A traditional Chinese medicine composition, its preparation method and uses

Abstract

The present invention belongs to the technical field of traditional Chinese medicine, and specifically relates to a traditional Chinese medicine composition, its preparation method and uses. A traditional Chinese medicine composition, calculated by mass parts, the traditional Chinese medicine composition is prepared from the following components: 0.5 - 1.5 parts of Cortex Moutan, 0.8 - 1.2 parts of Gardenia jasminoides Ellis and 0.3 - 1.7 parts of Rhizoma Coptidis. The present invention selects three traditional Chinese medicines, Cortex Moutan, Gardenia jasminoides Ellis and Rhizoma Coptidis, and prepares a traditional Chinese medicine dry powder through extraction, concentration and spray drying. Through in-vivo experiments on diabetic cardiomyopathy model mice, it is found that the traditional Chinese medicine dry powder of the present invention can alleviate the systemic metabolic disorder of diabetic mice and has a protective effect on the myocardial structure change of diabetic cardiomyopathy model mice; in addition, through immunoblotting detection, it is found that the traditional Chinese medicine dry powder of the present invention can inhibit the expression and activation of IL-6, MAPK1 and PRKCA in diabetic cardiomyopathy model mice. Therefore, the traditional Chinese medicine composition of the present invention provides support for the development of traditional Chinese medicine drugs for treating diabetic cardiomyopathy.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and particularly relates to a traditional Chinese medicine composition, a preparation method thereof and uses thereof. Background Art

[0002] Diabetes has developed into the most prevalent disease in the world. As a chronic metabolic disorder, hyperglycemia and insulin resistance are two prominent features of diabetes. Diabetes can damage the functions of other organs in the body and develop into various complications during the in-depth development of diabetes. Diabetic cardiomyopathy is one of the most severe of these complications, characterized by changes in the myocardial structure and ventricular systolic and diastolic dysfunction in diabetic patients. Diabetic cardiomyopathy ultimately develops into heart failure, leading to the death of patients. As a unique existence, there is a lack of specialized drugs for the treatment of diabetic cardiomyopathy. Although effective treatment can be achieved using sodium-glucose cotransporter 2 inhibitors, there is myocardial lipotoxicity and a lack of effective solutions. Therefore, new drugs or treatment methods are needed in the future.

[0003] Diabetic cardiomyopathy is characterized by abnormal heart structure and dysfunction, and ultimately develops into heart failure, affecting the life and health of patients. The pathogenesis of diabetic cardiomyopathy involves multiple pathways, including oxidative stress, calcium overload, mitochondrial dysfunction, activation of the protein kinase C signaling pathway, myocardial lipid accumulation, and low-grade inflammation of the myocardium. Therefore, the treatment of diabetic cardiomyopathy should also focus on its multiple pathogenic pathways. Traditional Chinese medicine has the characteristics of multi-targets, multi-components, and low toxicity and side effects. Based on this, searching for drugs for the treatment of diabetic cardiomyopathy from traditional Chinese medicine is undoubtedly a superior treatment strategy.

[0004] Moutan Cortex is derived from the dried root bark of Paeonia suffruticosa Andr. It has the effects of clearing heat and cooling blood, promoting blood circulation to remove stasis. Based on these effects, Moutan Cortex has been applied to traditional Chinese medicine prescriptions for anti-diabetes and anti-diabetic complications such as Bawei Dihuang Pills, Liuwei Dihuang Pills, and Sheng Puhuang Decoction. Modern pharmacological studies also show that Moutan Cortex has a therapeutic effect on diabetes, diabetic nephropathy, and heart failure.

[0005] Gardenia Fruit is the dried ripe fruit of Gardenia jasminoides Ellis. It has the effects of purging fire and relieving restlessness, clearing heat and promoting diuresis, cooling blood and detoxifying; and reducing swelling and alleviating pain when used externally. It also has a history of application in the treatment of consumptive thirst (a traditional Chinese medicine term for diabetes). Modern pharmacological studies show that the components in Gardenia Fruit have anti-inflammatory and anti-diabetic effects.

[0006] Coptis Rhizome is the dried rhizome of Coptis chinensis Franch., Coptis deltoidea C. Y. Cheng & Hsiao, or Coptis teeta Wall. The Chinese Pharmacopoeia (2020 Edition) states that Coptis Rhizome has the effects of clearing heat and drying dampness, purging fire and detoxifying. Similarly, there are also records and studies on the treatment of diabetes with Coptis Rhizome.

[0007] The information disclosed in this background section is only intended to enhance the overall understanding of the background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0008] The object of the present invention is to provide a traditional Chinese medicine composition, its preparation method and uses, which have a therapeutic effect on diabetic cardiomyopathy and can provide support for the development of traditional Chinese medicine drugs for treating diabetic cardiomyopathy.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A traditional Chinese medicine composition, calculated by mass parts, the traditional Chinese medicine composition is prepared from the following components: 0.5 - 1.5 parts of Cortex Moutan, 0.8 - 1.2 parts of Fructus Gardeniae, and 0.3 - 1.7 parts of Rhizoma Coptidis.

[0011] Preferably, calculated by mass parts, the traditional Chinese medicine composition is prepared from the following components: 1 part of Cortex Moutan, 1 part of Fructus Gardeniae, and 1 part of Rhizoma Coptidis.

[0012] The second object of the present invention is to provide a traditional Chinese medicine dry powder, which is prepared from the above traditional Chinese medicine composition.

[0013] The preparation method of the traditional Chinese medicine dry powder is as follows: Weigh Cortex Moutan, Fructus Gardeniae, and Rhizoma Coptidis according to the mass ratio, extract twice with distilled water, combine the filtrates, concentrate, and spray dry to obtain the traditional Chinese medicine dry powder; wherein, for the first extraction, add 8 times the mass of water, soak for 30 min, and reflux extract for 60 min; for the second extraction, add 6 times the mass of water and reflux extract for 40 min.

[0014] The third object of the present invention is to provide the application of the above traditional Chinese medicine composition or the traditional Chinese medicine dry powder in the preparation of drugs for treating diabetic cardiomyopathy.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention selects three traditional Chinese medicines, Cortex Moutan, Fructus Gardeniae, and Rhizoma Coptidis, and prepares a traditional Chinese medicine dry powder through extraction, concentration, and spray drying. Through in - vivo experiments on diabetic cardiomyopathy model mice, it is found that the traditional Chinese medicine dry powder of the present invention can alleviate the systemic metabolic disorders of diabetic mice and has a protective effect on the myocardial structure changes in diabetic cardiomyopathy model mice; in addition, through immunoblotting detection, it is found that the traditional Chinese medicine dry powder of the present invention can inhibit the expression and activation of IL - 6, MAPK1, and PRKCA in diabetic cardiomyopathy model mice. Therefore, the traditional Chinese medicine composition of the present invention provides support for the development of traditional Chinese medicine drugs for treating diabetic cardiomyopathy. Brief Description of the Drawings

[0017] Figure 1It is the preparation flow chart of the traditional Chinese medicine dry powder of the present invention;

[0018] Figure 2 It is the chromatogram of the component analysis of the traditional Chinese medicine dry powder of the present invention;

[0019] Figure 3 It is the mixed chromatogram of 13 standard chemical components;

[0020] Figure 4 It is the structural diagram of 13 chemical components;

[0021] Figure 5 It is the representative HE and Masson staining pictures of the longitudinal section of the myocardial tissue in the evaluation center of the diabetic cardiomyopathy model;

[0022] Figure 6 It is the influence of the traditional Chinese medicine dry powder of the present invention on the systemic metabolic abnormalities of diabetic mice;

[0023] Figure 7 It is the therapeutic effect of the traditional Chinese medicine dry powder of the present invention on the heart injury of diabetic cardiomyopathy model mice;

[0024] Figure 8 It is the representative images of HE and Masson staining of the longitudinal section of the heart tissue in the influence of the pathological changes of the myocardial tissue of mice;

[0025] Figure 9 It is the "traditional Chinese medicine - active ingredient - target" network diagram;

[0026] Figure 10 It is the intersection target of the component targets of Cortex Moutan, Gardenia jasminoides and Rhizoma Coptidis and the disease targets of diabetic cardiomyopathy;

[0027] Figure 11 It is the "traditional Chinese medicine chemical component - potential therapeutic target - diabetic cardiomyopathy" network diagram;

[0028] Figure 12 It is the protein - protein interaction network diagram of potential therapeutic targets;

[0029] Figure 13 It is the protein - protein interaction network diagram of core targets;

[0030] Figure 14 It is the top 20 GO analysis results of Cortex Moutan - Gardenia jasminoides - Rhizoma Coptidis in the treatment of diabetic cardiomyopathy;

[0031] Figure 15 It is the top 20 KEGG analysis results of Cortex Moutan - Gardenia jasminoides - Rhizoma Coptidis in the treatment of diabetic cardiomyopathy;

[0032] Figure 16It is the intersection of the targets and core targets on the "AGE-RAGE signaling pathway in diabetic complications" and "Diabetic cardiomyopathy" signaling pathways;

[0033] Figure 17 It is the result of Western blot analysis.

[0034] Description of main reference numerals:

[0035] Figure 6 Among them, body weight (a), fasting blood glucose (FB) (b), heart index (c), insulin level (INS) (d), total cholesterol (TC) (e), triglyceride (TG) (f), high-density lipoprotein (HDL) (g), low-density lipoprotein (LDL) (h). The results are expressed as mean ± standard deviation (n = 6). # P < 0.05, ## P < 0.01, ### P < 0.001, and #### P < 0.0001 compared with the normal control group; * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 compared with the model control group;

[0036] Figure 7 Among them, representative images of M-mode echocardiogram (a); left ventricular ejection fraction (LVEF) (b); left ventricular fractional shortening (LVFS) (c); left ventricular end-diastolic volume (LVEDV) (d); left ventricular end-systolic volume (LVESV) (e); the values are expressed as mean ± standard deviation (n = 6), ## P < 0.01, and ### P < 0.001 compared with the control group; * P < 0.05, and ** P < 0.01 compared with the model group;

[0037] Figure 14 Among them, representative bands of β-actin, IL-6, MAPK1, and PRKCA (a); the histograms show the quantitative expression changes of IL-6, MAPK1, and PRKCA (b - d); before performing relative quantitative analysis, all proteins were normalized to β-actin protein. The values are expressed as mean ± standard deviation of three different experiments. # P < 0.05 compared with the normal control group; *P < 0.05 compared with the model group. Detailed implementation manners

[0038] The following clearly and completely describes the technical solutions of the present invention patent. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0039] Embodiment

[0040] I. Materials and methods

[0041] 1.1 Chemicals and reagents

[0042] 85% phosphoric acid (high performance liquid chromatography grade) was purchased from Tianjin Damao Chemical Reagent Factory;

[0043] Acetonitrile (high performance liquid chromatography grade) was purchased from Bengbu Puredil Biotechnology Co., Ltd.;

[0044] Gallic acid (purity ≥ 98%), 6α-hydroxygeniposide (purity ≥ 98%), oxypaeoniflorin (purity ≥ 98%), genipin-1-β-d-gentiobioside (purity ≥ 98%), geniposide (purity ≥ 98%), magnoflorine (purity ≥ 98%), groenlandicine (purity ≥ 98%), columbamine (purity ≥ 98%), epiberberine (purity ≥ 98%), jatrorrhizine (purity ≥ 98%), coptisine (purity ≥ 98%), palmatine (purity ≥ 98%), berberine hydrochloride (purity ≥ 98%) were purchased from Baoji Chenguang Biotechnology Co., Ltd.;

[0045] Common breeding feed was purchased from Beijing Keao Xieli Feed Co., Ltd.;

[0046] High-sugar and high-fat feed was purchased from RESEARCH DIETS (New Jersey, America);

[0047] Streptozotocin was purchased from Shanghai Rujie Biotechnology Development Co., Ltd.;

[0048] Citric acid / sodium citrate buffer (0.01 mol / L, pH = 6.0) was purchased from Beijing Laigen Biotechnology Co., Ltd.;

[0049] Dapagliflozin was purchased from AstraZeneca Pharmaceuticals Ltd.;

[0050] Anti-protein kinase Cαtype (PRKCA), mitogen-activated protein kinase 1 (MAPK1), and interleukin 6 (IL-6) proteins were purchased from Wuhan Saiwei Biotechnology Co., Ltd.

[0051] 1.2 Preparation of Peony Root, Gardenia and Coptis Chinensis Extract Powder

[0052] Peony bark was purchased from Shaanxi Fengdan Zhengyuan Biotechnology Co., Ltd., Gardenia jasminoides and Coptis chinensis were purchased from Shaanxi Duoyao Chinese Medicine Pieces Co., Ltd., and the Chinese medicinal materials were identified by Professor Xie Yanhua of Northwest University.

[0053] Weigh 1 kg each of peony bark, gardenia and coptis root, and extract twice with distilled water: add 8 times the amount of water for the first time, soak for 30 minutes, and reflux for 60 minutes; add 6 times the amount of water for the second time, and reflux for 40 minutes. Combine the two filtrates with a high-speed centrifugal spray dryer for medicine, concentrate, and spray dry to obtain Chinese medicine dry powder. The extract yield of Chinese medicine dry powder is 20.5%, and its preparation process is as follows: Figure 1 shown.

[0054] 1.3 High performance liquid phase component analysis

[0055] Dissolve 0.25g of Chinese medicine powder in 50mL of distilled water and oscillate ultrasonically for 30min. Then, filter the supernatant through a 0.45μm water microporous filter membrane to obtain the sample to be tested. 18 The components of the dried Chinese medicine powder were analyzed using a chromatographic column (250 mm × 4.6 mm, 5 μm; Shaanxi Lamdo Technology Co., Ltd.).

[0056] The mobile phase was 0.17% phosphoric acid (A)-acetonitrile (B), and the gradient elution program was: 0-25 min, 90%-80% (A); 20-50 min, 80%-73% (A); 50-70 min, 73%-71.5% (A). The spectra were recorded at 254 nm (0-45 min) and 345 nm (45-70 min), and the chromatograms were obtained by K2050 HPLC (Shandong Wukong Instrument Co., Ltd., Shandong, China) with UV detector. The column temperature was 25 °C, the flow rate was 0.6 mL / min, the injection volume was 10 μL, and the external standard method was used for quantification.

[0057] 1.4 Animals

[0058] Male C57BL / 6 mice without specific pathogens and weighing between 20 - 24 g were purchased from the Animal Center of Air Force Medical University (No. SCXK(ARMY)2019 - 001, Shaanxi).

[0059] First, the mice were fed with ordinary breeding feed and water for two weeks to adapt to the environment. The culture conditions were: temperature 18 - 22 °C, humidity 50 - 55%, light / dark cycle 12 h. All laboratory experimental procedures complied with the UK Animals (Scientific Procedures) Act 1986 and related guidelines, and were approved by the Welfare and Ethics Committee of the Experimental Animal Center of Northwestern University (approval number: NWUAWC - 20220908M; approval date: September 30, 2022).

[0060] 1.5 Animal intervention

[0061] Forty mice were randomly divided into 4 groups (n = 10): normal control group, diabetic cardiomyopathy model group, positive drug dapagliflozin group (0.65 mg / kg), and traditional Chinese medicine dry powder group (800 mg / kg). During the experiment, the body weight and fasting blood glucose (FBG) (fasting for 12 h) of the mice were continuously measured.

[0062] To establish a diabetic cardiomyopathy mouse model, the diabetic cardiomyopathy model group, traditional Chinese medicine dry powder group, and positive drug dapagliflozin group were first fed with a high - sugar and high - fat diet for 2 weeks. Then, the three groups of mice were intraperitoneally injected with streptozotocin dissolved in citric acid / sodium citrate buffer at a dose of 60 mg / kg on an empty stomach for 3 consecutive days to destroy pancreatic islet B cells. One week later, if the fasting blood glucose of the mice > 11.1 mmol / L, it was considered that a type 2 diabetes model was formed. Then, after continuing to feed the high - sugar and high - fat diet for 1 month, 2 mice were randomly selected from each group to take the heart for hematoxylin - eosin staining and Masson staining. Under an optical microscope, if the heart sections of the mice showed myocardial fiber rupture and collagen deposition, it was considered that the diabetic cardiomyopathy model was formed. The traditional Chinese medicine dry powder group and the positive drug group were respectively given traditional Chinese medicine dry powder and dapagliflozin by gavage intervention for 8 weeks. The traditional Chinese medicine dry powder and dapagliflozin were dissolved in distilled water. The diabetic cardiomyopathy model group was given an equal volume of distilled water. Throughout the experiment, the mice in the above three groups were fed a high - sugar and high - fat diet. The normal control group was fed with ordinary feed, injected with an equal amount of citric acid / sodium citrate buffer, and given an equal amount of distilled water.

[0063] At the end of the experiment, the cardiac function of the mice was measured by transthoracic echocardiography after anesthesia with 1.0% isoflurane. After that, all the mice were weighed, and blood was collected by eye socket after re - anesthesia with isoflurane. The heart tissue was quickly separated and weighed, and the heart index was calculated. After weighing and recording the tissue, 3 heart tissues were randomly selected and fixed with 4% paraformaldehyde for histopathological observation, and the remaining 5 heart tissues were stored at - 80 °C for Western blot detection.

[0064] 1.6 Determination of fasting blood glucose

[0065] The fasting blood glucose of mice was measured every two weeks by taking blood from the tip of the tail, using a OneTouch Verio Flex blood glucose meter and blood glucose test strips of the OneTouch Verio Flex model, purchased from LifeScan (Guangzhou) Medical Devices Co., Ltd.

[0066] 1.7 Cardiac function detection

[0067] Transthoracic echocardiography was used to measure cardiac function with a VisualSonics Vevo 3100 ultrasound system (Toronto, Canada). Computer algorithms were used to determine the left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), and left ventricular end-systolic volume (LVESV).

[0068] 1.8 Calculation of cardiac index

[0069] The cardiac index was calculated based on the recorded body weight and heart weight of the mice:

[0070] Cardiac index (%) = heart weight (g) / body weight (g) at the time of mouse sacrifice × 100%.

[0071] 1.9 Hematoxylin-eosin staining and Masson staining

[0072] The heart tissues were paraffin-embedded and sectioned at a thickness of 5 μm. Routine paraffin sections were stained with HE to observe the morphological and structural changes of the myocardium. Masson staining was used to observe the degree of extracellular collagen deposition in the myocardial tissue. Subsequently, the sections were observed and captured with a Leica DM3000 LED optical microscope.

[0073] 1.10 Determination of biochemical indicators

[0074] Blood samples were centrifuged at 3500 rpm for 15 min at 4 °C to collect serum and stored at -80 °C. Insulin (INS), total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were detected according to the kit instructions (Shanghai Sangon Biotech Co., Ltd.).

[0075] 1.11 Screening of chemical components contained in Cortex Moutan, Fructus Gardeniae, and Rhizoma Coptidis

[0076] The chemical components of these three traditional Chinese medicines were obtained from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) by setting the oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18. Meanwhile, the results of high-performance liquid analysis were added to the dataset of chemical components. The targets of these chemical components were also obtained from TCMSP. If the corresponding targets of a component could not be collected from TCMSP, we obtained the compound structure from the PubChem database and then predicted the potential targets in the Swiss Target Prediction database (Posibility>0).

[0077] 1.12 Screening of DCM-related genes

[0078] Using the GeneCard database, disease genes related to diabetic cardiomyopathy were collected with the keyword “Diabetic cardiomyopathy”, and the corresponding disease genes were obtained by setting score ≥ 5 and species as human.

[0079] 1.13 Construction of the “traditional Chinese medicine-chemical component-potential therapeutic target-disease” network

[0080] The targets matching the chemical components and the targets corresponding to the disease were imported into the Venny platform to obtain the intersecting targets, which could be considered as potential therapeutic targets. Subsequently, a network diagram of “traditional Chinese medicine-chemical component-therapeutic target-diabetic cardiomyopathy” was established using Cytoscape (version 3.9.1).

[0081] 1.14 Construction of the protein-protein interaction network among potential therapeutic target proteins and screening of core targets

[0082] The potential therapeutic targets were input into the String database to construct a protein-protein interaction network, with the species set as “human” and the highest confidence level as “0.9”. The PPI network of potential therapeutic targets was imported into the Cytoscape “3.9.1” software for analysis, and the core targets were obtained by setting degree > 9, betweenness centrality > 299, eigenvector centrality > 0.05, and closeness centrality > 0.35.

[0083] 1.15 GO and KEGG enrichment analysis

[0084] The potential therapeutic targets were imported into the Metasscape database for GO and KEGG enrichment analysis. GO functional analysis included biological process (BP), cellular component (CC), and molecular function (MF). The parameters were set as "human", "Min Overlap = 3", "Pvalue Cutoff = 0.01", and "Min Enrichment = 1.5".

[0085] 1.16 Screening of targets to be verified

[0086] The targets on the "AGE-RAGE signaling pathway in diabetic complications" and "Diabetic cardiomyopathy" pathways obtained from KEGG enrichment analysis, and the core targets were input into the Venny network platform to form the intersection dataset of the above three gene datasets. The genes contained in the intersection dataset could be used as targets for protein immunoblotting verification.

[0087] 1.17 Protein immunoblotting verification

[0088] RIPA lysis buffer consisted of 1% phenylmethylsulfonyl fluoride and 1% protease inhibitor. An appropriate amount of heart tissue was taken, washed, homogenized in RIPA lysis buffer at 4°C for 30 min, and the supernatant was collected by centrifugation. The protein concentration was measured using a BCA protein assay kit (Wuhan Saiwei Biotechnology Co., Ltd.). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane, blocked with 5% non-fat milk at room temperature for 2 h, and then washed. Then, the membrane was incubated with the primary antibody against the target protein overnight at 4°C. On the second day, the membrane was washed and incubated with the secondary antibody at room temperature for 2 h, and the bands were detected using an enhanced chemiluminescence gel imaging system. The gray values of the protein bands were analyzed using Image J software.

[0089] 1.18 Data analysis

[0090] All experimental results were statistically analyzed using GraphPad Prism statistical software (version 9.0). Data processing was performed using one-way analysis of variance, expressed as mean ± standard deviation, and the statistical significance level was set at p < 0.05.

[0091] 2. Results

[0092] 2.1 Analysis of the dry powder components of the traditional Chinese medicine of the present invention

[0093] The results are shown in Figure 2 and Figure 3 .

[0094] Figure 2 and Figure 3As shown in Figure 2, 13 compounds were identified from the dry powder of traditional Chinese medicine by external standard method and absorption spectrum analysis, including gallic acid (1), 6α-hydroxygeniposide (2), genipin-1-β-d-gentiobioside (3), oxypaeoniflorin (4), geniposide (5), magnoflorine (6), groenlandicine (7), columbamine (8), epiberberine (9), jatrorrhizine (10), coptisine (11), palmatine (12) and berberine hydrochloride (13). The chemical structures of these 13 compounds are shown in Figure 2. Figure 4 .

[0095] 2.2 Evaluation of diabetic cardiomyopathy model

[0096] After the fasting blood glucose of the diabetic cardiomyopathy model group, the peony bark-gardenia-coptis extract group and the positive drug dapagliflozin group was >11.1mMol / L, they continued to be fed with a high-sugar and high-fat diet for 2 months. Two mice were randomly selected from each group, and the heart tissues were taken for HE and Masson staining. Figure 5 .

[0097] like Figure 5 As shown, myocardial fiber rupture can be seen in the HE staining results, and extracellular collagen deposition can be observed in the Masson staining results.

[0098] 2.3 The Chinese medicine dry powder of the present invention improves systemic metabolic abnormalities in diabetic mice

[0099] Results Figure 6 .

[0100] Depend on Figure 6 It can be seen that the body weight, fasting blood sugar, cardiac index, total cholesterol, triglyceride, and low-density lipoprotein levels of the model group rats were significantly higher than those of the control group (p<0.05). In addition, the serum insulin and high-density lipoprotein levels of the model group rats were significantly reduced (p<0.05). This shows that diabetic mice have systemic metabolic abnormalities. After treatment with the Chinese medicine dry powder of the present invention, compared with the model group, the body weight, fasting blood sugar, cardiac index, total cholesterol, triglyceride, and low-density lipoprotein levels were reduced, and the insulin and high-density lipoprotein levels were increased (p<0.05). It shows that the Chinese medicine dry powder of the present invention can improve the systemic metabolic abnormalities of diabetic mice caused by high-sugar and high-fat diet and streptozotocin intervention.

[0101] 2.4 The Chinese medicine dry powder of the present invention improves the cardiac function damage in diabetic patients

[0102] To evaluate the improving effect of Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract on diabetic cardiac function damage, the cardiac function (including LVEF, LVFS, LVEDV, LVESV) of rats in each group was detected and recorded by echocardiography. Figure 7 .

[0103] like Figure 7 As shown in the figure, compared with the control group, the LVEF and LVFS in the model group were significantly decreased (p<0.05), and the LVEDV and LVESV were significantly increased (p<0.05). After treatment with Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract, LVEF and LVFS increased, and LVEDV and LVESV showed a downward trend, but the improvement of LVEDV was not very obvious. These results indicate that Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract treatment can alleviate cardiac dysfunction in DCM mice.

[0104] 3.5. Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract improves myocardial structural damage in diabetic patients

[0105] In the development of diabetic cardiomyopathy, there are changes in the morphology and structure of the myocardium. Therefore, HE staining was used to observe the effect of Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract on the pathological changes of myocardial tissue.

[0106] like Figure 8 As shown in the figure, the pathological changes of myocardial fiber rupture in the group treated with Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract were significantly reduced compared with the diabetic cardiomyopathy model group. Masson staining was used to observe the myocardial fibrosis in rats in each group. Figure 8 In the experiment, we found that the collagen deposition in the heart of the diabetic cardiomyopathy model group mice increased significantly compared with the control group. After intervention with Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis extract, the degree of liver fibrosis and collagen content were reduced.

[0107] 2.6 Potential active ingredients in Paeonia suffruticosa-Gardenia jasminoides-Coptis chinensis and their network construction

[0108] Under the conditions of OB ≥ 30% and DL ≥ 0.18, a total of 44 compounds were obtained from the TCMSP database and HPLC analysis. Among them, quercetin is a common component of Paeonia suffruticosa, Gardenia jasminoides and Coptis chinensis, and kaempferol is a common component of Paeonia suffruticosa and Gardenia jasminoides. Subsequently, 1480 target genes were collected from 44 potential active ingredients in the TCMSP and Swiss TargetPrediction databases. After deleting duplicate genes, there were 475 target genes. The active ingredient information is shown in Table 1.

[0109] Table 1 Active ingredient information

[0110]

[0111]

[0112] The "Traditional Chinese Medicine - Active Ingredient - Target" network of Cortex Moutan - Gardenia jasminoides - Coptis chinensis is as Figure 9 shown.

[0113] 2.7 Disease genes of diabetic cardiomyopathy

[0114] Using "Diabetic cardiomyopathy" as the keyword in the GeneCards database, with the settings of score ≥ 5 and species human, a total of 2391 indicators were collected.

[0115] 2.8 Venny analysis and construction of the "Traditional Chinese Medicine - Compound - Potential Therapeutic Target - Diabetic Cardiomyopathy" network

[0116] The results of the Venny analysis of the target genes related to chemical components and the disease genes related to diabetic cardiomyopathy are shown in Figure 10 , and a total of 186 intersection targets were obtained, as shown in Table 2. The "Traditional Chinese Medicine - Compound - Potential Therapeutic Target - Diabetic Cardiomyopathy" network is as Figure 11 shown.

[0117] Table 2 186 intersection targets

[0118]

[0119]

[0120]

[0121] 2.9 Construction of the PPI network and screening of core targets

[0122] The PPI network of potential therapeutic targets obtained from the string database is as Figure 12 shown. Then, according to the preset parameters, the PPI network of potential therapeutic targets was input into Cytoscape (version 3.9.1) software to obtain the core targets. A total of 31 core targets were obtained: AKT1, HSP90AA1, SRC, TP53, MAPK1, EP300, CTNNB1, EGFR, ESR1, TNF, CAV1, HRAS, MAPK14, IL6, FOS, MYC, MAPK8, PRKCZ, PRKCA, EGF, VEGFA, IL10, HIF1A, RB1, AR, TGFB1, PTEN, PPARG, CASP8, NOS2, PPARA. The topological parameters of the core targets are shown in Table 3. The PPI network of the core targets obtained from the string database is as Figure 13 shown.

[0123] Table 3 Topological parameters of core targets

[0124]

[0125]

[0126] 2.10 GO and KEGG enrichment analysis

[0127] The potential therapeutic targets were imported into the Metascape database for GO and KEGG analyses. 426 items were obtained from the GO enrichment analysis, among which 212 items were included in BP, 94 items in CC, and 120 items in MF. The top 20 items of BP, CC, and MF in the GO analysis are listed in Table 4 and Figure 14 . 197 pathways for the treatment of diabetic cardiomyopathy by Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis were obtained through KEGG analysis, and the top 20 items are listed in Table 5 and Figure 15 .

[0128] Table 4 Top 20 items of GO analysis

[0129]

[0130]

[0131]

[0132] Table 5 Top 20 items of KEGG analysis

[0133]

[0134] 2.11 Screening of target genes to be verified

[0135] The "AGE - RAGE signaling pathway in diabetic complications" signaling pathway contains a total of 51 target genes, including AGTR1, AKT1, AKT2, BAX, CCND1, BCL2, CASP3, CDK4, COL1A1, COL3A1, MAPK14, F3, HRAS, ICAM1, IL1A, IL1B, IL6, CXCL8, MMP2, NOS3, SERPINE1, PRKCA, PRKCB, PRKCZ, MAPK1, MAPK8, MAPK10, CCL2, SELE, STAT1, TGFB1, TNF, VCAM1, VEGFA, CALM1, CAV1, CTNNB1, FOS, GSTM1, GSTP1, HMOX1, HSP90AA1, IFNG, IKBKB, KDR, MMP9, NFE2L2, PLAT, SRC, TP53, NCF1.

[0136] The "Diabetic cardiomyopathy" signaling pathway contains a total of 36 target genes, including PARP1, AGTR1, AKT1, AKT2, COL1A1, COL3A1, MAPK14, CTSD, ACE, MTOR, G6PD, GAPDH, SK3B, INSR, MMP2, MMP9, NOS3, PPARA, PRKCA, PRKCB, PRKCZ, MAPK8, MAPK10, PTEN, SLC2A4, TGFB1, NCF1, IKBKB, IL6, NFKBIA, PTPN1, PTPN11, PYGM, RPS6KB1, TNF, MAPK1. Subsequently, the targets and core targets included in the above two pathways were input into the Venny data platform to obtain an intersection target dataset, as Figure 16 shown. A total of 9 targets were screened, including AKT1, MAPK1, TNF, MAPK14, IL6, MAPK8, PRKCZ, PRKCA, and TGFB1, for the next step of verification.

[0137] 2.12 Western Blot

[0138] The results are shown in Figure 17 .

[0139] As Figure 17 shown, the protein expression levels of IL-6, MAPK1, and PRKCA in the diabetic cardiomyopathy model group were significantly higher than those in the control group (p < 0.05). After treatment with the traditional Chinese medicine dry powder, the expression levels of the above three proteins were all decreased (p < 0.05). These results indicate that the traditional Chinese medicine dry powder of the present invention may play a therapeutic role in diabetic cardiomyopathy by regulating the "AGE-RAGE signaling pathway in diabetic complications" / "Diabetic cardiomyopathy" signaling pathway.

[0140] Network pharmacology is an effective method that combines the multi-component and multi-target characteristics of traditional Chinese medicine with diseases. There have been relevant studies on using network pharmacology to explore the treatment of diabetic cardiomyopathy with traditional Chinese medicine, such as Yunu Decoction, Zuogui Jiangtang Shuxin Prescription, and Erzhi Pills. Through network pharmacology analysis in this invention, 186 intersection targets, 426 GO enrichment terms, and 197 KEGG enrichment terms were obtained. In the BP term analysis, there are some items related to diabetic cardiomyopathy, such as the response to endotoxin, the response to oxygen level, the regulation of apoptotic signaling pathway, heart development, etc. These results are related to the pathogenesis of diabetic cardiomyopathy. In addition, KEGG enrichment analysis indicates that the "AGE-RAGE signaling pathway in diabetic complications" and "Diabetic cardiomyopathy" signaling pathways may play a key role in the process of treating diabetic cardiomyopathy with Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis. Therefore, we collected the targets on these two signaling pathways and obtained the intersection targets with the core targets for verification.

[0141] Insulin decreases in both type 1 diabetes and type 2 diabetes patients, which leads to hyperglycemia and hyperlipidemia. Chronic hyperglycemia and hyperlipidemia are two main characteristics of diabetes, and both promote the development of cardiac insufficiency. In in vivo experiments, after 8 weeks of treatment with the extract of Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis (i.e., the traditional Chinese medicine dry powder of this invention), the body weight, INS, and high-density lipoprotein levels increased significantly, while the fasting blood glucose, total cholesterol, triglyceride, and low-density lipoprotein levels decreased significantly. These results indicate that the extract of Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis can alleviate the systemic metabolic disorders in diabetic mice. Diabetic cardiomyopathy, as a unique cardiac manifestation in diabetes patients, is characterized by left ventricular hypertrophy, diastolic dysfunction, and reduced systolic function. Diabetic cardiomyopathy eventually develops into heart failure, leading to the death of advanced patients. After treatment with the extract of Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis, the cardiac index and LVDEV decreased, while LVES, LVFS, and LVESV increased. In HE and Masson staining, we can find that the cardiac lesions were alleviated. These results indicate that the extract of Cortex Moutan - Fructus Gardeniae - Rhizoma Coptidis has a protective effect on the myocardial structural changes in diabetic cardiomyopathy model mice.

[0142] According to the results of KEGG network pharmacology experiments, Western blotting was used to detect the expression of IL-6, MAPK1, and PRKCA in myocardial tissues. The expressions of the above three proteins were upregulated in the brains of DCM model mice, while their expression levels were downregulated after intervention with the Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis extract. The development of myocardial tissue inflammation promoted the progression of pathological changes such as myocardial hypertrophy and myocardial fibrosis. As a pro-inflammatory mediator, IL-6 can exacerbate the development of insulin resistance and heart failure. The Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis extract treatment group could effectively reduce the level of IL-6. The activation of the MAPK pathway affects the pathogenesis of diabetic cardiomyopathy and heart failure, leading to myocardial hypertrophy, myocardial fibrosis, and myocardial damage. The Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis extract can downregulate the expression of MAPK1 in diabetic cardiomyopathy model mice. An increase in the expression of protein kinase α subtype can lead to myocardial cell hypertrophy, cardiac microvascular lesions, and interstitial fibrosis, eventually developing into heart failure. From the results of Western blot analysis, it can be seen that the expression of PRKCA decreased after intervention with the Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis extract. The above results indicate that Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis can inhibit the expression of IL-6, MAPK1, and PRKCA in diabetic cardiomyopathy model mice.

[0143] Through experiments, the present invention found that Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis has a therapeutic effect on diabetic cardiomyopathy caused by diabetes. The exertion of the therapeutic effect of Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis may rely on alleviating the systemic metabolic disorder in diabetic mice and reducing the expression and activation of IL-6, MAPK1, and PRKCA through the "AGE-RAGE signaling pathway in diabetic complications" / "Diabetic cardiomyopathy" signaling pathway. These findings may further support the clinical treatment of diabetic cardiomyopathy. In addition, it is necessary for us to further study which components in Paeonia suffruticosa Gardenia jasminoides Rhizoma Coptidis play the main role.

[0144] The foregoing description of specific exemplary embodiments of the invention is for purposes of illustration and exemplification. These descriptions are not intended to limit the invention to the precise forms disclosed, and obviously, many modifications and variations are possible in light of the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the invention and its practical applications, so that those skilled in the art can implement and utilize various different exemplary embodiments of the invention as well as various different selections and modifications. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A Chinese medicine dry powder is used in the preparation of a drug for treating diabetic cardiomyopathy, characterized in that: The preparation method of the Chinese medicine dry powder is as follows: weigh 1 kg each of peony bark, gardenia and coptis root, extract twice with distilled water, combine the filtrate, concentrate, and spray dry to obtain the Chinese medicine dry powder; wherein, for the first extraction, add 8 times the mass of water, soak for 30 minutes, and reflux extraction for 60 minutes; for the second extraction, add 6 times the mass of water, and reflux extraction for 40 minutes.

Images