Traditional Chinese medicine composition for preventing and treating dyslipidemia and atherosclerosis as well as preparation method and application thereof

Abstract

The invention provides a traditional Chinese medicine composition and a preparation method and application thereof, the traditional Chinese medicine composition comprises the following functional components in parts by weight: 1-3 parts of selfheal, 1-3 parts of seaweed, 1-2 parts of motherwort, 1-2 parts of fructus forsythiae and 0.5-1.5 parts of thunberg fritillary bulb, and the traditional Chinese medicine composition is prepared by taking aqueous extracts as active ingredients. The traditional Chinese medicine composition has the effects of softening hardness to dissipate stagnation, clearing away heat and toxic materials, reducing phlegm and removing stasis, has the effects of improving hyperlipidemia and improving atherosclerotic plaque formation, promotes bile acid synthesis and cholesterol excretion by activating liver LXR-alpha, does not cause liver fatty degeneration, and has the advantages of no toxic or side effect and no toxic or side effect. The compound has potential clinical application value in the aspects of improving lipid metabolism disorder and dyslipidemia and preventing and treating atherosclerotic cardiovascular diseases.

Description

Technical Field

[0001] This invention relates to a traditional Chinese medicine composition, its preparation method, and its application, particularly to a traditional Chinese medicine composition for the prevention and treatment of dyslipidemia and atherosclerosis, its preparation method, and its application. Background Technology

[0002] Atherosclerosis (AS) is the primary pathological basis for coronary artery disease, ischemic stroke, and peripheral artery disease, posing a significant challenge to global public health. Extensive evidence indicates that hypercholesterolemia is a key factor in AS plaque formation and the development of atherosclerotic cardiovascular disease (ASCVD). Management of dyslipidemia, particularly by maintaining cholesterol homeostasis, is a major strategy for preventing and treating AS and can significantly reduce the incidence of cardiovascular disease (CVD). However, most patients fail to completely lower their low-density lipoprotein cholesterol (LDL-C) levels, leading to a persistent risk of recurrent cardiovascular events.

[0003] Currently, novel cholesterol-lowering therapies primarily focus on two strategies: inhibiting intestinal cholesterol reabsorption and promoting the transfer of excess cholesterol from peripheral tissues to the liver via reverse cholesterol transport (RCT). RCT is the core mechanism for lowering cholesterol levels and inhibiting the progression of ankylosing spondylitis (AS). Promoting RCT has become a research focus, especially regulating cholesterol absorption and excretion in the liver and intestines, which is considered one of the most promising therapeutic strategies.

[0004] Cholesterol cannot be metabolized by most cells and must be degraded and excreted in the liver. The liver breaks down cholesterol through bile acid synthesis and excretes it into the intestines; approximately 75% of bile acids are generated via the classical pathway. The hepatobiliary excretion pathway mediated by transport proteins accounts for about 65% of total cholesterol clearance in the human body, thus becoming a potentially important therapeutic target. However, current cholesterol-lowering drugs cannot directly promote cholesterol degradation or excretion. Hepatic X receptor α (LXR-α) can regulate the transcription and activity of various cholesterol transport proteins and bile acid synthases in hepatocytes, and has become an important target for the treatment of hypercholesterolemia and ankylosing spondylitis (AS). Targeting the LXR-α-mediated randomized controlled trials (RCTs) to promote cholesterol degradation and excretion is an effective strategy for treating AS.

[0005] Traditional Chinese medicine (TCM) has long played a vital role in the prevention and treatment of cardiovascular diseases. Many TCM formulas and natural products have been proven to effectively alleviate the progression of ankylosing spondylitis (AS) by regulating cholesterol homeostasis, lipid metabolism, and inflammatory responses. Furthermore, Western medicine treatments for dyslipidemia may cause adverse reactions such as abnormal liver function, muscle pain, and gastrointestinal discomfort. In-depth research into the role of TCM in the management of dyslipidemia in the prevention and treatment of AS has significant clinical value. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a traditional Chinese medicine composition for the prevention and treatment of dyslipidemia and atherosclerosis.

[0007] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned traditional Chinese medicine composition.

[0008] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned traditional Chinese medicine composition.

[0009] The technical solution adopted in this invention is:

[0010] A traditional Chinese medicine composition for the prevention and treatment of dyslipidemia and atherosclerosis, comprising Prunella vulgaris, Sargassum, Leonurus japonicus, Forsythia suspensa and Fritillaria thunbergii, wherein each component is expressed in parts as follows: Prunella vulgaris 1-3 parts, Sargassum 1-3 parts, Leonurus japonicus 1-2 parts, Forsythia suspensa 1-2 parts, and Fritillaria thunbergii 0.5-1.5 parts.

[0011] Preferably, in the above-mentioned traditional Chinese medicine composition, each component is expressed in parts as follows: 1.5 parts of Prunella vulgaris, 1.5 parts of Sargassum, 1.2 parts of Leonurus japonicus, 1.2 parts of Forsythia suspensa, and 1.0 part of Fritillaria thunbergii.

[0012] The above-mentioned traditional Chinese medicine composition is prepared by using the decoction pieces, fine powders or water extracts of the above-mentioned components as active ingredients.

[0013] Preferably, the preparation method of the above-mentioned traditional Chinese medicine composition includes the following specific steps:

[0014] (1) Weigh the raw medicinal materials accurately and mix them thoroughly. Add 10 times the volume of deionized water, soak them thoroughly and reflux for 1 hour, and extract twice.

[0015] (2) Combine the extracts from the two rounds and concentrate them using rotary evaporation;

[0016] (3) Freeze-dry the concentrated solution (-25℃, 5MPa) to obtain freeze-dried powder.

[0017] The application of the above-mentioned traditional Chinese medicine composition in the preparation of lipid-lowering drugs.

[0018] The application of the above-mentioned traditional Chinese medicine composition in the preparation of cholesterol-lowering drugs.

[0019] The application of the above-mentioned traditional Chinese medicine composition in the preparation of anti-inflammatory drugs.

[0020] The application of the above-mentioned traditional Chinese medicine composition in the preparation of drugs for treating atherosclerosis.

[0021] The application of the above-mentioned traditional Chinese medicine composition in the preparation of drugs for treating atherosclerotic cardiovascular diseases.

[0022] The beneficial effects of this invention are:

[0023] The aforementioned traditional Chinese medicine composition has the effects of softening and dispersing nodules, clearing heat and detoxifying, resolving phlegm and removing blood stasis.

[0024] A systematic evaluation was conducted on its effect on ApoE induced by a high-fat diet. - / - The study investigated the effects of this traditional Chinese medicine composition on blood lipid levels and atherosclerotic (AS) lesions in a mouse model. Combined with liver metabolomics analysis, the mechanism of its lipid-lowering effect was elucidated. The study revealed that this composition plays a key role in promoting bile acid synthesis and cholesterol excretion by activating hepatic LXR-α without inducing hepatic steatosis. Furthermore, the composition enhanced the liver's ability to absorb and degrade cholesterol through cholesterol reversal transport by promoting LXR-α activation, particularly by promoting bile acid synthesis and cholesterol excretion into bile and the intestines. This resulted in an anti-atherosclerotic effect without inducing hepatic steatosis, demonstrating the composition's ability to improve hyperlipidemia and atherosclerotic plaque formation. The study clarified its potential clinical application value in improving lipid metabolism disorders, dyslipidemia, and preventing and treating atherosclerotic cardiovascular diseases. Attached Figure Description

[0025] Figure 1 The chromatograms obtained by UPLC-Q-TOF-MS in negative (A) and positive (B) ion modes are for the mass spectrometric analysis of the chemical components of the QRSJF extract.

[0026] Figure 2 QRSJF-induced ApoE - / - Effects of the study on mouse body weight, blood lipids, and inflammatory factor levels, including (A) experimental design, (B) changes in body weight, (C) serum total cholesterol and triglycerides in each group (n=15), (D) serum LDL-C and HDL-C in each group (n=15), and (E) serum IL-6 (n=10–16) and IL-1β (n=14–16) in each group; all data are expressed as mean ± SD, compared with the control group (CTR). * P<0.05, ** P<0.01; ## P<0.01 compared with the HFD group; HFD: high-fat diet; CTR: control group.

[0027] Figure 3 QRSJF can reduce plaque formation and improve arterial stenosis. The following data were used to evaluate its effectiveness: (A) representative ultrasound images and lumen diameters of the LCCA in each mouse group (n=11–12); (B) LCCA stenosis rate in each mouse group; (C) IMT; (D) PWV (n=10–14); (E) aortic arch diameter in each mouse group (n=12); (F) hemodynamic parameters of the ascending and descending aorta in each group (n=6–7). Data are expressed as mean ± SD. *P<0.05, ** P<0.01 compared with the CTR group, ## P<0.01 compared with the HFD group.

[0028] Figure 4 QRSJF can improve atherosclerotic lesions and enhance plaque stability. The study included: (A) representative images and quantitative analysis of Oil Red O staining on the surface of the mouse aorta (n=3), scale bar: 500 μm; (B) representative images and quantitative analysis of H&E staining on the sinus lesions, necrotic core area, and fibrous cap area of ​​the aortic root (n=5-6), scale bar: 500 μm; (C) Oil Red O staining and Masson's trichrome staining on the surface of the mouse aorta; (D) CD68 and α-SMA; (E) representative images and quantitative analysis of stained aortic root sections (n=5-6), scale bar: 500 μm; and (E) plaque vulnerability index. Data are expressed as mean ± SD. * P<0.05, ** P<0.01 compared with the CTR group, ## P<0.01 compared with the HFD group.

[0029] Figure 5 For liver sample metabolomics analysis, (A) PCA score map of liver samples in each group (n=6), (B) volcano plot showing metabolite differences among groups, (C, E) heatmap of expression levels of major differential metabolites reversed by QRSJ-L and QRSJ-H treatments among groups, and (D, F) KEGG enrichment analysis of differential metabolites.

[0030] Figure 6 To improve ApoE induced by QRSJF - / - Expression of proteins related to RCT progression, bile acid synthesis, and excretion in mice: (A) Representative Western blot images and quantitative analysis of SR-B1 and LDLR expression in the liver (n=3); (B) Representative images of mouse liver H&E staining (scale bar: 100 μm, 50 μm) and Oil Red O (scale bar: 200 μm) (n=3); (C) TC (n=11–12) and TG levels in mouse liver (n=12); (D) Total bile acid levels in mouse liver (n=9) and feces (n=6); (EG) Representative Western blot images and quantitative analysis of CYP7A1, ABCG5, ABCG8, BESP, ABCA1, and LXR-α expression in the liver (n=3); Data are expressed as mean ± SD and compared with the CTR group. * P<0.05, ** P<0.01, compared with the HFD group # P<0.05, ## P<0.01.

[0031] Figure 7 LXR-α inhibition eliminated the effects of QRSJF on lipid accumulation and RCT protein expression in HepG2 cells. The data included: (A) Representative Oil Red O staining images of HepG2 cells, scale bar: 20 μm (n=6); (B) Representative images and quantification of BODIPY / dapi positive regions in HepG2 cells, scale bar: 20 μm; (CD) Representative Western blot images and relative quantification analysis of LXR-α, CYP7A1, and ABCG5 expression in HepG2 cells with or without FFA, QRSJF, and GSK2033, scale bar: 20 μm; (E) Representative images and quantification analysis of BODIPY / dapi positive regions in HepG2 cells with or without FFA, QRSJF, and GSK2033, scale bar: 20 μm. Data are expressed as mean ± SD and compared with the CTR group. * P<0.05, ** P<0.01, compared with the FFA group # P<0.05, ## P<0.01. Detailed Implementation

[0032] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0033] The herbs used in the following examples, including Prunella vulgaris, seaweed, Leonurus japonicus, Forsythia suspensa, and Fritillaria thunbergii, were all purchased from Tianjin Tongrentang Group Co., Ltd. The preparation process for tablets, granules, solutions, pills, ointments, or capsules made from the compositions of this invention is mature and can be carried out using conventional methods in the prior art.

[0034] Example 1

[0035] A traditional Chinese medicine composition (QRSJF) for the prevention and treatment of dyslipidemia and atherosclerosis has the following formula: 1.5 parts of Prunella vulgaris, 1.5 parts of Sargassum, 1.2 parts of Leonurus japonicus, 1.2 parts of Forsythia suspensa, and 1.0 part of Fritillaria thunbergii.

[0036] Medicinal material sources: Prunella vulgaris L. (Lamiaceae) is the dried fruit spike of Prunella vulgaris L.; Sargassum pallidum (Turn.) C.Ag. (Sargassumaceae) is the dried thallus of Sargassum pallidum (Turn.) C.Ag.; Forsythia suspensa (Thunb.) Vahl. (Oleaceae) is the dried fruit of Forsythia suspensa (Thunb.) Vahl.; Fritillaria thunbergii Miq. (Liliaceae) is the dried bulb of Fritillaria thunbergii Miq.; Leonurus japonicus Houtt. (Lamiaceae) is the dried aerial part of Leonurus japonicus Houtt. (Lamiaceae).

[0037] The preparation method is as follows:

[0038] (1) Weigh the raw medicinal materials according to the prescription and mix them thoroughly. After mixing, wrap the leafy medicinal materials in gauze, put them in a beaker, add 10 times the amount of deionized water, and soak them thoroughly for 1 hour.

[0039] (2) Heat the mixture to boiling using an electric heating mantle and reflux for 1 hour. After cooling slightly, filter the mixture through gauze. Extract the remaining residue once more using the same method.

[0040] (3) Combine the two extracts, rotary evaporate the extracts to the extract state, spread them evenly in a petri dish, and freeze dry them in a freeze dryer at -25℃ and 5MPa to obtain freeze-dried powder.

[0041] Example 2

[0042] A traditional Chinese medicine composition for clearing heat and dissipating nodules has the following formula: 1 part Prunella vulgaris, 3 parts Sargassum, 2 parts Leonurus japonicus, 1 part Forsythia suspensa, and 1.5 parts Fritillaria thunbergii. The preparation method is the same as in Example 1.

[0043] Example 3

[0044] A traditional Chinese medicine composition for clearing heat and dissipating nodules has the following formula: 3 parts Prunella vulgaris, 1 part Sargassum, 1 part Leonurus japonicus, 2 parts Forsythia suspensa, and 0.5 parts Fritillaria thunbergii. The preparation method is the same as in Example 1.

[0045] Example 4

[0046] 1. Materials and Methods

[0047] 1.1 Composition and Preparation of the Heat-Clearing and Stasis-Dissolving Formula QRSJF

[0048] Same as Example 1.

[0049] 1.2 UPLC-Q-TOF-MS Analysis of QRSJF Chemical Composition

[0050] The major components were chemically identified using an ACQUITY ultra-high performance liquid chromatography (UPLC) system (Waters, USA) combined with a quadrupole-electrostatic field orbital ion trap mass spectrometer (Orbitrap-MS) (Thermo Fisher, USA) equipped with an electrospray ionization (ESI) source. The mobile phase was 0.1% formic acid in water (A)-acetonitrile (B), with a flow rate of 0.4 mL / min, a column temperature of 35 °C, and a sample pan temperature of 10 °C. The elution gradients were as follows: 0–6 min, 99% A and 1% B; 6–14 min, 85%–70% A and 26%–95% B; 14–22 min, 70%–5% A and 26%–95% B; 22–25 min, 99% A and 1% B. The electrospray ionization source (ESI) was selected. + / ESI - Ion scanning mode, ion source parameters are set as follows: capillary voltage is ESI. + 3.5kV / ESI - 3.0kV, sheath gas velocity 35arb, auxiliary gas velocity 8arb; data acquisition mode: Full MS / ddMS 2 The acquisition mass range is 100–1500 Da; the normalized collision energy is 10⁻⁶⁰ V. The positive and negative ion chromatograms of RJSJF are shown below. Figure 1 As shown.

[0051] 1.3 Experimental Animals and Models

[0052] 6–8 week old SPF grade male C57BL / 6J mice and C57BL / 6 background male ApoE mice - / - Mice, weighing 18±2.0g, were purchased from the Beijing Vital River Animal Laboratory. All animal experimental procedures were performed according to the recommendations of the National Institutes of Health (NIH) Guidelines for the Care and Use of Laboratory Animals (1996), and were reviewed and approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine. All experimental animals were subjected to one week of acclimatization feeding under SPF conditions. Subsequently, ApoE... - / - Mice were fed a high-fat diet (MD12015A, Jiangsu Medison Biomedical Co., Ltd.), while C57BL / 6J mice continued to be fed a normal diet for 8 weeks. The general condition of the mice was observed daily, and the body weight of each group was recorded weekly.

[0053] 1.4 Drug intervention

[0054] After confirming the model is established, ApoE will be used. - / -Mice were randomly divided into groups of 10-15 mice each according to their body weight. They were given saline or medication via gavage for 12 weeks in addition to their original diet. The groups were: (1) Model group, (2) High-dose group of the Ruanjian Sanjie formula (RJSJ-H, 16.64g crude drug / kg / d), (3) Low-dose group of the Ruanjian Sanjie formula (RJSJ-L, 8.32g crude drug / kg / d), and (4) Simvastatin as the positive control drug, with the Simvastatin group (Simva, 2.6mg / kg / d). C57BL / 6J mice were used as the control group. Mice were administered 0.1mL / 10g body weight via gavage, while the control and model groups were administered the same volume of double-distilled water via gavage. Gavage was administered once daily for 12 weeks.

[0055] Following administration, mice were anesthetized and euthanized. Blood was collected using centrifuge tubes with or without heparin sodium, and serum or plasma samples were obtained after centrifugation and stored at -80°C. The mice were then perfused with pre-chilled PBS via the apex of the heart. The aorta, heart, and a portion of the liver were collected on ice. Some samples were preserved in 4% paraformaldehyde, while the remaining tissue samples were flash-frozen in liquid nitrogen and then stored at -80°C.

[0056] 1.5 Vascular ultrasound examination

[0057] After 12 weeks of drug intervention, vascular ultrasound examination was performed on mice using a high-resolution small animal color Doppler ultrasound imaging system. Mice were anesthetized with isoflurane (1 L / min oxygen flow rate, 2.5% isoflurane), and a maintenance dose of 1.0%–1.5% isoflurane was continued throughout the procedure to maintain the anesthetized state. Hair was removed from the neck, chest, and abdomen of the mice, and their limbs were fixed to the operating table.

[0058] Parameter detection for carotid artery ultrasound imaging: In B mode, adjust the depth key to 5-6 mm, and position the probe longitudinally and perpendicular to the projection position of the blood vessels on the left side of the mouse's neck. Use the long axis view to visualize the complete, clear, and continuous left common carotid artery and its anterior and posterior walls, using the bifurcation of the internal and external carotid arteries as the anatomical location. Avoiding the carotid sinus, measure the luminal diameter 1 mm from the branch of the internal and external carotid arteries on the left side of the mouse. Use a Vevo Vasc workstation to measure the intima-media thickness (IMT) of the anterior and posterior walls of the common carotid artery at the same location.

[0059] Assessment of vascular stenosis: Switch the ultrasound microscope to color Doppler mode. Determine the hemodynamic status of the common carotid artery, including blood flow direction, filling, velocity, and characteristics, based on the color Doppler blood flow color and brightness. Observe whether there are "filling defects" in the lumen caused by stenosis or obstruction. Select a clear long-axis color Doppler image of the vessel and freeze it. Measure the original lumen diameter (D1) at the abnormal site and the residual lumen diameter (D2) at the most severe stenosis point. Calculate the percentage of maximum stenosis using the formula (D1-D2) / D1×100%.

[0060] Vascular stiffness assessment: Switch the ultrasound microscope to spectral Doppler mode. Adjust the sampling volume length along the long axis of the carotid artery to 1 / 3-1 / 4 of the common carotid artery diameter. The angle correction line should be basically parallel to the vessel and have an angle ≤60° with the blood flow direction. Select the proximal and distal ends of the common carotid artery as measurement points, respectively. Start pulsed Doppler and simultaneously start EKV mode to record the electrocardiogram. Detect the pulse-wave velocity (PWV) of the common carotid artery based on the time-of-transmission (TT) method, calculated as PWV = ΔL / TT. After the mice have a stable heart rate and respiration, continuously measure the morphology of the spectrum and TT for 5 cardiac cycles at each measurement point, calculate the average value, and measure the ΔL distance in B mode. All data were measured and analyzed using Visual Sonics analysis software.

[0061] Aortic arch ultrasound imaging parameters were measured: Aortic arch images were recorded in B-mode, and the aortic arch diameter was measured. Spectral Doppler was activated, and the sampling volume length and angle correction line parameters were adjusted as before. The morphology of the spectrum of the ascending and descending aorta of mice was scanned for five cardiac cycles. Peak systolic velocity (PSV), end-diastolic velocity (EDV), mean flow velocity (MFV), resistance index (RI) = (PSV - EDV) / PSV, and pulsative index (PI) = (PSV - EDV) / MFV were measured to observe hemodynamic changes in mice. All data were measured and analyzed using Visual Sonics analysis software.

[0062] 1.6 Lipid Detection

[0063] Mouse serum was subjected to biochemical assays using a diagnostic kit (Nanjing Jiancheng Biotechnology Research Institute, China) to measure the levels of total cholesterol (TC), triglycerides (TG), LDL-C, and high-density lipoprotein cholesterol (HDL-C). A suitable amount of liver tissue was collected and homogenized using an ultrasonic homogenizer. The levels of TG and TC in the liver were then measured using the diagnostic kit.

[0064] 1.7 Enzyme-linked immunosorbent assay (ELISA) and total bile acid detection

[0065] Serum interleukin-1β (IL-1β) and interleukin-6 (IL-6) levels (88-7324, 88-7013, and 88-7064; Thermo Fisher Scientific) were determined by ELISA. Total bile acid levels in liver and fecal tissues were measured using a total bile acid assay kit (E003-2-1, Nanjing Jiancheng Bioengineering Institute). Lysis solutions were prepared by mixing liver or fecal samples with 0.9% physiological saline at a weight ratio of 1:9. Fecal or tissue samples were then lysed using an ultrasonic homogenizer on ice. After centrifugation, the supernatant was collected for further analysis.

[0066] 1.8 Tissue analysis of atherosclerotic lesions

[0067] Unrelated tissues around the aorta were cleaned under a stereoscope and fixed with 4% paraformaldehyde. Longitudinal sections were then prepared and stained with Oil Red O (C0157S; Beyotime Biotechnology Co., Ltd.). The stained aorta was photographed using a Leica camera (Germany), and the plaque surface area was quantified using ImageJ software.

[0068] To stain the aortic root, sections connected to the heart were fixed in 4% paraformaldehyde, then dehydrated with sucrose, embedded in OCT, and serially sectioned using a cryostat (Leica, Germany). Oil Red O staining was used to detect lipid accumulation levels. Additionally, the aortic valve was embedded in paraffin, cut into 5-6 μm sections, and stained using H&E (G1120, Solarbio) and Masson's trichrome (G1340, Solarbio). ImageJ software was used to measure cross-sectional plaque lesion area, fibrous cap thickness, necrotic core area, and collagen content.

[0069] Immunohistochemistry was used to assess positive areas of smooth muscle cells and macrophages within aortic root plaques. Primary antibodies against α-SMA (ab7817) and CD68 (ab125212) were purchased from Abcam. ImageJ software was used to calculate and analyze positive cells within the plaques. The plaque vulnerability index was calculated using the following formula: (percentage of macrophage staining + percentage of lipid staining) / (percentage of smooth muscle cells + percentage of collagen fibers).

[0070] 1.9 Liver Pathology

[0071] Mouse liver tissue was fixed in 4% paraformaldehyde, dehydrated with saturated sucrose solution, embedded in OCT, frozen sectioned, and stained with Oil Red O. Alternatively, the tissue was dehydrated, cleared, embedded in paraffin, sectioned, and stained with H&E. Finally, the sections were sealed with neutral glue and imaged using an optical microscope.

[0072] 1.10 Metabolomics Analysis

[0073] Mouse liver samples were deproteinized in acetonitrile and methanol, respectively. After centrifugation, the supernatant was analyzed using ultra-high performance liquid chromatography (UHPLC) combined with a quadrupole electrostatic field orbital trap mass spectrometer. The raw data format was converted using ABF Converter software before being imported into MS-DIAL software for peak extraction, identification, alignment, and normalization. The resulting three-dimensional data matrix included compound names, retention times, peak areas, and other relevant information. Metabolites were identified by comparing the data with MS-DIAL's internal database and the Human Metabolomics Database (HMDB). Multivariate statistical analysis was performed on the resulting data matrix using Simca-P14.1 software. Differentially metabolized metabolites were screened based on VIP values ​​> 1.0, fold change (FC) > 1.5, and P < 0.05, followed by enrichment analysis.

[0074] 1.11 Western blot detection

[0075] Total protein was extracted from liver tissue or cell samples using RIPA lysis buffer containing PMSF, and all procedures were performed according to standard laboratory protocols. The antibody information is as follows: anti-LXR-α (catalog number: 3284587), anti-ABCA1 (catalog number: 3327185), anti-SR-B1 (catalog number: 217318), and anti-CYP27A1 (catalog number: ab126785) were purchased from Abcam; anti-CYP7A1 (catalog number: bs-21429R) was purchased from Beijing Bio-Sensing; anti-ABCG5 (catalog number: 27722-1-AP) and anti-ABCB11 (catalog number: 67512-1-IG) were purchased from Wuhan Proteintech; anti-ABCG8 (catalog number: PA5-104397) was purchased from Invitrogen; and anti-GAPDH (catalog number: 5174S) was purchased from CST. Immunoreactive bands were visualized using ECL chemiluminescence, and images were acquired using a Bio-Rad gel imaging system. Finally, the protein bands were quantitatively analyzed using ImageJ software.

[0076] 1.12 Cell Culture and Inhibitor Treatment

[0077] Human hepatocellular carcinoma (HepG2) cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin-streptomycin mixture. All experiments used cells in the logarithmic growth phase. To construct a lipid accumulation cell model, a medium containing 10% fatty acid-free bovine serum albumin (Solepro, A8850) was used, with the addition of a free fatty acid (FFA) solution of sodium oleate (MedChemExpress, HY-N1446B) and sodium palmitate (Sigma, P9767) in a 2:1 ratio, bringing the final concentration to 500 μM. HepG2 cells were co-treated with 100 μg / mL, 200 μg / mL QRSJF, and 500 μM FFA for 24 hours, respectively; the inhibitor GSK2033 (MedChemExpress, HY-108688) was dissolved in DMSO at a concentration of 10 μM and pretreated for 12 hours. Using Oil Red O (Solarbio, G1260) and BODIPY TM Neutral lipids were stained using 493 / 503 (Thermo Fisher, D3922) to assess lipid accumulation, and imaged using a Leica Stellaris 8 confocal microscope.

[0078] 1.13 Statistical Analysis

[0079] SPSS 23.0 software was used for statistical analysis. Quantitative data variables are expressed as Mean ± SEM. For comparisons among multiple groups, one-way ANOVA and LSD test were used if the data conformed to a normal distribution and homogeneity of variance was verified. If the data did not conform to a normal distribution or the variances were unequal, nonparametric tests and the Kruskal-Wallis test were used for analysis. A p-value < 0.05 was considered statistically significant.

[0080] 2. Results

[0081] 2.1 Identification of major chemical components of QRSJF based on UPLC-Q-TOF-MS

[0082] like Figure 1 As shown, after preparing the test solution and injecting it for analysis, positive and negative ion full scans were used to obtain positive and negative ion pattern diagrams. After obtaining the molecular formula from high-resolution m / z, candidate structures were first identified using literature and public databases, and then confirmed by MS fragmentation. The identification results preliminarily characterized 140 major components of QRSJF (see Table 1). The identified compounds included 9 flavonoids and their glycosides, 32 organic acids and their derivatives, 11 alkaloids and their derivatives, 10 nucleic acid bases and their derivatives, 1 amino acid and its derivative, and 68 other polyphenols and glycosides.

[0083] Table 1. UPLC-Q-TOF-MS characterization of compounds in QRSJF

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] 2.2QRSJF improves ApoE - / - Abnormal blood lipids and serum inflammatory factor levels in mice

[0092] Experimental design such as Figure 2 As shown in Figure A, the weight change curves of each group of animals during the study period are as follows. Figure 2 As shown in Figure B, during the entire 12-week intervention period, the body weight of ApoE- / - mice fed a high-fat diet (HFD) was significantly higher than that of the control group (CTR), while the body weight gain was improved to varying degrees after treatment with QRSJF and simvastatin. Figure 2 B). Regarding blood lipids, the HFD group showed significant dyslipidemia, while low- and high-dose QRSJF treatment significantly reduced serum total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-C) levels, while increasing high-density lipoprotein cholesterol (HDL-C) levels. Figure 2 Simvastatin (CD) also exhibits a similar regulatory effect.

[0093] Atherosclerosis (AS) is a chronic inflammatory disease characterized by autoimmunity. This study also measured serum inflammatory factor levels and found that the concentrations of IL-6 and IL-1β in the HFD group were significantly higher than those in the CTR group. Figure 2 E) indicates a chronic low-grade inflammatory state. QRSJF treatment significantly reduced the above inflammatory markers, and simvastatin also showed a comparable effect.

[0094] In conclusion, QRSJF can effectively improve ApoE. - / - It induces dyslipidemia in mice and reduces systemic inflammatory responses induced by a high-fat diet.

[0095] 2.3 QRSJF can reduce atherosclerotic plaque formation and arterial stenosis.

[0096] To further investigate the impact of QRSJF on atherosclerotic plaque formation, vascular ultrasound was used to assess plaque and hemodynamics in real time. Carotid artery plaque formation was observed in mice in different groups. Color Doppler images showed "filling defects" caused by plaque within the vascular lumen, and the maximum stenosis percentage was calculated accordingly. Intraluminal thrombus thickness (IMT) is a clinical indicator used to assess the severity of carotid atherosclerosis and is also a predictor of cardiovascular event risk. By measuring the inner diameter, stenosis percentage, and IMT of the LCCA at 1 mm after the bifurcation, the results showed that the HFD group had a significantly reduced inner diameter, increased stenosis percentage, and elevated IMT. Figure 3 AC). After QRSJF intervention, both arterial stenosis and vessel wall thickening were significantly reduced (AC). Figure 3 (A–C).

[0097] Arterial stiffness (AS) is often accompanied by decreased arterial elasticity and compliance. To further evaluate whether QRSJF improves this phenotype, pulse wave velocity (PWV)—the gold standard for assessing arterial stiffness and elasticity—was measured. PWV was significantly increased in the HFD group, and QRSJF treatment significantly alleviated this trend. Figure 3 D). In addition, ApoE - / - Mice are prone to plaque formation at the aortic root and aortic arch, and luminal stenosis further leads to hemodynamic changes. The degree of stenosis was indirectly assessed using peak systolic velocity (PSV), end-diastolic velocity (EDV), and mean ventricular velocity (MFV). Significant abnormalities in aortic hemodynamic parameters were found in the HFD group, while both the high-dose QRSJF group (QRSJ-H) and the simvastatin group (Simva) significantly improved these parameters, with a marked decrease in PSV.

[0098] The results showed that QRSJF alleviated ApoE in patients fed with HFD. - / - In mice, carotid artery stenosis and intimal thickening reduced arterial stiffness, restored compliance, and improved abnormal aortic hemodynamic parameters.

[0099] 2.4QRSJF reduces atherosclerotic lesions and enhances plaque stability.

[0100] To further investigate the protective effect of QRSJF against atherosclerosis, an analysis of ApoE was conducted. - / - The formation of aortic plaques in mice was evaluated. Mice induced by a high-fat diet (HFD) showed significant plaque formation and a marked increase in lipid-positive areas; however, treatment with QRSJF and simvastatin significantly reduced the plaque area and lipid deposition in the aortic root. Figure 4 AB). H&E staining showed that the plaque area and necrotic core were significantly reduced in both treatment groups. Figure 4 C). To further assess plaque stability, Masson's trichrome staining was performed on aortic root sections (C). Figure 4 D), and immunohistochemical staining and quantitative analysis of CD68 (macrophages) and α-SMA (smooth muscle cells) were performed. Figure 4 E). Compared with the control group (CTR), the HFD group had reduced collagen content and α-SMA positive areas, and CD68... + Macrophage infiltration increased; QRSJF and simvastatin treatment significantly inhibited macrophage infiltration and increased collagen and smooth muscle content. Figure 4 DE). Furthermore, the plaque vulnerability index in the treatment group was significantly lower than that in the HFD group ( Figure 4 The results showed that QRSJF could effectively inhibit the progression of AS plaques and enhance their stability.

[0101] 2.5QRSJF alters HFD-induced ApoE - / - Metabolic characteristics of mouse liver

[0102] To evaluate the effect of QRSJF on ApoE in HFD-fed individuals - / - The effects of endogenous metabolic changes in mice were investigated, and non-targeted metabolomics analysis was performed on liver samples from each group of mice. Principal component analysis revealed significant separation of metabolomic profiles among the CTR, QRSJ-L, QRSJ-H, and HFD groups. Figure 5 A) indicates that QRSJF modulates abnormal metabolites induced by HFD in mice. The volcano plot shows the distribution of metabolites in both positive and negative ion modes. Figure 5 B). Differential metabolites were identified by integrating data from the HMDB and MS / MS fragment databases.

[0103] In liver samples, administration of low and high doses of QRSJF reversed the expression of 51 and 78 differentially expressed metabolites, respectively (Tables 2 and 3). Heatmaps of the top 30 disease-related metabolites show changes in abundance after treatment. Figure 5 C, 5E). Metabolites, such as choline, riboflavin, inosine, glutamine, allose, histidine, proline, threonine, methotrexate, taurodeoxycholic acid, glycocholic acid, and bile acids, returned to normal or near-normal levels after low- and high-dose QRSJF intervention. Subsequent KEGG pathway enrichment analysis using P<0.05 and enrichment analysis indicated that ABC transporter, aminoacyl-tRNA biosynthesis, and primary bile acid biosynthesis are key pathways affected by QRSJF treatment. Figure 5 D, 5F).

[0104] Liver metabolomics analysis showed that QRSJF prevents and treats AS by regulating ABC transporter and bile acid metabolism pathways.

[0105] Table 2 Differential metabolites in mouse liver samples after QRSJ-L intervention.

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[0109] Table 3 Differential metabolites in mouse liver samples after QRSJ-H intervention

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[0113] 2.6QRSJF activates ApoE - / - LXR-α in mouse liver promotes randomized controlled trials (RCTs), hepatic bile acid synthesis, and cholesterol excretion.

[0114] This study investigated whether the protective effect of QRSJF on arteriosclerosis (AS) is related to reverse cholesterol transport (RCT) and bile acid metabolism using non-targeted metabolomics analysis. The results showed that QRSJF increased ApoE induced by a high-fat diet. - / - Protein expression of SR-B1 and low-density lipoprotein receptor (LDLR) in mouse liver ( Figure 6 A). Despite enhanced hepatic lipid transport, neither Oil Red O nor H&E staining showed hepatic steatosis. Figure 6 BC); conversely, QRSJF treatment significantly reduced hepatic lipid accumulation and hepatocyte degeneration (BC); Figure 6 B), and significantly reduced liver TC and TG levels ( Figure 6 C) suggests an enhanced ability to convert cholesterol into bile acids. After QRSJF intervention, the levels of total bile acids in the liver and feces increased, with a particularly significant increase in the high-dose group. Figure 6 D). Further analysis showed that both low and high doses of QRSJF upregulated bile acid synthase CYP7A1 (D). Figure 6 E) and expression of bile salt output pump (BSEP) Figure 6 F). Simultaneously, the expression of ABCG5, ABCG8, and ABCA1 also increased, indicating enhanced cholesterol excretion into bile / intestinal lumen and transport to HDL particles. Figure 6 F) is one of the mechanisms by which QRSJF improves hepatic steatosis.

[0115] To investigate whether QRSJF targets LXR-α—a receptor that plays a key regulatory role in cholesterol transport and bile acid metabolism—liver expression was examined. The results showed that both low and high doses of QRSJF promoted LXR-α protein levels. Figure 6 G). In summary, QRSJF reduces plasma cholesterol levels by activating hepatic LXR-α, promoting hepatic RCT, accelerating cholesterol breakdown into bile acids and excretion into bile / intestine, and ultimately improving HFD-induced hepatic steatosis and AS progression.

[0116] 2.7 QRSJF promotes RCT and bile acid synthesis mediated by activation of the LXR-α pathway.

[0117] To verify whether QRSJF promotes reverse cholesterol transport (RCT) and bile acid synthesis and excretion by activating LXR-α, this study first stimulated HepG2 cells with 500 μM FFA and then intervened with QRSJF. After 24 hours of treatment, Oil Red O and BODIPY staining results showed that QRSJF alleviated FFA-induced lipid accumulation in a concentration-dependent manner. Figure 7 AB). Simultaneously, QRSJF increased the expression of LXR-α and its downstream targets CYP7A1, ABCG5, and ABCG8 in FFA-induced HepG2 cells (AB). Figure 7 C).

[0118] To further clarify whether the above effects depend on LXR-α activation, its specific antagonist GSK2033 was used to inhibit receptor expression. The results showed that GSK2033 could reverse the upregulation of CYP7A1, ABCG5, and ABCG8 by QRSJF. Figure 7 D), and eliminated the inhibitory effect of QRSJF on FFA-induced intracellular lipid droplets (D). Figure 7 E). The above results indicate that LXR-α plays a key role in the regulation of RCT-related protein expression and bile acid synthesis in HepG2 cells by QRSJF.

[0119] In summary, the chemical composition of QRSJF was systematically analyzed using UPLC-Q-TOF-MS. An atherosclerosis model was established in ApoE- / - mice induced by a high-fat diet, and high- and low-dose QRSJF were administered as interventions. A combination of biochemical assays, histopathology, immunohistochemistry, ELISA, non-targeted metabolomics, Western blot, and immunofluorescence techniques were used to evaluate the interventional effects and mechanisms of QRSJF on atherosclerosis (AS) in vitro and in vivo. Results showed that QRSJF effectively improved lipid abnormalities induced by a high-fat diet in ApoE- / - mice, reduced serum inflammatory factor levels, inhibited atherosclerotic plaque formation, alleviated arterial stenosis, and enhanced plaque stability. Non-targeted metabolomics analysis suggested that QRSJF can reverse liver metabolic disorders, and its effects are closely related to the regulation of ABC transporters and bile acid metabolism. Further experiments showed that QRSJF could reduce hepatic lipid accumulation and significantly upregulate the expression of proteins such as SR-B1, LDLR, ABCA1, CYP7A1, ABCG5 / G8, BSEP, and LXR-α in the liver. In HepG2 cells, QRSJF reduced free fatty acid-induced lipid accumulation by activating LXR-α and enhancing the expression of downstream CYP7A1 and ABCG5 / 8; however, the protective effect could be reversed by using the LXR-α inhibitor GSK2033.

[0120] The above results indicate that QRSJF promotes cholesterol transport to the liver, bile acid synthesis, and cholesterol excretion by activating the LXR-α / ABCG5 / G8 pathway, thereby improving lipid metabolism and inhibiting the development of atherosclerosis. This study reveals the mechanism of action of Qingre Sanjie formula in combating atherosclerosis at the molecular and metabolic levels through LXR-α / ABCG5 / G8-mediated reverse cholesterol transport and bile acid biosynthesis pathways.

[0121] The above-described embodiments 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 composition for the prevention and treatment of dyslipidemia and atherosclerosis, characterized in that: It is composed of Prunella vulgaris, seaweed, Leonurus japonicus, Forsythia suspensa and Fritillaria thunbergii. The proportions of each component are as follows: Prunella vulgaris 1-3 parts, seaweed 1-3 parts, Leonurus japonicus 1-2 parts, Forsythia suspensa 1-2 parts, and Fritillaria thunbergii 0.5-1.5 parts.

2. The traditional Chinese medicine composition according to claim 1, characterized in that: The components are as follows: Prunella vulgaris 1.5 parts, Sargassum 1.5 parts, Leonurus japonicus 1.2 parts, Forsythia suspensa 1.2 parts, and Fritillaria thunbergii 1.0 part.

3. The method for preparing the traditional Chinese medicine composition according to claim 1 or 2, characterized in that: It is prepared from the active ingredients of each component, such as sliced ​​medicinal materials, fine powder, or water extract of raw materials.

4. The method for preparing the traditional Chinese medicine composition according to claim 3, characterized in that: The specific steps are as follows: (1) Weigh the raw medicinal materials according to the formula and mix them thoroughly. Add deionized water, soak them thoroughly and reflux to extract. (2) The extract was concentrated by rotary evaporation; (3) Freeze-dry the concentrated solution to obtain freeze-dried powder.

5. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of lipid-lowering drugs.

6. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a cholesterol-lowering drug.

7. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of anti-inflammatory drugs.

8. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating atherosclerosis.

9. The use of the traditional Chinese medicine composition according to claim 1 or 2 in the preparation of a medicament for treating atherosclerotic cardiovascular diseases.

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