disease.

8. Use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in improving left ventricular ejection fraction and left ventricular fractional shortening.

9. Use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in alleviating myocardial pathological damage and myocardial fibrosis.

10. Use of the traditional Chinese medicine composition according to any one of claims 1 to 5 in reducing the release of lactate dehydrogenase and creatine kinase isoenzymes.

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