A traditional Chinese medicine compound preparation for protecting cardiovascular function and application thereof

Abstract

The application discloses a traditional Chinese medicine compound preparation with cardiovascular protection and application thereof, and belongs to the technical field of traditional Chinese medicine preparations. The traditional Chinese medicine compound preparation is composed of the following traditional Chinese medicinal materials in parts by weight: chrysanthemum 5-10, hawthorn 9-12, medlar 6-12, turmeric 3-10, and schisandra chinensis 2-6. Animal experiments show that the traditional Chinese medicine compound preparation can significantly improve aortic elasticity, carotid hemodynamics and cardiac systolic function, reduce learning and memory impairment, resist inflammation and oxidative stress, and has no adverse effect on liver and kidney functions. The application provides a safe and multi-target new selection for cardiovascular protection and prevention and treatment of related cardiovascular diseases.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, and in particular relates to a traditional Chinese medicine compound preparation with cardiovascular protective effects and its application. Background Technology

[0002] The incidence of obesity and hyperlipidemia continues to rise, becoming the leading risk factor for metabolic cardiovascular diseases. Long-term lipid metabolism disorders can persistently damage vascular endothelial function, promote abnormal proliferation and migration of vascular smooth muscle, ultimately leading to aortic wall thickening and vascular remodeling. Simultaneously, the cumulative lipid toxicity induces myocardial ischemia, myocardial interstitial damage, and progressive decline in cardiac function, seriously threatening patients' lives and health. Currently, mainstream lipid-lowering drugs such as statins, ezetimibe, and PCSK9 inhibitors can effectively regulate blood lipid levels, but their effects on improving vascular endothelial damage, reversing pathological vascular remodeling, and protecting against myocardial damage are limited, making it difficult to comprehensively block the multi-faceted cardiovascular pathological damage mediated by obesity and hyperlipidemia.

[0003] Given the aforementioned challenges, the development of a safe and effective drug to protect the cardiovascular system is urgently needed. Traditional Chinese medicine (TCM) compound formulas, with their unique advantages of multiple components, multiple pathways, and holistic regulation, represent a new direction for solving these problems. The various components in TCM compound formulas can not only improve dyslipidemia, protect vascular endothelium, inhibit vascular remodeling, and reduce myocardial ischemia damage, but also intervene in the progression of obesity-related cardiovascular diseases from multiple angles. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes a traditional Chinese medicine compound preparation with cardiovascular protective effects and its application, providing a new option for drugs with cardiovascular protective effects.

[0005] To achieve the above objectives, the present invention provides a traditional Chinese medicine compound preparation for protecting the cardiovascular system, which is composed of the following traditional Chinese medicinal materials in parts by weight: chrysanthemum 5-10, hawthorn 9-12, wolfberry 6-12, turmeric 3-10, and schisandra 2-6.

[0006] Preferably, the dosage form is decoction, pill, powder, capsule, tablet, granule or drop pill.

[0007] Preferably, the decoction is prepared by the following method: after washing the Chinese medicinal materials in the specified weight proportions, soak them in water for 6-8 hours, bring to a boil over high heat, then simmer over low heat for 30-60 minutes, filter and collect the juice; then add water and decoct a second time, bring to a boil over high heat, then simmer over low heat for 20-40 minutes, combine the two decoctions and concentrate to obtain the final product.

[0008] Furthermore, the present invention also provides the application of the above-mentioned traditional Chinese medicine compound preparation in the preparation of drugs for protecting the cardiovascular system.

[0009] Preferably, the drug is used to improve vascular structure and elasticity; the improvement in vascular structure and elasticity is manifested by increasing the difference between the systolic and diastolic diameters of the aortic arch, reducing the thickness of the intima to media of the aortic sinus, and / or reducing the carotid pulse wave velocity.

[0010] Preferably, the drug is used to improve cardiac function; the improvement of cardiac function is manifested as increasing the left ventricular ejection fraction and / or shortening the ejection fraction.

[0011] Preferably, the drug is used to inhibit oxidative stress in cardiac tissue; the inhibition of oxidative stress is manifested by reducing malondialdehyde content and / or increasing superoxide dismutase activity.

[0012] Preferably, the drug is used to target and activate the PI3K-AKT-eNOS signaling pathway and / or inhibit the STAT3 signaling pathway.

[0013] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention is the first to discover that a traditional Chinese medicine compound composed of chrysanthemum, hawthorn, wolfberry, turmeric and schisandra can improve the structure and function of blood vessels such as the aortic arch and carotid artery, and enhance the heart's pumping function.

[0014] (2) The present invention initially revealed the mechanism of action of the compound, and found that it can upregulate the ratio of p-PI3K / PI3K, p-AKT / AKT and p-eNOS / eNOS in the aorta and heart tissue, and downregulate the ratio of p-STAT3 / STAT3, providing a new drug target for lowering blood lipids and protecting the cardiovascular system.

[0015] (3) It was found that TMAO-induced endothelial dysfunction in HUVEC cells promoted NO release, and its effect was related to inhibiting abnormal STAT3 activation and upregulating PI3K / AKT / eNOS axis activity.

[0016] (4) The formulation has a certain inhibitory effect on LPS-induced inflammatory response in RAW264.7 cells, reduces NO and TNF-α levels, and downregulates the expression of iNOS, COX-2 protein and IL-6 mRNA.

[0017] (5) The traditional Chinese medicine compound of the present invention can be prepared into various dosage forms as needed to meet the medication needs of different patients. Moreover, the raw materials are widely available, the preparation process is simple, the cost is controllable, and it has good application prospects. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a network diagram of protein-protein interactions (PPIs) between the target sites of the traditional Chinese medicine compound and cardiovascular disease-related targets in this invention. Figure 2 This is a GO functional enrichment analysis chart of the traditional Chinese medicine compound for the treatment of cardiovascular diseases in this invention; Figure 3 This is a KEGG pathway enrichment analysis diagram of the traditional Chinese medicine compound for the treatment of cardiovascular diseases in this invention; Figure 4 The figure shows the effect of the traditional Chinese medicine compound of the present invention on the weight changes of obese mice before and after modeling and after drug administration; where A is the baseline weight before modeling; B is the weight after 6 weeks of high-fat feeding; C is the weight after 4 weeks of drug administration; and D is the overall weight change trend. Figure 5 This diagram illustrates the effects of the traditional Chinese medicine compound of this invention on inflammation-related markers in obese mice; where A represents LDL-C level, B represents TC level, C represents TG level, D represents HDL-C level, E represents LDH level, F represents CK level, G represents Hcy level, H represents CK-MB level, I represents IL-6 level, and J represents TNF-α level; the model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, * P <0.05,** P <0.01, *** P <0.001; Figure 6 This is a graph showing the effects of the traditional Chinese medicine compound of this invention on liver and kidney function-related indicators in obese mice; where A represents UA level; B represents UREA level; C represents Cr level; D represents ALT level; and E represents AST level. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 7 These are representative ultrasound images of the aortic arch, carotid artery, heart, liver, and kidneys of obese mice, obtained using the traditional Chinese medicine compound of this invention; the model group was compared with the normal control group.### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 8 This diagram illustrates the effects of the traditional Chinese medicine compound of this invention on the aortic arch diameter difference and vessel wall thickness in obese mice; where A represents the difference in aortic arch diameter between systole and diastole, and B represents the thickness of the intima to media of the aortic sinus; the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 9 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on the Doppler pulse velocity of the carotid artery in obese mice; the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 10 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on left ventricular ejection fraction (LVEF) and left ventricular fraction of shortening (LVFS) in obese mice; compared with the normal control group, ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 11 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on the liver-kidney echo ratio in obese mice; the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, ***P <0.001; Figure 12 This diagram illustrates the effect of the traditional Chinese medicine compound of this invention on spatial learning and memory abilities in obese mice; where A is the trajectory diagram; B is the escape latency statistics; C is the target quadrant dwell time statistics; and D is the platform crossing frequency statistics. The model group is compared with the normal control group. ## P <0.01, ### P <0.001; Comparison between the drug group and the model group, * P <0.05, ** P <0.01, *** P <0.001; Figure 13 HE staining images of various organs (liver, kidney, fat, aorta, colon, lung, and heart) of obese mice by the traditional Chinese medicine compound of this invention; Figure 14 This is a statistical chart comparing the aortic wall thickness of mice in different treatment groups; the model group is compared with the normal control group. ## P <0.01, ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 15 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on the MDA and SOD content in the heart tissue of obese mice; where A represents MDA content; B represents SOD content; and the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, ** P <0.01, *** P <0.001; Figure 16 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on the expression of PI3K / AKT / eNOS pathway proteins in the heart of obese mice; where A is the WB band of PI3K / AKT / eNOS pathway proteins; B is the p-PI3K / PI3K ratio; C is the p-AKT / AKT ratio; and D is the p-eNOS / eNOS ratio; the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, ** P <0.01, *** P <0.001; Figure 17 This is a graph showing the effect of the traditional Chinese medicine compound of this invention on the expression of PI3K / AKT / eNOS pathway proteins in the aorta of obese mice; where A is the WB band of PI3K / AKT / eNOS pathway proteins; B is the p-PI3K / PI3K ratio; C is the p-AKT / AKT ratio; and D is the p-eNOS / eNOS ratio; the model group is compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, ** P <0.01, *** P <0.001; Figure 18 This diagram illustrates the effect of the traditional Chinese medicine compound of this invention on STAT3 protein expression in the heart and aorta of obese mice. In the diagram, A represents the Western blot (WB) band of STAT3 protein in the heart, B represents the p-STAT3 / STAT3 ratio in the heart, C represents the WB band of STAT3 protein in the aorta, and D represents the p-STAT3 / STAT3 ratio in the aorta. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 19 This invention relates to the effects of a traditional Chinese medicine compound on HUVEC endothelial cells. Specifically, A represents the effect of different concentrations of the traditional Chinese medicine compound on HUVEC cell activity, and B represents the effect of the traditional Chinese medicine compound on TMAO-induced NO production in HUVEC endothelial cells. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 20 This diagram illustrates the effect of the traditional Chinese medicine compound of this invention on TMAO-induced HUVEC endothelial cells. In the diagram, A represents the Western blot (WB) bands of pathway proteins in TMAO-induced HUVEC endothelial cells, B represents the relative protein expression levels of p-PI3K / PI3K, C represents the relative protein expression levels of p-AKT / AKT, and D represents the relative protein expression levels of p-eNOS / eNOS. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 21This is a graph showing the effect of the traditional Chinese medicine compound of this invention on STAT3 protein expression in HUVEC endothelial cells; where A is the WB band of STAT3 protein in HUVEC endothelial cells, and B is the p-STAT3 / STAT3 ratio in endothelial cells; compared with the normal control group, ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 22 This invention relates to the effects of a traditional Chinese medicine compound on RAW264.7 cells. Specifically, A represents the effect of different concentrations of the traditional Chinese medicine compound on the activity of RAW264.7 cells; B represents the effect of the traditional Chinese medicine compound on LPS-induced NO production in RAW264.7 cells; and C represents the effect of the traditional Chinese medicine compound on LPS-induced TNF-α production in RAW264.7 cells. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 23 This invention relates to the effects of a traditional Chinese medicine compound on the expression of iNOS and COX-2 proteins induced by LPS in RAW264.7 cells. In the figure, A represents the Western blot (WB) bands of iNOS and COX-2 proteins, B represents the relative protein expression levels of COX-2 / β-actin in LPS-induced RAW264.7 cells, and C represents the relative protein expression levels of iNOS / β-actin in LPS-induced RAW264.7 cells. The model group was compared with the normal control group. ### P <0.001; Comparison between the drug group and the model group, *** P <0.001; Figure 24 LPS-induced IL-6 mRNA expression in RAW264.7 cells; compared with the normal control group, ### P <0.001; Comparison between the drug group and the model group, *** P <0.001. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the term "parts" in this invention refers to parts by weight.

[0026] Example 1 A traditional Chinese medicine compound consists of 5g chrysanthemum, 9g hawthorn, 6g wolfberry, 3g turmeric, and 2g schisandra.

[0027] After washing the above-mentioned Chinese medicinal materials, soak them in 200mL of water for 8 hours. Bring to a boil over high heat, then simmer over low heat for 60 minutes. Filter and collect the juice. Add another 200mL of water and decoct a second time. Bring to a boil over high heat, then simmer over low heat for 20 minutes. Combine the two decoctions and concentrate to 100mL to prepare a Chinese herbal decoction.

[0028] Example 2 A traditional Chinese medicine compound is composed of 5g chrysanthemum, 10g hawthorn, 8g wolfberry, 5g turmeric, and 3g schisandra; the preparation method of the decoction is the same as in Example 1.

[0029] Example 3 A traditional Chinese medicine compound is composed of 10g chrysanthemum, 12g hawthorn, 10g wolfberry, 8g turmeric, and 4g schisandra; the preparation method of the decoction is the same as in Example 1.

[0030] Example 4 A traditional Chinese medicine compound is composed of 10g of chrysanthemum, 12g of hawthorn, 12g of wolfberry, 10g of turmeric, and 6g of schisandra; the preparation method of the decoction is the same as in Example 1.

[0031] Test Example 1 The traditional Chinese medicine decoction prepared in Example 1 was analyzed by ultra-high performance liquid chromatography-quadrupole electrostatic field orbital trap tandem mass spectrometry (UPLC-MS / MS) to analyze the phytochemicals contained therein.

[0032] Liquid chromatography system: Thermo Vanquish UHPLC. Mobile phase: 0.1% formic acid aqueous solution (A), pure acetonitrile (B); injection volume 2 μL; flow rate 0.3 mL / min; column temperature: 30℃; column: Zorbax Eclipse C18 column (100 mm × 2.1 mm, 1.8 μm). Gradient elution program: 0–2 min, 95% A; 2–6 min, 95% → 90% A; 6–12 min, 90% → 80% A; 12–19 min, 80% → 70% A; 19–23 min, 70% → 55% A; 23–27 min, 55% → 40% A; 27–29 min, 40% → 20% A; 29–32 min, 20% → 5% A; 32–38 min, 5% → 95% A.

[0033] Mass spectrometry system: Q-Exactive HF (Thermo Fisher Scientific), ESI thermal spray ionization source. Ion source parameters: spray voltage 3.5kV (+) / 3.5kV (-); sheath gas 45 arb; capillary temperature 330℃; auxiliary gas flow rate 15 arb; purge gas flow rate 1 arb; probe heater temperature 325℃.

[0034] Mass spectrometry scanning parameters: Scan mode: Full MS-ddMS2; Resolution: 120,000 (Level 1 mass spectrometry) & 60,000 (Level 2 mass spectrometry); Scan range: 100-1500 m / z; Maximum dwell time: 50 ms (Full MS) and 100 ms (ddMS2 / dd-SIM); AGC target: 1e6 (Full MS) and 2e5 (ddMS2 / dd-SIM); Minimum AGC target: 8.00e3. Mass spectrometry data were processed using Compound Discoverer 3.3 and compared and identified using the mzVault and mzCloud databases.

[0035] UPLC-MS / MS identified 276 compounds, which can be divided into 13 categories based on their chemical structure characteristics. The main categories are phenols (38), alkaloids (33), flavonoids (29), terpenes (27), and fatty acids (24). In addition, the categories include organic acids, amino acids, nucleosides, sugars, coumarins, lignans, quinones, and other compounds.

[0036] Test Example 2 The role of traditional Chinese medicine formulas in cardiovascular diseases.

[0037] Using "Cardiovascular disease" as the keyword, cardiovascular disease-related targets were searched in the GeneCards and OMIM databases. The obtained target information was integrated into an Excel spreadsheet, duplicates were removed, and gene names were standardized using the UniProt database to finally obtain the disease-related target information.

[0038] like Figure 1 As shown, after taking the intersection of all drug targets with cardiovascular disease target genes, 423 intersection target genes of cardiovascular disease and drugs were obtained, which are interactive target genes for drug treatment of cardiovascular disease.

[0039] Drug-disease intersection targets were imported into the DAVID database. The gene identifier was selected as OFFICIAL_GENE_SYMBOL, and the species was set as Homo sapiens. KEGG pathway enrichment analysis was performed. GO analysis elucidated the potential biological function of the formulation in treating cardiovascular diseases from three levels: biological process (BP), cellular component (CC), and molecular function (MF). KEGG pathway enrichment analysis was used to reveal the main signaling pathways involved by related targets. Significantly enriched GO items and KEGG pathways were screened, and the top 10 items for BP, CC, and MF, as well as the top 20 KEGG pathways related to cardiovascular diseases, were selected for visualization analysis to predict the potential mechanism of action of the formulation in treating cardiovascular diseases. Figure 2 ).

[0040] Pathway enrichment analysis using the DAVID database identified 209 pathways related to the treatment of cardiovascular diseases using the formulation. P <0.01 identified 185 pathways related to the formulation and treatment of cardiovascular diseases, and the results are as follows: Figure 3The pathways shown are: lipids and atherosclerosis, fluid shear stress and atherosclerosis, apoptosis, tumor necrosis factor signaling pathway, endocrine resistance, PI3K-AKT signaling pathway, cell senescence, FOXO signaling pathway, neurotrophic factor signaling pathway, and vascular endothelial growth factor signaling pathway.

[0041] Test Example 3 An obesity model was established using C57BL / 6 mice. All C57BL / 6 mice were male, weighing approximately 20g, and SPF grade. The mice were fed a high-fat diet (purchased from Dyets, product model HF60) for 6 weeks to establish the obesity model. Mice fed a normal diet served as the control group.

[0042] The experimental groups are as follows: Normal control group: After being fed with normal feed for six weeks, they were given physiological saline (0.9%) by gavage for four weeks.

[0043] Model group: fed a high-fat diet without any drug intervention.

[0044] Low-dose group: After six weeks of feeding on a high-fat diet, patients were given a four-week course of traditional Chinese medicine formula (25 mg / (kg·d)) by gavage.

[0045] High-dose group: After six weeks of feeding on a high-fat diet, patients were given a four-week course of traditional Chinese medicine formula (50 mg / (kg·d)) by gavage.

[0046] Positive control group: After being fed a high-fat diet for six weeks, they were given atorvastatin (10 mg / (kg·d)) by gavage for four weeks.

[0047] Mouse weight changes as follows Figure 4 As shown, the baseline weight of mice in each group before modeling was approximately 20g. Figure 4 In group A, compared with the normal control group, the weight of mice in all other groups significantly increased after being fed a high-fat diet for 6 weeks. Figure 4 (Middle B), indicating that the mouse obesity model was successfully constructed. P <0.001). Following this, a 4-week intervention involving gavage administration was performed. Compared to the model group, the body weight of mice in each administration group showed varying degrees of decrease, with significant overall changes. Figure 4 (C). After the administration period, the body weight of each treatment group was lower than that of the model group, and the difference was statistically significant. P <0.001). Furthermore, the overall weight change trend chart shows ( Figure 4 In the D group, the body weight continued to increase, while the body weight growth trend in each drug-treated group was somewhat suppressed, suggesting that the formulation intervention can improve the abnormal weight gain in mice induced by a high-fat diet to some extent.

[0048] Test Example 4 The levels of triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in mouse serum were detected using corresponding kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. TG was detected using the single-reagent GPO-PAP method, LDL-C and HDL-C were detected according to the respective kit instructions, and TC was detected and calculated according to the kit instructions.

[0049] The levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB), lactate dehydrogenase (LDH), and homocysteine ​​(Hcy) in mouse serum were detected using kits from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. CK-MB was detected using an immunosuppression method, while the other indicators were detected according to the respective kit instructions. The levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cell supernatant were detected using enzyme-linked immunosorbent assay (ELISA).

[0050] Results of blood lipid, myocardial damage and related biochemical indicators are as follows Figure 5 As shown, compared with the model group, the levels of TG, LDL-C, Hcy, IL-6, and TNF-α in each treatment group were significantly reduced ( P <0.001); TC was significantly reduced in the low-dose group ( P <0.01%, and all other treatment groups showed significant reductions ( P <0.001). HDL-C levels showed some variation, but the difference was not statistically significant. CK levels showed no significant difference between the low-dose group and the model group, while they were significantly lower in the high-dose group and the positive control group. P <0.001. LDH levels were not significantly different between the low-dose group and the model group, but were significantly lower in both the high-dose group and the positive control group. P <0.01. CK-MB levels showed no significant difference between the low-dose group and the model group, but decreased in both the high-dose group and the positive control group, with a statistically significant difference in the high-dose group. P <0.05, the difference was more significant in the positive drug group ( P <0.01).

[0051] The above results suggest that this formula can play a certain protective role against obesity-related cardiovascular damage by regulating lipid metabolism, reducing tissue damage, and inhibiting inflammatory responses.

[0052] Test Example 5 Liver and kidney function-related indicators in mouse serum were detected using appropriate kits. Liver function indicators, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were detected using kits from Nanjing Jiancheng Biotechnology Co., Ltd. Kidney function indicators, including uric acid (UA), creatinine (Cr), and urea (UREA), were detected using kits from Nanjing Jiancheng Biotechnology Co., Ltd., and UREA using kits from Beyotime Biotechnology Co., Ltd. All tests were performed and results were calculated according to the respective kit instructions. Figure 6 As shown in the figure, no significant abnormalities were observed in serum liver and kidney function-related indicators in each group. Therefore, this formula did not cause significant liver and kidney function damage and has a certain degree of preliminary safety.

[0053] Test Example 6 In Test Case 3, mice underwent ultrasound examination after the last administration. Prior to the examination, hair was removed from the chest, neck, and upper abdomen. Mice were placed on a constant-temperature heating table to maintain a body temperature of approximately 37°C, and heart rate was continuously monitored. Inhalation anesthesia (1-2% isoflurane) was used to induce and maintain the depth of anesthesia. Mice were fixed in a supine position, and pre-warmed ultrasound coupling gel was applied to reduce artifacts. All parameters were recorded continuously for at least 3-5 cardiac cycles, and the average value was used for statistical analysis.

[0054] (1) With the mouse in a supine position, the probe was placed in a suitable section above the suprasternal notch or beside the sternum to obtain the long axis image of the aortic arch. After the boundary between the vessel lumen and the vessel wall was clearly displayed in 2D B-mode, the image was frozen. The aortic arch diameter (Ds and Dd) was measured at end-systole and end-diastole, respectively. The difference between the systolic and diastolic diameters was calculated, and the vessel wall thickness was measured at the same time.

[0055] (2) High-frequency ultrasound was used to locate the long axis section of the common carotid artery in two-dimensional B-mode, ensuring clear vascular course and stable images. Carotid pulse waveforms were recorded in M-mode or pulsed Doppler (PW Doppler) mode, and 3-5 consecutive and stable cardiac cycles were selected for analysis. Carotid PWV was calculated as "distance / time difference": two measurement points (Δx) were selected at a fixed distance in the same vascular segment, and the time difference (Δt, transit time) of the pulse wave reaching the two points was measured. The calculation was: PWV = Δx / Δt (m / s).

[0056] (3) Keep the mouse in a supine position and obtain a clear image of the left ventricle in the long axis section next to the sternum. Under the guidance of two-dimensional B mode, obtain M-mode ultrasound images at the level of the papillary muscles and measure the left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD). The left ventricular ejection fraction (LVEF) and ventricular shortening fraction (LVFS) can be calculated directly from the data obtained by the system.

[0057] (4) The probe is placed in the upper right abdomen to acquire two-dimensional B-mode images of the liver and right kidney on the same screen (or separately acquired under the same parameter settings). To ensure comparability, the imaging parameters such as gain, depth, dynamic range, and TGC are kept consistent for each group during acquisition. ROIs of the same size are selected on the images of the liver parenchyma and renal cortex, and the average gray value is measured to calculate the liver-kidney echo ratio L / K: L / K = average gray value of liver ROI / average gray value of renal cortex ROI.

[0058] The ultrasound images of the mice in tests (1)-(4) are as follows Figure 7 As shown.

[0059] The results of the blood vessel wall thickness test are as follows Figure 8 As shown, A represents the difference in aortic arch diameter between systole and diastole, and B represents the thickness of the intima to media of the aortic sinus. The results showed that, compared with the model group, the difference in aortic arch diameter between systole and diastole was significantly increased in the high-dose group and the positive control group, with statistically significant differences. P <0.001, suggesting that the formula can improve aortic elasticity loss under high-fat conditions. Compared with the normal group, the model group showed a significant increase in the thickness of the intima to media in the aortic sinus region ( ). P <0.01, suggesting that a high-fat diet can lead to thickening of the blood vessel wall. Compared with the model group, the high-dose formulation group and the positive control group can reduce the thickness of the intima to media in the aortic sinus (…). P <0.05 indicates that the formula has a certain ameliorative effect on blood vessel wall thickening caused by obesity.

[0060] Carotid Doppler pulse velocity test results as follows Figure 9 As shown, the results indicated that carotid Doppler pulse velocity was significantly improved after the intervention, with a particularly significant difference in the low-dose group. P <0.01%, with more significant improvement in the high-dose group and the positive control group ( P <0.001), indicating that the formulation can improve hemodynamic abnormalities in obese mice.

[0061] The results of left ventricular ejection fraction (LVEF) and ventricular shortening fraction (LVFS) tests are as follows: Figure 10 As shown, the results indicated that, compared with the model group, the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) of mice increased after intervention with the formulation, with statistically significant differences observed in the low-dose group and the positive control group. P <0.05%, the high-dose group showed a more significant improvement ( P The value is <0.001, suggesting that the formula can improve the decreased cardiac contractile function caused by obesity to a certain extent.

[0062] Liver-kidney echo ratio test results as follows Figure 11 As shown in the figure. Compared with the model group, the liver-kidney ratios in each treatment group were significantly improved, and the differences were all statistically significant. P The value was <0.001, indicating that the formulation also has a good regulatory effect on abnormal changes in liver and kidney ultrasound under high-fat conditions.

[0063] In summary, the traditional Chinese medicine formula intervention of this invention can improve obesity to varying degrees.

[0064] The study investigated abnormalities in vascular structure and function, decreased cardiac contractile function, and liver and kidney-related ultrasound indicators in mice, with the most significant improvements observed in changes in aortic arch diameter, carotid Doppler velocity, and liver-kidney ratio.

[0065] Test Example 7 The Morris water maze apparatus consisted of a circular pool, 120 cm in diameter and 50 cm high, with a water depth of approximately 30 cm and a water temperature maintained at 22 ± 1 °C. A suitable amount of non-toxic white pigment was added to the water to make it opaque. The pool was divided into four quadrants, with a hidden platform approximately 10 cm in diameter placed in one fixed quadrant, its top about 1 cm below the water surface. Stable spatial reference objects were placed around the pool and remained unchanged throughout the experiment.

[0066] The orientation and navigation experiment was conducted continuously for 4 days, with each mouse trained 4 times per day, entering the water from a different entry point each time. The maximum swimming time per session was set at 60 seconds. After finding the platform, the mouse was allowed to stay on the platform for 10 seconds; if it could not find the platform within the specified time, the experimenter guided it to the platform and allowed it to stay there for 10-15 seconds. The escape latency of each group of mice was recorded. On the 5th day, a spatial exploration experiment was conducted. After removing the platform, the mice were placed in the water from a fixed entry point in the quadrant opposite to the original platform and allowed to swim freely for 60 seconds. The percentage of time spent in the target quadrant and the number of times the mice crossed the original platform location were recorded to evaluate the mice's spatial memory ability.

[0067] The results of the water maze experiment are as follows Figure 12 As shown, compared with the normal group, the escape latency period on day 4 was relatively prolonged in the model group ( P <0.01, the percentage of time spent in the target quadrant and the number of times the platform was traversed decreased significantly ( P <0.001 indicates that spatial learning and memory abilities are significantly impaired in obese model mice. Compared with the model group, the escape latency on day 4 was shortened in the low-dose group ( P <0.01), increased number of platform crossings ( P <0.01); Both the high-dose group and the positive group significantly shortened the escape latency period ( P <0.001) and significantly increased the number of platform crossings ( P <0.001); the percentage of time spent in the target quadrant was significantly increased in the high-dose group (P <0.001, the percentage of time spent in the target quadrant in the positive group also showed an increasing trend ( P The concentration of the drug (<0.05) indicates that the formulation can improve spatial learning and memory impairment in obese mice, with a more significant improvement in the high-dose group. Further trajectory plots show that the model group exhibited chaotic search paths and poor swimming purpose, while the drug-treated and positive control groups showed more concentrated search trajectories and reduced ineffective swimming, suggesting a possible improvement in their spatial orientation and search abilities.

[0068] Test Example 8 Mice were euthanized in accordance with animal experimentation ethics requirements, and pathological examinations were performed on the aorta, heart, liver, kidneys, fat, colon, and lung tissue of each group of mice.

[0069] (1) After tissue fixation, routine dehydration, clearing and paraffin embedding were performed. The section thickness was 4-5 μm, and the sections were baked at 60℃ for 30-60 min.

[0070] (2) Dewaxing the sections to water: xylene for 10 min × 2, followed by 100% ethanol for 5 min, 95% ethanol for 2 min, 80% ethanol for 2 min, 70% ethanol for 2 min, and distilled water for 2 min.

[0071] (3) Hematoxylin-eosin staining: Stain with hematoxylin solution for 5 min, rinse with distilled water for 10 min, differentiate with 1% hydrochloric acid alcohol for 10 s, rinse with distilled water for 1 min, repeat once, and then blue with 0.1-0.2% ammonia water or Scott's blue solution for 30-60 s, and then rinse with running water for 1 min. Stain with eosin solution for 1 min, and rinse quickly for a few seconds. Dehydrate successively with 95% ethanol for 1 min, 100% ethanol for 1 min × 2, clear with xylene for 5-10 min × 2, mount with neutral resin, air dry, and observe and acquire images under an optical microscope.

[0072] The thickness of the aortic wall was measured using ImageJ / Fiji software on HE-stained sections. Regions with relatively intact and regular cross-sections of the blood vessel were selected, and multiple measurements were taken at different locations on the blood vessel wall. The average value was taken as the thickness of the blood vessel wall for that sample.

[0073] HE staining results of various tissues are as follows Figure 13As shown, compared with the normal group, the obesity model group exhibited more significant pathological changes in the liver, kidneys, adipose tissue, and aortic vessels, mainly manifested as hepatocyte vacuolar degeneration, disordered kidney tissue structure, adipocyte hypertrophy, and thickening of the vessel walls. Focal aggregation of deeply stained cells was observed in the colon tissue, considered to be lymphocyte infiltration or lymphoid follicle-like structures, suggesting a local inflammatory response, but the overall glandular structure remained intact. The lung tissue showed some degree of structural abnormalities, while the overall morphological changes in the heart tissue were relatively less pronounced. Compared with the model group, all the above-mentioned pathological changes in the treatment groups showed varying degrees of improvement, with more significant improvements in the liver, kidneys, adipose tissue, and aortic vessels. The colon tissue also showed some degree of relief from pathological changes, but the improvement was not as significant as in the aforementioned tissues. This suggests that the formula has a certain protective effect against obesity-related vascular and multi-organ tissue damage.

[0074] To further assess vascular remodeling, Fiji software was used to measure the vessel wall thickness of HE-stained sections of the aorta. First, a relatively intact and structurally regular aortic region was selected under a 10× microscope. Then, under a 40× microscope, multiple measurements were taken at five different angles. For each angle, five measurement points (d1-d5) were selected to measure the vessel wall thickness, and the average value was taken as the wall thickness value for that angle. Finally, the average of the five angles was taken as the final aortic vessel wall thickness for that sample.

[0075] The results of HE staining of the aorta are as follows Figure 14 As shown, compared with the normal group, the blood vessel wall thickness in the model group was significantly increased ( P <0.001, suggesting that a high-fat diet can induce vascular wall thickening and structural remodeling. Compared with the model group, the vascular wall thickness in both the treatment group and the positive group was significantly reduced ( P <0.001), suggesting that drug intervention can alleviate vascular wall thickening and improve vascular structural changes.

[0076] Test Example 9 Detection of oxidative stress indicators.

[0077] MDA activity assay: 1) Weigh 10 mg of heart tissue, cut it into small pieces with scissors, and place it in a 1.5 mL centrifuge tube. Add 100 μL of 1×PBS buffer and homogenize the tissue using a motorized homogenizer. Keep the process on ice as much as possible. Centrifuge at 13,000 rpm for 15 min at 4°C. Collect the supernatant and transfer it to a new 1.5 mL centrifuge tube. Determine the protein concentration using a BCA protein assay kit for subsequent calculations.

[0078] 2) Referring to Table 1, add the sample to be tested and other solutions prepared according to the instructions to a 1.5 mL centrifuge tube, and mix thoroughly.

[0079] Table 1 Sample Dosage for MDA Detection

[0080] 3) Place the centrifuge tubes in a metal bath and heat at 100°C for 15 minutes.

[0081] 4) Cool to room temperature, centrifuge at 1000g for 10 min, and add 200µL of supernatant to each well plate.

[0082] 5) Measure the absorbance of each well at a wavelength of 532 nm using an ELISA reader; substitute the absorbance into the standard curve and calculate the MDA content of each sample according to the instructions.

[0083] SOD activity assay: 1) Weigh 10 mg of heart tissue, cut it into small pieces with scissors, and place it in a 1.5 mL centrifuge tube. Add 100 μL of SOD sample preparation solution and homogenize the tissue using an electric homogenizer. Perform the operation on ice if possible. Centrifuge at 13000 rpm for 15 min at 4°C. Collect the supernatant and transfer it to a new 1.5 mL centrifuge tube. Determine the protein concentration using a BCA protein assay kit. Dilute the sample appropriately with SOD detection buffer based on the protein concentration.

[0084] 2) Referring to Table 2, add the test sample and other solutions prepared according to the instructions to the 96-well plate in sequence, and mix thoroughly.

[0085] Table 2 SOD detection sample dosage

[0086] 3) After incubation at 37℃ for 30 minutes, measure the OD value of each well at a wavelength of 450nm using a microplate reader and export the absorbance value (A). Calculate the SOD enzyme activity according to the instructions.

[0087] Test results as follows Figure 15 As shown, A represents the MDA content and B represents the SOD content.

[0088] like Figure 15 As shown in Figure A, compared with the normal control group, the MDA content in the heart tissue of the model group mice was significantly increased, and the difference was statistically significant. P <0.001); Compared with the model group, the MDA content in the cardiac tissue of the drug-treated group and the positive group was significantly reduced, and the difference was statistically significant. P <0.001). This indicates that the formulation can inhibit the increase of MDA content in the heart tissue of obese mice, inhibit the generation of reactive oxygen species (ROS), affect the peroxidation process, alleviate the degree of tissue oxidative damage, and has the effect of preventing cardiovascular diseases.

[0089] like Figure 15As shown in Figure B, compared with the normal control group, the SOD content in the heart tissue of the model group mice was significantly reduced, and the difference was statistically significant. P <0.001); Compared with the model group, the SOD content in the tissues of the drug-treated group and the positive group was significantly increased, and the difference was statistically significant ( P <0.01, P <0.001). This indicates that the formulation can inhibit the reduction of SOD content in the heart tissue of obese mice, thereby reducing the degree of oxidative damage in the body and having a preventive effect on cardiovascular diseases.

[0090] Test Case 10 The expression of PI3K / AKT / eNOS pathway proteins in cardiac and aortic tissues was detected.

[0091] 1) Extraction of total cellular protein: Place a 6-well plate on ice, discard the original culture medium, and wash 2-3 times with pre-chilled 1× PBS buffer. Add 50 μL of cell lysis buffer (RIPA:PMSF:phosphatase inhibitor = 100:1:1) to each well, place the plate on ice, and lyse on a shaker for 20 min. Collect the lysis buffer with a cell scraper and transfer it to a 1.5 mL pre-chilled centrifuge tube. Determine the protein concentration using a BCA protein kit and measure the OD value at 562 nm using a microplate reader. Calculate the required protein volume and double-distilled water volume. Add 5× SDS-PAGE protein loading buffer, vortex thoroughly, and place in a 100℃ metal bath for 10 min to allow for complete protein denaturation.

[0092] 2) Gel preparation: Place the glass plate into the gel preparation tank. The concentration of the separating gel used needs to be selected according to the molecular weight of the target protein. Then, prepare the electrophoresis gel according to the formula in Table 3 (8% separating gel and 5% stacking gel are used in this embodiment).

[0093] Table 3 Formulation of separating gel and stacking gel

[0094] Pour in double-distilled water to check the seal, pour out the double-distilled water, let it dry, then pour the separating gel into the glass plate to the horizontal line, add anhydrous ethanol and press it flat, let it stand at room temperature for half an hour, then discard the anhydrous ethanol, pour in the prepared concentrated gel, insert the comb, and let it stand for half an hour.

[0095] 3) Electrophoresis: Carefully insert the gel plate onto the electrophoresis electrode and place it in the electrophoresis tank. Add fresh 1× electrophoresis buffer to the inner tank. Slowly and vertically pull out the comb. Add 3 μL of pre-stained protein marker to the side wells, then add the protein samples sequentially. Fill the side comb wells with 1× SDS-PAGE protein loading buffer. Add recycled 1× electrophoresis buffer to the outer tank. Correctly connect the positive and negative electrodes of the electrophoresis apparatus. Perform the process in two steps: set the voltage to 70V for 30 min; set the voltage to 100V for 60 min. Transfer the membrane when the bromophenol blue indicator reaches the bottom.

[0096] 4) Transfer: Cut nitrocellulose (NC) membranes of uniform size according to the required molecular weight range. Pour the pre-cooled 1× rapid transfer solution into the transfer tray and prepare the transfer "sandwich" in the tray in the following order: black plastic clip-sponge-three filter papers-glue-NC membrane-three filter papers-sponge. Be careful not to generate air bubbles. Place the sandwich in the transfer tank of the transfer apparatus, paying attention to the positive and negative electrodes. Pour in an appropriate amount of 1× rapid transfer solution and place an ice pack in the tray. Place the tray on ice and transfer the membrane for 25 minutes under a constant current of 400mA.

[0097] 5) Blocking: After the transfer is complete, remove the NC membrane, place it in the antibody incubation box, add an appropriate amount of 5% skim milk blocking solution, and place it on a shaker at room temperature for 2 hours.

[0098] 6) Incubation with primary antibody: After blocking, wash with 1× Tris(hydroxymethyl)aminomethane buffer (TBST) for 10 min each time, 3 times, add the primary antibody diluted with 1×TBST buffer, and incubate overnight at 4°C.

[0099] 7) Incubation of secondary antibody: Wash with 1×TBST buffer for 10 min / time, 3 times the next day. Select the appropriate secondary antibody according to the species of primary antibody and incubate at room temperature for 1-2 hours.

[0100] 8) Development: After incubation with the secondary antibody, wash with 1×TBST buffer for 10 min each time, 3 times, and then develop using a dual near-infrared fluorescence molecular imaging system.

[0101] Western blot results of cardiac tissue as follows Figure 16 As shown, the Western blot results of the aortic vascular tissue are as follows: Figure 17 As shown. Compared with the model group, the ratios of p-PI3K / PI3K, p-AKT / AKT, and p-eNOS / eNOS in cardiac tissue and aortic vascular tissue were significantly increased in the formulation and after intervention with the positive drug. P<0.001). Among them, the changes in aortic vascular tissue suggest that the formulation can enhance endothelial protection-related signal transduction and improve obesity-related endothelial dysfunction; the same trend in cardiac tissue suggests that the formulation may play a certain protective role against hyperlipidemia-related myocardial injury by upregulating PI3K / AKT / eNOS axis activity.

[0102] Test Example 11 The expression of STAT3 protein in cardiac and aortic tissues was detected using the method described in Example 10. Western blot results are shown below. Figure 18 As shown, A represents the Western blot (WB) band of STAT3 protein in cardiac tissue, B represents the p-STAT3 / STAT3 ratio in cardiac tissue, C represents the WB band of STAT3 protein in aortic tissue, and D represents the p-STAT3 / STAT3 ratio in aortic tissue. Compared with the normal group, the p-STAT3 / STAT3 ratio in cardiac and vascular tissues of the model group was significantly increased ( P The value <0.001 indicates that obesity can promote STAT3 phosphorylation activation. Compared with the model group, the p-STAT3 / STAT3 ratio was significantly decreased after the intervention of the formulation and the positive drug (p-STAT3 / STAT3 ratio). P The value <0.001 indicates that the formulation can inhibit abnormally activated STAT3 signaling, thereby playing a certain role in intervening in obesity-related inflammatory damage.

[0103] Test Example 12 Human umbilical vein endothelial cells (HUVECs) were cultured in a 37°C, 5% CO2 incubator. The culture medium used was ECM complete endothelial cell culture medium containing a mixture of 5% fetal bovine serum (FBS), 1% endothelial growth factor, and 1% penicillin-streptomycin.

[0104] Once the HUVEC cells adhered and grew to approximately 80% confluence, the culture supernatant was discarded. Except for the normal group, all other groups were stimulated with trimethylamine oxide (TMAO) to establish a cell damage model; subsequently, different concentrations of the formulation or positive control drug were added according to the groupings for treatment.

[0105] HUVEC cells were treated with formulations at concentrations of 0, 50, 100, 200, 400, and 600 μg / mL for 24 h. The effect of different concentrations of the formulation on HUVEC cell viability was determined using the MTT assay. The results are as follows: Figure 19 As shown in Figure A, the results indicate that cell viability was above 80% at concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, while cell viability was below 80% at 400 μg / mL and 600 μg / mL. This suggests that the formulation at a concentration of 200 μg / mL has no toxic effect on endothelial cells. Therefore, in this embodiment, 50 μg / mL, 100 μg / mL, and 200 μg / mL were selected as the low, medium, and high concentration groups of the formulation.

[0106] The experiment was divided into a normal group, a model group (TMAO group), a low-dose TMAO+ formulation group, a medium-dose TMAO+ formulation group, a high-dose TMAO+ formulation group, and a TMAO+ positive control group, where the positive control group was L-arginine. The final concentration of TMAO was 500 μM, and the final concentration of L-arginine was 1 mM.

[0107] NO release assay: Cells were injected at a concentration of 1×10⁻⁶ 4 Cells were seeded per well in a 96-well plate and cultured for 24 hours. The next day, cells were observed; when approximately 80% adhered, the supernatant was discarded, and drug treatment was initiated, with 5 replicates per group. 24 hours after drug treatment, 50 μL of supernatant per well was transferred to a new 96-well plate. Griess Reagent I was added: 50 μL to each well, gently mixed, and incubated at room temperature for 5 minutes. Griess Reagent II was added: another 50 μL to each well, gently mixed, and incubated at room temperature in the dark for 5 minutes. The absorbance was measured at 540 nm using a microplate reader.

[0108] The results are as follows Figure 19 As shown in Figure B, compared with the normal group, TMAO stimulation significantly reduced the NO release level in HUVEC cells ( P <0.001); Compared with the model group, the NO release level gradually increased with increasing concentration, indicating that this formula has a significant ameliorative effect on NO release during endothelial dysfunction at concentrations of 50, 100, and 200 μg / mL. P <0.001).

[0109] Western blot results of TMAO-induced HUVEC cells are as follows: Figure 20 As shown, the method was the same as in test example 10. Compared with the normal group, the ratios of p-PI3K / PI3K, p-AKT / AKT, and p-eNOS / eNOS in the HUVEC cell model group stimulated with TMAO were significantly decreased. P <0.001 indicates that TMAO successfully induced an endothelial cell injury model. Compared with the model group, the traditional Chinese medicine formula significantly increased the p-PI3K / PI3K, p-AKT / AKT, and p-eNOS / eNOS ratios at all dosage groups (50, 100, 200 μg / mL) and in the positive control group. P <0.001).

[0110] The expression of STAT3 protein in HUVEC cells was detected using the same method as in Test Example 10. The results are as follows: Figure 21As shown, compared with the normal group, the p-STAT3 / STAT3 ratio increased in the HUVEC cell model group after TMAO stimulation, indicating that TMAO can promote the activation of STAT3 signaling. Compared with the model group, the p-STAT3 / STAT3 ratio was reduced in all dose groups of the traditional Chinese medicine formula (50, 100, 200 μg / mL) and the positive group. P <0.001).

[0111] Test Example 13 RAW264.7 cells were cultured in a 37°C, 5% CO2 incubator. The culture medium was DMEM basal medium supplemented with a mixture of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. When the RAW264.7 cells adhered and grew to approximately 80% confluence, the culture supernatant was discarded. Except for the normal control group, all other groups were stimulated with LPS to establish an inflammation model. Subsequently, different concentrations of formulations or positive control drugs were added according to the group. The experiment consisted of a normal control group, a model group (LPS group), a low-dose LPS + formulation group, a medium-dose LPS + formulation group, a high-dose LPS + formulation group, and an LPS + positive control drug group, where the positive control drug was dexamethasone. The final LPS concentration was 1 μg / mL, and the low, medium, and high doses of the formulations were 25 μg / mL, 50 μg / mL, and 100 μg / mL, respectively. The final dexamethasone concentration was 20 μM.

[0112] 1) RNA extraction: Remove the 6-well plate and place it on ice. Wash 2-3 times with 1× PBS buffer. Add 1 mL of TRNzol Universal reagent to each well, and pipette the mixture thoroughly. Transfer the liquid to an enzyme-free centrifuge tube, then add 200 μL of chloroform. Manually invert the plate rapidly for 2 min to mix. Let it stand for 10 min, then centrifuge at 4℃ and 13000 rpm for 15 min. Transfer the supernatant to a new 1.5 mL centrifuge tube, add an equal volume of isopropanol, mix thoroughly, place on ice for 10-20 min, and centrifuge at 4℃ and 13000 rpm for 10 min. Carefully discard the supernatant, add 1 mL of 75% ethanol, wash thoroughly, centrifuge at 4℃ and 13000 rpm for 5 min, discard the supernatant, and air dry at room temperature. Once the precipitate becomes clear, add 30 μL of... RNase-Free ddH2O was mixed to fully dissolve the RNA. After incubation at 72°C for 10 min, the RNA was quickly cooled on ice and centrifuged. The RNA concentration and purity were measured using a full-function microplate reader (the A260 / A280 ratio should be between 1.9 and 2.1). Reverse transcription was then performed.

[0113] 2) cDNA synthesis: Calculate the required RNA volume, bring the volume to 13 μL with enzyme-free sterile water, add the required reaction system reagents in Table 4, for a total volume of 20 μL, and perform reverse transcription according to the program set in Table 5.

[0114] Table 4 Reagents for Reverse Transcription Reaction System

[0115] Table 5 Reverse transcription reaction conditions

[0116] 3) RT-qPCR: Design and synthesize the corresponding primers according to the target gene. The primer sequences are shown in Table 6. Prepare a 20 μL system according to Table 7 and place it in an eight-tube PCR array. Perform amplification according to the reaction program in Table 8.

[0117] 4) Data Analysis: After the reaction is complete, save the CT value data and use the relative quantification method (2) -ΔΔCT (Method) to calculate the expression level of the target gene.

[0118] Table 6 RT-qPCR Primer Sequences

[0119] Table 7 RT-qPCR amplification system

[0120] Table 8 RT-qPCR amplification reaction program

[0121] The effect of different concentrations of the formulation on the activity of RAW264.7 was determined using the MTT assay. Figure 22 As shown in Figure A, the results indicate that cell viability was above 80% at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL, while it was below 80% at concentrations of 200 μg / mL and 400 μg / mL. This suggests that the formulation at a concentration of 100 μg / mL had no toxic effect on RAW264.7 cells. Therefore, 25 μg / mL, 50 μg / mL, and 100 μg / mL were selected as the low, medium, and high concentration groups of the formulation in this embodiment.

[0122] The NO generation amount of each group was measured. The detection method is described in Test Example 12. The detection results are as follows: Figure 22 As shown in Figure B, compared with the normal group, the NO level in the supernatant of RAW264.7 cells was significantly increased after LPS stimulation, indicating that the inflammation model was successfully established. Compared with the LPS model group, the NO production level in the formulated intervention showed a decreasing trend, and the decreasing effect gradually increased with increasing drug concentration. P <0.001). The results suggest that this formulation has a certain inhibitory effect on LPS-induced inflammatory response in RAW264.7 cells.

[0123] TNF-α ELISA test results showed that... Figure 22 As shown in Figure C, compared with the normal group, the TNF-α level in the supernatant of RAW264.7 was significantly increased after LPS stimulation ( P <0.001 indicates successful construction of the inflammation model. Compared with the LPS model group, the TNF-α secretion level decreased after the intervention, with significant reductions observed in low, medium, and high concentration groups. P <0.001). The results suggest that this formulation can, to some extent, inhibit the release of LPS-induced pro-inflammatory factor TNF-α, thereby alleviating the inflammatory response in RAW264.7.

[0124] Western blot was used to detect the protein expression of COX-2 and iNOS in RAW264.7 cells. The results are shown in Figure 23. Compared with the normal group, the expression levels of COX-2 and iNOS proteins in the model group were significantly increased. P <0.001); Compared with the model group, the low, medium and high dose groups of the formulation could inhibit LPS-induced iNOS protein expression in RAW264.7 cells, and the differences were statistically significant at concentrations of 25, 50 and 100 μg / mL. P <0.001); Simultaneously, the formulation also inhibited COX-2 protein expression, and the differences at concentrations of 25, 50, and 100 μg / mL were all statistically significant. P <0.001), and shows a certain concentration dependence.

[0125] The mRNA expression level of the pro-inflammatory cytokine IL-6 in cells after LPS induction was detected by RT-qPCR, and the results are as follows: Figure 24 As shown, compared with the normal group, the mRNA expression level of IL-6 in the model group was significantly increased ( P <0.001); Compared with the model group, the low, medium, and high groups of the formulation significantly inhibited the mRNA expression level of IL-6 ( P <0.001), showing a drug concentration-dependent relationship.

[0126] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A traditional Chinese medicine compound preparation with cardiovascular protective effects, characterized in that, It is composed of the following Chinese medicinal materials in parts by weight: chrysanthemum 5-10, hawthorn 9-12, wolfberry 6-12, turmeric 3-10, and schisandra 2-6.

2. The traditional Chinese medicine compound preparation according to claim 1, characterized in that: Dosage forms include decoctions, pills, powders, capsules, tablets, granules, or drop pills.

3. The traditional Chinese medicine compound preparation according to claim 2, characterized in that, The decoction is prepared by the following method: after washing the Chinese medicinal materials in the specified weight proportions, soak them in water for 6-8 hours, bring to a boil over high heat, then simmer over low heat for 30-60 minutes, filter and collect the juice; then add water and decoct a second time, bring to a boil over high heat, then simmer over low heat for 20-40 minutes, combine the two decoctions and concentrate to obtain the final product.

4. The use of a traditional Chinese medicine compound preparation as described in any one of claims 1-3 in the preparation of a drug for protecting the cardiovascular system.

5. The application according to claim 4, characterized in that: The drug is used to improve vascular structure and elasticity; the improvement in vascular structure and elasticity is manifested by increasing the difference between the systolic and diastolic diameters of the aortic arch, reducing the thickness of the intima to media of the aortic sinus, and / or reducing the carotid pulse wave velocity.

6. The application according to claim 4, characterized in that: The drug is used to improve cardiac function; the improvement in cardiac function is manifested by increasing the left ventricular ejection fraction and / or shortening the ejection fraction.

7. The application according to claim 4, characterized in that: The drug is used to inhibit oxidative stress in cardiac tissue; the inhibition of oxidative stress is manifested by reducing malondialdehyde content and / or increasing superoxide dismutase activity.

8. The application according to claim 4, characterized in that: The drug is used to target and activate the PI3K-AKT-eNOS signaling pathway and / or inhibit the STAT3 signaling pathway.

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