A compound in salvia miltiorrhiza and its pharmaceutical composition and anti-heart failure application

Abstract

The application belongs to the field of medicine, and relates to a kind of salvia miltiorrhiza extract and pharmaceutical composition and application thereof, specifically to a kind of novel structure compound in salvia miltiorrhiza and preparation method thereof and application of the compound in preparation of medicine for preventing and treating heart failure disease, especially application in medicine for preventing and treating heart failure caused by myocardial fibrosis. Pharmacological test proves that the compound has obvious protective effect on H9c2 myocardial cell hypoxia injury, and has obvious inhibitory effect on proliferation, migration and activation of myocardial fibroblast AC16 caused by isopropyl epinephrine, and can be used for preparing medicine for preventing and treating heart failure disease.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to the application of a class of compounds isolated from the traditional Chinese medicine Danshen and their pharmaceutical compositions in the prevention and treatment of heart failure. Background Technology

[0002] Heart failure (HF) has long been a serious cardiovascular disease affecting human health, ranking alongside cerebrovascular disease and cancer as one of the three major killers. With the advent of an aging society, the incidence and mortality rates of HF are showing a year-on-year upward trend. The latest clinical statistics show that there are approximately 13.7 million HF patients in China, placing a heavy economic burden on society. In recent years, although there have been some breakthroughs in the treatment of HF, its prognosis remains poor, with a five-year mortality rate still as high as 60-80%. This necessitates in-depth research into the molecular pathogenesis of HF to find more effective therapeutic targets and develop safer and more effective anti-heart failure drugs.

[0003] By reducing myocardial oxygen consumption and energy expenditure, current chemotherapy can rapidly improve hemodynamic parameters and alleviate heart failure symptoms in patients, improving survival rates within the first 2-3 years. However, frequent complications such as electrolyte imbalances, hypotension, and bradycardia, along with poor prognosis and lower quality of life, place a significant burden on patients and their families. Heart failure patients in developing countries like China tend to be younger than those in developed countries. Therefore, future research on anti-heart failure drugs should focus more on comprehensive treatment, striving to reverse abnormal myocardial remodeling and restore energy supply and demand balance to achieve the therapeutic goal of reducing mortality and improving quality of life.

[0004] Abnormal tissue remodeling is a major cause and pathological basis of organ failure, and fibrosis, as an important pathological manifestation of tissue remodeling, is a significant cause of disability and death in many diseases. Epidemiological surveys show that nearly 45% of deaths in heart failure patients can be attributed to fibrotic diseases, clinically manifested as increased ventricular diameter and altered ventricular geometry. Injury stimuli, including ischemia, pressure overload, and cardiotoxic substances, can induce apoptosis and necrosis of cardiomyocytes, leading to myocardial cell loss. The remaining myocardium thickens to compensate for insufficient cardiac function, while surrounding connective tissue proliferates to fill the gaps left by the loss of myocardial cells, resulting in changes in the morphology and structure of the heart—this process is myocardial remodeling. Over time, the hypertrophied myocardium decompensates, resulting in a large loss of myocardial cells and an increase in reactive extracellular matrix, leading to significant myocardial fibrosis. The latter increases ventricular wall stiffness, significantly affecting cardiac systolic and diastolic function, ultimately leading to chronic heart failure.

[0005] The molecular mechanisms involved in the development of myocardial fibrosis include inflammation, mitochondrial damage, cardiomyocyte apoptosis, collagen deposition, myocardial fibroblast proliferation, and the transformation of fibroblasts into myofibroblast phenotypes. Among these, myocardial fibroblasts, as the core effector cells in myocardial fibrosis, play a crucial role in the progression of myocardial fibrosis through proliferation, migration, and activation.

[0006] Traditional drug treatments primarily target hemodynamic changes. "Inotropic agents, diuretics, and vasodilators" have long been the standard and classic treatments for heart failure. Numerous clinical studies have shown that while these drugs can improve hemodynamics and alleviate symptoms in the short term, long-term use does not reduce the mortality rate of heart failure and may even increase it. Furthermore, inotropic drugs (such as digoxin) and diuretics (such as hydrochlorothiazide) commonly used to treat heart failure caused by myocardial infarction are generally not considered to have anti-myocardial fibrosis effects.

[0007] Danshen (Salvia miltiorrhiza Bunge), a perennial herb belonging to the genus Salvia of the family Lamiaceae, is the dried root and rhizome of the plant. It is distributed throughout most parts of my country. With a long history of medicinal use, it is primarily used for symptoms such as blood stasis causing chest pain, abdominal pain, irregular menstruation, amenorrhea, dysmenorrhea, postpartum abdominal pain due to blood stasis, carbuncles and boils, palpitations, and insomnia. Modern research has found that Danshen mainly contains two types of components: tanshinones and salvianolic acids, both of which exhibit significant activity. Tanshinones primarily possess antitumor, antibacterial, and cardiovascular activities, while salvianolic acids mainly exhibit cardiovascular and cerebrovascular and antioxidant activities. Furthermore, Danshen has also shown good activity in pharmacological studies related to anti-inflammatory, anti-ulcer, neuroprotective, and hepatoprotective effects. Summary of the Invention

[0008] The applicant's research revealed the isolation of a novel class of compounds—Dilithospermic acid A–B—from Salvia miltiorrhiza, with the following chemical structures:

[0009]

[0010] Pharmacological experiments have demonstrated that compound 1 (dilithospermic acid A) can effectively alleviate hypoxic damage to H9c2 cardiomyocytes while significantly inhibiting isoproterenol (ISO)-induced proliferation, migration, and activation of AC16 cardiomyocytes. It possesses good potential for the prevention and treatment of heart failure, particularly remodeling heart failure characterized by myocardial fibrosis, with an effective dose reaching 0.01 μmol / L. Currently, there are no research reports, preparation methods, or patent literature regarding the use of this type of compound in the treatment of heart failure and myocardial fibrosis.

[0011] The technical problem solved by this invention is to provide a method for preparing compounds isolated and extracted from the traditional Chinese medicine Danshen and their application in the preparation of drugs for treating heart failure.

[0012] To solve the technical problem of this invention, the present invention provides the following technical solution:

[0013] The first aspect of the present invention is to provide a class of compounds or pharmaceutically acceptable salts thereof, characterized in that the structure of the class of compounds is as follows:

[0014]

[0015] The pharmaceutically acceptable salts mentioned above are selected from salts formed by compounds and inorganic or organic bases. The organic bases include methylamine, ethylamine, diethylamine, triethylamine, propylamine, butylamine, octylamine, hexamethylenediamine, ethylenediamine, propylenediamine, butylamine, benzylamine, phenethylamine, o-phenylenediamine, and p-phenylenediamine. The inorganic bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, copper hydroxide, iron hydroxide, ammonium hydroxide, sodium bicarbonate, and potassium bicarbonate.

[0016] Compounds 1 and 2 were isolated and extracted from Salvia miltiorrhiza. The dilithospermicacid A described in this invention is compound 1, and the dilithospermic acid B is compound 2. Both compounds have novel basic skeletal structures, and no literature or patents have reported compounds of this type.

[0017] The second aspect of the technical solution of the present invention is to provide a method for preparing the compounds described in the first aspect. The preparation method is as follows: Salvia miltiorrhiza is extracted by reflux with 80% ethanol. After concentration, the extract is purified by organic solvent extraction, silica gel column chromatography, reversed-phase silica gel column chromatography, and preparative HPLC to obtain the above-mentioned compounds dilithospermic acid A to B. The structure of the compounds is identified by spectroscopic methods such as UV, IR, NMR, MS, and CD. They are identified as a class of decarboxylated coupling dimers of shikonin. No such substances have been reported to date.

[0018] A third aspect of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient. The dosage form of the pharmaceutical composition includes tablets, capsules, pills, granules, oral liquids, or suspensions.

[0019] The fourth aspect of this invention is the use of the compound described in the first aspect in the preparation of a medicament for the prevention and / or treatment of heart failure. Heart failure is characterized primarily by myocardial ischemia-hypoxia injury, abnormal myocardial tissue remodeling, and myocardial fibrosis.

[0020] Beneficial technical effects:

[0021] 1. The novel tanshinone compounds of this invention have significant anti-heart failure effects, especially anti-heart failure with significant myocardial fibrosis. Firstly, they have a significant protective effect against hypoxic damage to H9c2 cardiomyocytes; secondly, they have a significant inhibitory effect on the proliferation, migration and activation of AC16 in cardiomyocytes induced by ISO; and thirdly, they have relatively clear target sites.

[0022] 2. The novel structure of the new Danshen compound of this invention is novel and has not been reported in the literature, and has the potential to be further developed into a drug for the prevention and treatment of heart failure. Attached Figure Description

[0023] Figure 1 Venn diagram results of the intersection of disease-gene databases (Genecard and DisGeNET) and compound target prediction databases (Targetnet and Swiss TargetPrediction).

[0024] Figure 2 To detect MMP9 expression in AC16 cells treated with isoproterenol and compound 1 (dilithospermic acid A) by immunoblotting. Representative images and statistical results are shown from left to right for the normal group, isoproterenol model group, and compound 1 treatment group (0.01 μM, 0.1 μM, 1 μM, 5 μM).

[0025] Figure 3 The Transwell migration assay was used to detect the migration of AC16 cells treated with isoproterenol and compound 1 (dilithospermicacid A). From left to right, the images show representative figures and statistical results of the normal control, isoproterenol model group, and compound 1 treatment group (0.1 μM).

[0026] Figure 4 To detect the activation of AC16 cells treated with isoproterenol and compound 1 (dilithospermic acid A) using laser confocal scanning microscopy, representative images and statistical results are shown from left to right for the normal group, the isoproterenol model group, and the compound 1 treatment group (0.1 μM). Detailed Implementation

[0027] The following examples and pharmacological activity experiments are used to further illustrate the present invention, but they do not imply any limitation on the present invention.

[0028] The anhydrous solvent was prepared by removing water from commercially available analytical grade reagents using the Pure Solv. solvent purification system, and all other reagents were commercially available analytical grade.

[0029] Unless otherwise specified, all compounds used in the experiments were purchased from Sigma.

[0030] The PBS mentioned in the examples refers to phosphate buffer solution with a concentration of 0.1M and a pH of 7.2.

[0031] The room temperature described in the embodiments is the conventional room temperature in the art, preferably 15-30°C.

[0032] Experimental results are expressed as mean ± standard error. After parametric or nonparametric variance tests, a p-value < 0.05 was considered statistically significant, and a p-value < 0.01 was considered extremely statistically significant. Unless otherwise specified, # represents the comparison between the model group and the normal group, and * represents the comparison between the model + compound treatment group and the model group.

[0033] Example 1: Preparation and identification of monomeric compounds dilithospermic acid A-B (compounds 1-2) from Danshen.

[0034] 70 kg of Salvia miltiorrhiza was extracted three times by reflux with 80% ethanol for 2 hours each time. After the extract was concentrated, it was extracted three times with ethyl acetate. The solvent was recovered from the ethyl acetate fraction to obtain the extract. The extract was further subjected to silica gel column chromatography and eluted with a petroleum ether-acetone solvent system. The elution ratios were: 100:0, 80:1, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 3:1, 2:1, 1:1, 0:100. Finally, pure methanol was used as the elution. After concentration of all eluted components under reduced pressure, a total of 13 fractions (Fr.1-Fr.13) were obtained. Fra.11 was selected for preparative high performance liquid chromatography purification. The liquid chromatography conditions were: (1) Column: YMC-Triart C18 (250mm×20mm, 5μm); (2) Mobile phase: 0.2% acetic acid aqueous solution / methanol (60 / 40); (3) Flow rate: 5.0mL / min; (4) Column temperature: 25℃; (5) Detection wavelength: 320nm. The above compounds dilithospermic acid A and dilithospermic acid B were obtained. Their structures were identified by UV, IR, NMR, MS and CD spectroscopic methods. They are a class of novel shikonin decarboxylation coupling dimers.

[0035] The spectral information and NMR signal assignments of the above-mentioned new compounds are as follows:

[0036] Dilithospermic acid A (Compound 1): White amorphous powder; (c0.02, MeOH); UV (MeOH) λ max (logε) 254 (2.37), 291 (2.34), 310 (2.36) nm; IR ν max 3339, 2957, 2921, 1699, 1610, 1509, 1440, 1261, 1116, 1041, 976, 805 cm -1 ; CD (MeOH) 235 (Δε 39.67), 256 (Δε -6.51), 268 (Δε -1.12), 295 (Δε -16.87), 329 (Δε 25.44) nm; 1 1H NMR (500 MHz, Acetone-d6) δ H : 7.38 (1H, d, J = 16.0 Hz, H-7 / 7′), 7.25 (1H, d, J = 8.5 Hz, H-6 / 6′), 6.93 (1H, d, J = 8.5 Hz, H-5 / 5′), 6.74 (1H, d, 2.0 Hz, H-13 / 13′), 6.66 (1H, d, J = 8.0 Hz, H-25 / 25′), 6.59 (1H, d, J = 8.0 Hz, H-16 / 16′), 6.52 (1H, s, H-22 / 22′), 6.49 (1H, dd, J = 8.0, 2.0 Hz, H-17 / 17′), 6.26 (1H, d, J = 8.5 Hz, H-26 / 26'), 6.22 (1H, d, J = 16.0 Hz, H-8 / 8'), 5.47 (1H, s, H-20 / 20'), 5.10 (1H, dd, J = 7.5, 5.5 Hz, H-10 / 10′), 4.07 (1H, s, H-19 / 19′), 3.60 (3H, s, H-18 / 18′-OCH3), 3.0 (2H, m, H-11 / 11′); 13 13C NMR (125 MHz, Acetone-d6) δ C: 170.8 (C-18 / 18′), 166.2 (C-9 / 9′), 148.6 (C-3 / 3′), 145.8 (C-24 / 24′), 145.7 (C-23 / 23′), 145.6 (C-14 / 14′), 144.7 (C-15 / 15′), 144.4 (C-4 / 4′), 141.6 (C-7 / 7′), 133.9 (21 / 21′), 129.1 (C-2 / 2′), 128.7 (C-12 / 12′), 124.4 (C-1 / 1′), 121.6 (C-17 / 17′), 121.2 (C-6 / 6′), 118.5 (C-5 / 5′), 117.3 (C-26 / 26′), 117.2 (C-13 / 13′), 116.4 (C-8 / 8′), 116.1 (C-25 / 25′), 115.9 (C-16 / 16′), 112.9 (C-22 / 22′), 87.2 (C-20 / 20′), 73.9 (C-10 / 10′), 55.3 (C-19 / 19′), 52.2 (C-18 / 18′-OCH3), 37.2 (C-11 / 11′); HR-ESI-MS m / z 1015.2648 [M+H] + (calcd for C 54 H 47 O 32 , 1015.2655).

[0037] Dilithospermic acid B (Compound 2): White amorphous powder; (c0.02, MeOH); UV (MeOH) λ max (logε) 254 (2.64), 290 (2.61), 313 (2.62) nm; IR ν max 3375, 2923, 2851, 1686, 1611, 1509, 1443, 1262, 1116, 1180, 977, 806 cm -1 ; CD (MeOH) 235 (Δε -35.41), 256 (Δε 0.97), 267 (Δε -1.34), 296 (Δε 15.77), 333 (Δε -13.45) nm; 1 1H NMR (500 MHz, Acetone-d6) δ H: 7.22 (1H, d, J = 16.0 Hz, H-7 / 7'), 7.12 (1H, d, J = 8.5 Hz, H-6 / 6′), 6.82 (1H, d, 2.0 Hz, H-13 / 13′), 6.77 (1H, d, J = 8.5 Hz, H-5 / 5′), 6.74 (1H, d, J = 8.0 Hz, H-16 / 16′), 6.67 (1H, d, J = 8.0 Hz, H-25 / 25′), 6.63 (1H, dd, J = 8.0, 2.0 Hz, H-17 / 17'), 6.55 (1H, s, H-22 / 22'), 6.33 (1H, d, J = 7.5 Hz, H-26 / 26′), 6.16 (1H, d, J = 16.0 Hz, H-8 / 8′), 5.49 (1H, s, H-20 / 20′), 5.07 (1H, dd, J = 4.5, 2.0 Hz, H-10 / 10′), 3.96 (1H, s, H-19 / 19′), 3.55 (3H, s, H-18 / 18′-OCH3), 3.01 (2H, m, H-11 / 11′); 13 13C NMR (125 MHz, Acetone-d6) δ C : 170.9 (C-18 / 18′), 166.3 (C-9 / 9′), 148.2 (C-3 / 3′), 145.8 (C-24 / 24′), 145.6 (C-14 / 14′), 145.6 (C-23 / 23′), 144.7 (C-15 / 15′), 144.4 (C-4 / 4′), 141.8 (C-7 / 7′), 133.8 (21 / 21′), 129.1 (C-12 / 12′), 129.1 (C-2 / 2′), 124.6 (C-1 / 1′), 121.7 (C-17 / 17′), 121.1 (C-6 / 6'), 118.6 (C-5 / 5′), 117.4 (C-13 / 13′), 117.3 (C-26 / 26′), 116.4 (C-8 / 8′), 116.0 (C-25 / 25′), 116.0 (C-16 / 16′), 112.8 (C-22 / 22′), 87.5 (C-20 / 20′), 74.1 (C-10 / 10′), 55.3 (C-19 / 19′), 52.2 (C-18 / 18′-OCH3), 37.3 (C-11 / 11′); HR-ESI-MS m / z 1015.2659 [M+H] + (calcd for C 54 H 47 O 32 , 1015.2655).

[0038] Pharmacological experiments

[0039] Example 1: Protective effect of compounds 1-2 from Danshen on hypoxic injury of H9c2 cardiomyocytes

[0040] H9c2 rat cardiomyocytes (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, hereinafter referred to as the Union Cell Bank) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U / ml) in a 5% CO2 incubator at 37°C. At passage, cells were seeded at a density of 8000 cells / well in 96-well plates. After overnight adhesion, cells were treated with the corresponding compounds according to their experimental groups. After 24 hours, 10% CCK8 reagent was added, and the cells were incubated at 37°C for 2 hours. The OD value at 450 nm was then measured using a microplate reader, with 6 replicates per group.

[0041] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0042] As: Experimental wells (containing cell culture medium, CCK8, and the substance to be tested)

[0043] Ac: Control wells (containing cell culture medium and CCK8, but without the analyte).

[0044] Ab: Blank wells (containing no cells, CCK8, or analyte)

[0045] The results are shown in Table 1. As shown in Table 1, doxorubicin had a significant damaging effect on H9c2 cardiomyocytes, reducing the cell survival rate to 26.55%. Compound 1 significantly increased the survival rate of H9c2 cardiomyocytes treated with doxorubicin, with the best improvement effect observed at 10 μM. Compound 2, however, had a weaker protective effect against doxorubicin-induced damage to H9c2 cardiomyocytes.

[0046] Table 1: Protective effects of compounds 1-2 against hypoxic injury in H9c2 cardiomyocytes

[0047]

[0048] Experimental Example 2: Inhibitory effect of compounds 1-2 from Danshen on isoproterenol-induced proliferation of AC16 myocardial fibroblasts

[0049] AC16 cardiac fibroblasts (purchased from Shanghai Zeye Biotechnology Co., Ltd.) were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (10,000 U / ml) at 37°C with 5% CO2. At passage, cells were seeded at a density of 8000 cells / well in 96-well plates. After overnight adhesion, cells were treated with the corresponding compounds according to their experimental groups. After 24 hours, 10% CCK8 reagent was added, and the cells were incubated at 37°C for 2 hours. The OD value at 450 nm was measured using a microplate reader, with 6 replicates per group.

[0050] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0051] As: Experimental wells (containing cell culture medium, CCK8, and the substance to be tested)

[0052] Ac: Control wells (containing cell culture medium and CCK8, but without the analyte).

[0053] Ab: Blank wells (containing no cells, CCK8, or analyte)

[0054] The results are shown in Table 2. As shown in Table 2, isoproterenol promoted the proliferation of AC16 cardiomyocytes, increasing their proliferation rate to approximately 119.61%. Compound 1 significantly inhibited AC16 cell proliferation, with 1 μM of compound 1 showing the best inhibitory effect, restoring the cells to normal levels. Compound 2, however, had a weaker inhibitory effect on isoproterenol-induced AC16 cell proliferation.

[0055] Table 2: Inhibitory effects of compounds 1-2 from Danshen on isoproterenol-induced proliferation of AC16 myocardial fibroblasts

[0056]

[0057] Example 3: Target search for compound 1 in Danshen.

[0058] Genecard (https: / / www.genecards.org / ) and DisGeNET (https: / / www.disgenet.org / search) are commonly used disease-gene association databases. These databases were used to screen for genes related to heart failure. Targetnet (http: / / targetnet.scbdd.com / home) and SwissTargetPrediction (http: / / swisstargetprediction.ch / ) are commonly used databases for predicting compound targets. These databases were used to obtain potential targets for compound 1. UniProt (https: / / www.uniprot.org / ) was used to normalize the targets. Venn diagrams were used to cross-reference heart failure-related targets and compound-predicted targets, yielding MMP9 as a potential target for compound 1 against heart failure (see appendix). Figure 1 ).

[0059] Experimental Example 4: Immunoblotting confirms the target MMP9 of compound 1 in Danshen.

[0060] The specific experimental steps are as follows:

[0061] 1) Protein extraction

[0062] AC16 cells were evenly seeded into 6-well plates and allowed to adhere overnight. Reagents were added according to Table 3. After 24 hours, the culture medium was aspirated from the 6-well plates, and the cells were washed twice with PBS. 200 μL of RIPA lysis buffer (pre-added according to a RIPA:PMSF:Cocktail ratio of 50:1:1) was added to each well. The plates were incubated on ice for 5 minutes. Cells were scraped from each well using a cell scraper and placed into pre-labeled 1.5 mL centrifuge tubes. Lysis was performed on ice, with the tubes tapped every 10 minutes for 3 taps. Protein was then centrifuged at 12000 rpm, 4°C, for 30 minutes. After centrifugation, the supernatant protein was carefully aspirated and transferred to new labeled centrifuge tubes for protein quantification.

[0063] 2) Protein concentration determination

[0064] Protein quantification was performed using the BCA protein quantification kit.

[0065] ① Protein sample dilution: Prepare a blank centrifuge tube, add 47.5 μL of ultrapure water to the tube, and after the protein supernatant is mixed, take 2.5 μL of the supernatant and add it to ultrapure water to prepare a 20-fold diluted protein sample.

[0066] ②Preparation of protein standards: Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of protein standards (0.5 mg / mL) sequentially to a 96-well plate, and make up the total volume to 20 μL with ultrapure water.

[0067] ③ Take 20 μL of sample dilution and add it to each well of the 96-well plate. Make one duplicate well for each sample.

[0068] ④ Prepare the working solution by mixing solutions A and B from the BCA protein quantification kit at a ratio of 50:1. Add 200 μL of the prepared working solution to each standard well and sample well.

[0069] ⑤ Place the 96-well plate at 37°C and incubate for 30 minutes.

[0070] ⑥ Set the wavelength of the microplate reader to 562 nm, scan the plate, and record the absorbance value. Plot a standard curve based on the concentration and absorbance value of the protein standard, and calculate the protein sample concentration based on the standard curve.

[0071] ⑦ Adjust the protein sample to a uniform concentration using RIPA lysis buffer, add 5× loading, and boil in a 98℃ metal bath for 5-10 minutes to denature the protein, preparing for electrophoresis.

[0072] 3) SDS-PAGE electrophoresis

[0073] ① Protein electrophoresis: Remove the comb from the pre-prepared 10% SDS-polyacrylamide gel, blow away any residual gel strands, and add 10 μL of the denatured protein sample to the sample wells. Electrophoresis is performed using a constant voltage DC mode at 80V for 30 minutes, then adjusted to 120V and continued for 60 minutes.

[0074] ② Transfer: After electrophoresis, the protein on the gel was transferred to a PVDF membrane using the wet transfer method. The constant current mode was set to 220mA, and the membrane was transferred in an ice bath for 90 minutes.

[0075] ③ Milk blocking: Immerse the PVDF membrane after transfer in 5% skim milk prepared with TBST buffer, and block on a shaker at room temperature for 30 minutes to block non-specific binding regions.

[0076] ④ Primary antibody incubation: Dilute the primary antibody 1:1000 with the primary antibody dilution buffer. Cut out the target protein region according to the molecular weight based on the marker, and incubate the protein band with the corresponding primary antibody at 4°C overnight.

[0077] ⑤ Secondary antibody incubation: Recover the primary antibody on the second day. Wash the protein bands with TBST buffer, washing 3 times for 10 min on a shaker at room temperature. Dilute the corresponding secondary antibody 1:2000 with TBST buffer. Incubate the protein bands with the corresponding secondary antibody in the hybridization zone at room temperature for 2 h. Wash the protein bands with TBST buffer, washing 3 times for 10 min on a shaker at room temperature to remove unbound secondary antibody.

[0078] ⑥ Exposure and color development: The target band is developed using a pre-prepared color developing solution, and the developed images are recorded and saved using a gel imaging system (see appendix). Figure 2 And use ImageJ to perform grayscale value analysis.

[0079] The results are shown in Table 3. As shown in Table 3, ISO significantly increased the protein expression of MMP9 in AC16 cells by approximately 67.5%, while dilithospermic acid A significantly decreased the protein expression of MMP9 in a dose-dependent manner.

[0080] Table 3: Detection of differences in MMP9 protein expression in AC16 cells under different treatments using Western blotting

[0081]

[0082] Experimental Example 5: The real-time fluorescence quantitative nucleic acid amplification detection system confirmed the target MMP9 of compound 1 in Danshen. The specific experimental steps are as follows:

[0083] 1) Total RNA was extracted using the Trizol method:

[0084] AC16 cells were evenly seeded into 6-well plates and allowed to adhere overnight. Reagents were added according to Table 4. After 24 hours, the culture medium was aspirated from the 6-well plates. 500 μL of Trizol was added to each well to release RNA. After standing on ice for 5 minutes, the cells were transferred to labeled 1.5 mL centrifuge tubes. 100 μL of chloroform was added, and the tubes were vortexed for 15 seconds. The cells were then allowed to stand for 3 minutes to denature the protein. The tubes were centrifuged at 12000 rpm and 4°C for 20 minutes to remove the protein. 200 μL of the supernatant (containing RNA) was carefully aspirated into a clean centrifuge tube. An equal volume of isopropanol was added, and the mixture was stirred. The mixture was allowed to stand for 10 minutes to precipitate RNA. The precipitate was centrifuged at 12000 rpm and 4°C for 10 minutes. The precipitate was RNA. The supernatant was discarded. The precipitate was flicked with 1 mL of 75% ethanol (prepared with DEPC water) to dissolve impurities. The precipitate was centrifuged at 12000 rpm and 4°C for 10 minutes to obtain pure RNA precipitate. Discard the supernatant, air-dry the precipitate in an ultra-clean bench, dissolve the RNA in 20 μL of DEPC water, and determine the concentration.

[0085] 2) Determine the concentration of RNA:

[0086] Using the nucleic acid quantification mode of the ELISA reader, after calibrating with 2 μL of DEPC water, take 2 μL of sample for measurement.

[0087] 3) Reverse transcription:

[0088] RNA was reverse transcribed and amplified according to the instructions of the All Gold Reverse Transcription Kit to obtain cDNA.

[0089] 4) qPCR:

[0090] A 20 μL system was prepared using FastStart Universal SYBR Green Master (KAPA Biosystems, KK4601) enzyme. After mixing and centrifugation, real-time PCR analysis of MMP9 and the internal control GAPDH was performed using a LineGene 9620 PCR instrument. Formula 2 was used. -ΔΔCt Calculate relative expressions.

[0091] The primer sequences used for qPCR are as follows:

[0092]

[0093] The results are shown in Table 4. As shown in Table 4, isoproterenol significantly increased the mRNA level of Mmp9 in AC16 cells by 2.8-fold. Compound 1 reduced the mRNA level of Mmp9 to normal levels.

[0094] Table 4: Differences in Mmp9 mRNA in AC16 cells under different treatments detected by real-time quantitative nucleic acid amplification detection system

[0095]

[0096] Note: 1 The results show a comparison between the compound 1 treatment group and the normal group; 2 The results show the comparison between the isoproterenol + compound 1 group and the isoproterenol model group.

[0097] Example 6: Inhibitory effect of compound 1 from Danshen on isoproterenol-induced AC16 cardiomyocyte migration.

[0098] The specific experimental steps are as follows:

[0099] 1) Open the Transwell (8μm pore size) in a clean bench, invert it onto a large dish, and spread 40μL of fibronectin (2mg / mL stock solution, diluted 100 times with serum-free medium) on the lower surface of each chamber, spreading it evenly and paying attention to the edges. Do not turn on the fan; allow it to air dry at room temperature before placing it back into the well plate.

[0100] 2) Pre-starve AC16 cells overnight to make them more sensitive to stimulation, digest, centrifuge, wash 1-2 times with serum-free medium, and count to approximately 5 × 10⁶ cells. 5 Cells / mL, add the corresponding compounds according to the experimental groups (see Table 5), add 200 μL of cell suspension (containing compounds) to the upper chamber, add 1 mL of culture medium to the lower chamber, and incubate at 37℃ for 6 h.

[0101] 3) After 6 hours, remove the chamber, aspirate the cell suspension from the upper chamber, wash the chamber once in a well plate containing 500 μL PBS, fix with 4% paraformaldehyde for 10 min, wash off the paraformaldehyde with PBS, and stain with crystal violet for 20 min. After staining, gently wipe away unmigrated cells from the inside of the chamber with a cotton swab. Invert the chamber and randomly select 6 fields of view for photographic observation under a bright-field microscope (see appendix). Figure 3 ), and count the number of cells that migrated.

[0102] The results are shown in Table 5. As shown in Table 5, isoproterenol treatment significantly promoted the migration of AC16 cells, while treatment with compound 1 significantly reduced the number of migrating cells.

[0103] Table 5: Results of the inhibitory effect of compound 1 from Danshen on isoproterenol-induced AC16 cardiomyocyte migration.

[0104]

[0105] Example 7: Inhibitory effect of compound 1 from Danshen on isoproterenol-induced activation of AC16 cardiomyocytes

[0106] The specific experimental steps are as follows:

[0107] 1) Cell culture: After soaking the cell spreaders in anhydrous ethanol and air-drying them, place them in 24-well plates and wash them three times with PBS. After digestion, AC16 cells were seeded into 24-well plates with cell spreaders and allowed to adhere overnight. Then, according to the experimental groups, the corresponding reagents (see Table 6) were added and the cells were treated for 24 hours.

[0108] 2) Production:

[0109] ① Fixation: After 24 hours, discard the supernatant, wash twice with PBS, add 1 mL of 4% paraformaldehyde to completely soak the cell slide, fix on a shaker for 10 minutes, and wash away excess paraformaldehyde with PBS for 10 minutes × 3 times.

[0110] ② Membrane permeation: Add 1 mL of 0.5% Triton to soak the cell smear for 10 min to permeate the membrane, and wash away excess Triton with PBS for 10 min × 3 times.

[0111] ③ Blocking: Prepare 3% BSA with PBS, add 1 mL to each well to block nonspecific sites, and wash with PBS after 1 hour.

[0112] ④ Primary antibody incubation: Dilute the primary antibody with PBS at a ratio of 1:300, add 40 μL of the diluted primary antibody to each cell smear, place in a humidified chamber, and incubate overnight at 4°C.

[0113] ⑤ Secondary antibody incubation: Wash cell slides with PBS for 10 min × 3 times. Dilute Alexa Fluor 488 or Alexa Fluor 647-conjugated secondary antibodies with PBS at a ratio of 1:300. Add 40 μL of the correspondingly diluted secondary antibody to each cell slide and incubate in a dark box at room temperature for 2 h in the dark. Wash cell slides with PBS for 10 min × 3 times.

[0114] ⑥ Mounting: Add 10 μL of mounting medium (containing DAPI) to a glass slide, invert the cell slide and fix it on the glass slide, and let it air dry at room temperature in the dark.

[0115] 3) Observation:

[0116] Select the appropriate excitation light and randomly select 10 fields of view for photographic observation using an FV3000 immunofluorescence confocal microscope (see appendix). Figure 4 The fluorescence intensity was statistically analyzed using ImageJ.

[0117] The results are shown in Table 6. As shown in Table 6, isoproterenol significantly increased the expression of α-SMA and Collagen I in AC16 cells by 1.655-fold, and morphological changes indicating fibroblast activation were observed. Compound 1 reduced α-SMA and Collagen I to normal levels.

[0118] Table 6-1 Results of the inhibitory effect of compound 1 from Danshen on isoproterenol-induced AC16 cardiomyocyte activation (α-SMA)

[0119]

[0120] Table 6-2 Results of the inhibitory effect of compound 1 from Danshen on isoproterenol-induced AC16 cardiomyocyte activation (Collagen I)

[0121]

[0122] As shown in Examples 1-7, compound 1 exhibited a strong protective effect in the H9c2 doxorubicin injury model and a strong inhibitory effect in the isoproterenol-induced AC16 proliferation model of cardiomyocytes, in a dose-dependent manner. During the target identification phase, we identified the most likely active protein, MMP9, and confirmed this target at both the protein and RNA levels. In migration experiments, compound 1 inhibited isoproterenol-induced AC16 migration, and laser confocal scanning results showed that compound 1 inhibited isoproterenol-induced AC16 activation. These results suggest that compound 1 has a strong cardioprotective effect, inhibiting the proliferation, activation, and migration of cardiomyocytes, and has the potential to be developed into a therapeutic drug for cardiac fibrosis and heart failure.

Claims

1. A class of compounds or pharmaceutically acceptable salts thereof, characterized in that, The structure of this type of compound is as follows: 。 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The pharmaceutically acceptable salt is selected from salts formed by compounds and inorganic or organic bases.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, characterized in that, The organic bases are methylamine, ethylamine, diethylamine, triethylamine, propylamine, butylamine, octylamine, hexamethylenediamine, ethylenediamine, propylenediamine, butylamine, benzylamine, phenethylamine, o-phenylenediamine, and p-phenylenediamine. The inorganic bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, copper hydroxide, iron hydroxide, ammonium hydroxide, sodium bicarbonate, and potassium bicarbonate.

4. The method for preparing the compound of claim 1, characterized in that, The preparation method is as follows: Salvia miltiorrhiza is extracted by reflux with 80% ethanol, the concentrated extract is extracted with ethyl acetate, the solvent is recovered from the ethyl acetate fraction to obtain the extract, the extract is further subjected to silica gel column chromatography, eluted with a petroleum ether-acetone solvent system, and the eluted fraction containing the target compound is purified by preparative high performance liquid chromatography to obtain compounds 1 and 2 as described in claim 1.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of any one of claims 1-3 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that, The dosage form of the pharmaceutical composition is tablets, capsules, pills, granules, oral liquids, or suspensions.

7. Use of the compound according to any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of heart failure.

8. The application as described in claim 7, characterized in that, The heart failure described is induced by hypoxic damage to myocardial cells.

9. The application as described in claim 7, characterized in that, The heart failure described is induced by cardiac remodeling characterized by myocardial fibrosis.

10. The application as described in claim 9, characterized in that, The aforementioned myocardial fibrosis involves the excessive proliferation, migration, and activation of myocardial fibroblasts.

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