Use of an ethyl acetate effective part of salix gmelinii

Abstract

The application provides the use of the ethyl acetate effective part of the total extract of Salix viminalis flowers, screening of an active part (ScL) of the total extract of Salix viminalis flowers for separation, chemical component analysis and structure identification; construction of a cell model related to inflammation, oxidative stress and lipid deposition of atherosclerosis, and systematic evaluation of the in-vitro biological activity of ScL; establishment of an atherosclerosis animal model by feeding ApoE- / - mice with high-fat feed, and exploration of the mechanism of ScL for improving atherosclerosis of ApoE- / - mice by serum metabolomics, liver tissue proteomics analysis and WB verification. The research shows that ScL can improve the level of oxidative stress, reduce the level of inflammation, reduce the level of blood lipids, reduce the area of atherosclerotic plaques, reduce cholesterol accumulation, and enhance the efflux capacity of intracellular cholesterol. The technical scheme of the application comprehensively discusses the intervention effect and possible mechanism of ScL on the progression of atherosclerotic lesions, so as to lay a pharmacodynamic foundation for further development and utilization of ScL.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the field of plant extraction, and more specifically to the field of applications of extracts from willow catkins. Background Technology

[0002] Atherosclerosis is a chronic, progressive vascular disease characterized by lipid deposition in the arterial intima, inflammatory cell infiltration, and fibrosis. It is the most common pathological basis of cardiovascular disease. The disease primarily affects important vascular beds such as the coronary arteries, aorta, and cerebral arteries. As the disease progresses, plaques gradually enlarge or rupture, leading to luminal narrowing or even occlusion, which in turn causes serious clinical events such as myocardial infarction, ischemic stroke, and limb ischemic necrosis, posing a significant threat to human health. In recent years, with the deepening of research on natural products, many natural compounds with good anti-atherosclerotic potential have been discovered. Current research results show that hydroxysaffron yellow A can regulate the levels of inflammatory factors and lipid indicators in the serum of ApoE gene knockout (ApoE- / -) mice fed a high-fat diet, and regulate the activation of Akt / mTOR and NF-κB by inhibiting PI3K in macrophages. Cyperine can significantly reduce blood pressure, improve metabolic indicators in hyperlipidemic states, and alleviate vasodilatory dysfunction, thereby inhibiting atherosclerosis. Artesunate, alone or in combination with rosuvastatin, can significantly reduce the levels of inflammatory factors such as TNF-α and IL-6 in the serum of ApoE⁻ / ⁻ mice fed a high-fat diet, and alleviate the progression of atherosclerotic lesions.

[0003] In recent years, research on plant-derived natural products in the adjuvant treatment of cardiovascular diseases has received increasing attention. Flavonoids, as the main active components of these natural products, possess excellent antioxidant properties and show potential application value in the prevention of oxidative stress-related diseases such as cardiovascular disease, diabetes, and neurodegenerative diseases. Salix caprea L. has long been used in Xinjiang for the prevention and treatment of cardiovascular diseases. Its flavonoids and polysaccharides have been proven to have pharmacological activities such as lowering blood pressure, regulating blood lipids, and improving vascular function; related antihypertensive functions have also been reported. Studies have shown that total flavonoids from Salix caprea L. flowers have a significant therapeutic effect on a rat model of myocardial ischemia-reperfusion injury.

[0004] Therefore, atherosclerosis is a disease involving multiple factors such as lipid deposition, oxidative stress, chronic inflammation, and endothelial damage. Plant-derived natural products often exert multiple effects simultaneously, including antioxidant, anti-inflammatory, smooth muscle cell proliferation and migration inhibition, and autophagy regulation, making them more suitable for the complexity of the disease than single-target drugs. Discovering new plant-derived natural products for the treatment of atherosclerosis, and expanding the applications of the effective components related to *Gynostemma pentaphyllum* flowers, is crucial for the treatment of cardiovascular diseases. Summary of the Invention

[0005] To address the technical problems existing in the prior art, the purpose of this invention is to provide the use of the effective fraction of ethyl acetate from *Salvia splendens*. The invention involves isolating the more active fractions from the total extract of *Salvia splendens* through screening, identifying their chemical composition and structure, constructing cell models of inflammation, oxidative stress, and lipid deposition related to atherosclerosis, systematically evaluating the in vitro bioactivity of ScL, establishing an animal model of atherosclerosis using ApoE- / - mice fed a high-fat diet, and verifying the effect through Western blotting using serum metabolomics and liver tissue proteomics analysis. The invention explores the mechanism by which ScL improves atherosclerosis in ApoE- / - mice. ScL can improve oxidative stress levels, reduce inflammation levels, reduce blood lipid levels, reduce atherosclerotic plaque area, reduce cholesterol accumulation, and enhance intracellular cholesterol efflux. This application comprehensively explores the interventional effect of ScL on the progression of atherosclerotic lesions and its possible mechanisms, aiming to lay a pharmacodynamic foundation for the further development and utilization of ScL.

[0006] To achieve this technical objective, the present invention adopts the following technical solution: This application provides the use of the effective fraction of ethyl acetate from *Gynostemma pentaphyllum* in the preparation of drugs for the prevention or treatment of atherosclerosis.

[0007] The atherosclerosis mentioned includes those induced by high fat content.

[0008] The effective component of ethyl acetate from *Gynostemma pentaphyllum* can prevent or treat atherosclerosis by improving oxidative stress levels, reducing inflammation levels, lowering blood lipid levels, reducing the area of ​​atherosclerotic plaques, reducing cholesterol accumulation, and enhancing the ability of intracellular cholesterol to be excreted.

[0009] The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* also improves oxidative stress by increasing the activity levels of antioxidant enzymes SOD, GSH, and CAT; reduces inflammation by decreasing the levels of inflammatory factors IL-6, TNF-α, and IL-1β; and lowers blood lipid levels by decreasing the levels of TC, TG, and LDL-C; thereby preventing or treating atherosclerosis.

[0010] The effective component of the ethyl acetate of *Gynostemma pentaphyllum* also promotes the conversion of cholesterol into bile acids and steroid hormones by upregulating the expression of CYP11A1, the rate-limiting enzyme in steroid hormone synthesis, and CYP7A1, the rate-limiting enzyme in bile acid synthesis, thereby reducing cholesterol accumulation in the body and preventing or treating atherosclerosis.

[0011] The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* may further enhance the efflux of intracellular cholesterol by activating the expression of membrane transport proteins ABCG1 and ABCA1, thereby preventing or treating atherosclerosis.

[0012] Furthermore, the applied drugs include single-component or compound preparations.

[0013] The dosage form of the drug includes one of the following: tablets, capsules, oral liquids, syrups, pills, injections, or lyophilized powder for injection.

[0014] This application achieves the following technical effects through the above technical solution: (1) The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* provided in this invention exhibits the best performance in terms of polyphenol and flavonoid enrichment and antioxidant capacity. An in vitro cell model was constructed to investigate the anti-inflammatory and anti-lipid accumulation activities of each component. Comprehensive evaluation results showed that the component with a dichloromethane-methanol ratio of 10:1 showed the best performance in activity testing. This component was named ScL. UPLC-HRMS was used to analyze the composition and structure of ScL, revealing 1841 compounds from 20 major categories. The compounds with the largest number of species included terpenes, phenylpropanoids, organic heterocyclic compounds, flavonoids, and phenols. Among these, compounds with a large peak area included apigenin, (+)-abscisic acid, cyclohexanoic acid, enantio-kauridine-16B,17-diol, and 6-gingerol. Furthermore, analysis of the distribution of the types and categories of compounds identified in ScL provides crucial material clues for elucidating the mechanism by which ScL regulates lipid metabolism through anti-inflammatory and antioxidant pathways.

[0015] (2) The application of the effective fraction of ethyl acetate from *Salix babylonica* provided in this invention in the preparation of drugs for the prevention or treatment of atherosclerosis, and the effect of ScL on improving oxidative stress levels in mice. Compared with the Con group, the serum activity levels of antioxidant enzymes SOD and GSH in the HFD group mice were significantly reduced (p<0.01), while CAT activity was significantly reduced (p<0.05), and the level of lipid peroxidation product MDA was significantly increased (p<0.05), indicating that there was a significant oxidative stress state in the model mice. After ScL intervention, compared with the HFD group, the serum SOD and GSH levels in ScL-H mice were significantly restored (p<0.01), the serum CAT activity in ScL-M and ScL-H groups was increased (p<0.01), while the MDA content was significantly reduced in a dose-dependent manner (p<0.01). ScL can effectively improve the redox imbalance in atherosclerosis model mice, and its mechanism may be related to enhancing antioxidant enzyme activity and inhibiting lipid peroxidation.

[0016] (3) The application of the effective fraction of ethyl acetate from *Salix babylonica* provided in this invention in the preparation of drugs for the prevention or treatment of atherosclerosis, and the effect of ScL on improving the level of inflammation in mice. Compared with the Con group, the serum levels of inflammatory factors IL-6, TNF-α, and IL-1β in the HFD group mice were significantly increased (p<0.01), indicating that there was a significant inflammatory response in the atherosclerosis model mice. After ScL intervention, the levels of each inflammatory factor showed a decreasing trend to varying degrees. Specifically, compared with the HFD group, the serum IL-6 content in the ScL-M and ScL-H groups was significantly reduced (p<0.01); the serum TNF-α level in each ScL intervention group was significantly reduced (p<0.01); while the IL-1β level was significantly reduced in the ScL-L group (p<0.05), and extremely significantly reduced in the ScL-M and ScL-H groups (p<0.01). ScL can effectively inhibit the inflammatory response in the atherosclerosis model mice, and its effect is dose-dependent.

[0017] (4) The application of the effective fraction of ethyl acetate from *Gynostemma pentaphyllum* provided in this invention in the preparation of drugs for the prevention or treatment of atherosclerosis, and the effect of ScL on blood lipid levels in mice. Compared with the Con group, the serum TC, TG, and LDL-C levels in the HFD group mice were significantly increased (p<0.01), while the HDL-C level was significantly decreased (p<0.01), indicating that the HFD group mice had obvious lipid metabolism disorders. After ScL intervention, the above blood lipid indicators all showed varying degrees of improvement. Specifically, compared with the HFD group, the serum TC level in the ScL-H group was significantly lower (p<0.01); the TG level in mice in each ScL intervention group decreased in a dose-dependent manner, with a highly significant difference between the ScL-M and ScL-H groups (p<0.01); the LDL-C level was significantly lower in the low-dose ScL group (p<0.05), and significantly lower in the ScL-M and ScL-H groups (p<0.01); while the HDL-C level only showed a significant increase in the ScL-H group (p<0.01). ScL can effectively improve lipid metabolism disorders in atherosclerotic model mice, and its effect may involve reducing atherogenic lipid components and increasing protective lipoprotein levels.

[0018] (5) Application of the effective component of ethyl acetate from *Salix viminalis* provided in this invention in the preparation of drugs for the prevention or treatment of atherosclerosis, and the effect of ScL on atherosclerotic plaques in mice. Oil Red O staining results showed that, compared with the Con group, the overall plaque area of ​​the aorta in the HFD group mice was significantly increased (p<0.01); while after ScL intervention, the aortic plaque area in the ScL-M group and the ScL-H group mice was reduced to varying degrees (p<0.05, p<0.01). Further H&E staining analysis of aortic valve sections revealed that the necrotic core area at the valve of the HFD group mice was significantly increased (p<0.01p), while the necrotic core area of ​​the ScL-M group and the ScL-H group mice was significantly reduced (p<0.01); at the same time, the fibrous cap thickness in the HFD group was significantly increased compared with the normal group (p<0.05), while the fibrous cap thickness at the plaque of the ScL-H group mice was significantly reduced (p<0.05), suggesting that the plaque structure tended to be stable. Oil Red O staining of aortic valves showed a significant increase in plaque area in the HFD group (p<0.01), while all ScL intervention groups showed varying degrees of plaque area reduction, with the ScL-M and ScL-H groups showing particularly significant effects (p<0.01). Masson staining further confirmed that the collagen fiber thickness at the plaque site was significantly reduced in the HFD group (p<0.01), while the ScL-M and ScL-H groups significantly increased collagen fiber deposition (p<0.01). ScL can effectively reduce aortic plaque area in atherosclerotic model mice, inhibit the formation of necrotic cores at the valves, and increase plaque collagen fiber content, thereby improving plaque stability and exerting a protective effect in delaying the progression of atherosclerosis.

[0019] (6) The application of the effective fraction of ethyl acetate of *Gynostemma pentaphyllum* provided by this invention in the preparation of drugs for the prevention or treatment of atherosclerosis. Serum metabolomics analysis results showed that there were 946 differential metabolites in the Con group and the HFD group, of which 271 differential metabolites were upregulated and 675 differential metabolites were downregulated. There were 971 differential metabolites in the ScL-H group and the HFD group, of which 757 differential metabolites were upregulated and 214 differential metabolites were downregulated. According to the p value from small to large and p<0.05, 10 metabolic pathways mainly involved by the metabolic markers in the serum of mice with atherosclerosis induced by high-fat diet were screened. Among them, 7 pathways were highly correlated with atherosclerosis, and steroid bile acids were selected. Liver tissue proteomics analysis revealed 265 differentially expressed proteins in the Con and HFD groups, with 176 upregulated and 89 downregulated. In the ScL-H and HFD groups, 338 differentially expressed proteins were identified, with 144 upregulated and 194 downregulated. Based on KEGG enrichment analysis, this study selected the steroid biosynthesis pathway, highly associated with atherosclerosis, as the research subject. Metabolomics and proteomics analyses, along with experimental validation, suggest that ScL may promote the conversion of cholesterol to bile acids and steroid hormones by upregulating the expression of steroid hormone synthesis rate-limiting enzymes CYP11A1 and CYP7A1, thereby reducing cholesterol accumulation in the body. Simultaneously, ScL may activate the expression of membrane transport proteins ABCG1 and ABCA1, further enhancing the efflux of intracellular cholesterol, thus playing a role in improving atherosclerosis. Attached Figure Description

[0020] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 Figures showing the total polyphenol and total flavonoid content of different extraction fractions (Figure a shows the total polyphenol content; Figure b shows the total flavonoid content).

[0022] Figure 2The effect of different components from the ethyl acetate fraction of *Salix viminalis* on the activity of HUVECs was investigated (Figure a: dichloromethane; Figure b: dichloromethane-methanol 80:1; Figure c: dichloromethane:methanol 60:1; Figure d: dichloromethane-methanol 40:1; Figure e: dichloromethane-methanol 20:1; Figure f: dichloromethane-methanol 10:1; Figure g: dichloromethane-methanol 5:1; Figure h: dichloromethane-methanol 1:1; Figure i: methanol. n=3).

[0023] Figure 3 The effects of different components of the ethyl acetate fraction of *Salix viminalis* on the viability of RAW264.7 cells were investigated (Figure a: dichloromethane; Figure b: dichloromethane-methanol 80:1; Figure c: dichloromethane:methanol 60:1; Figure d: dichloromethane-methanol 40:1; Figure e: dichloromethane-methanol 20:1; Figure f: dichloromethane-methanol 10:1; Figure g: dichloromethane-methanol 5:1; Figure h: dichloromethane-methanol 1:1; Figure i: methanol. n=3).

[0024] Figure 4 The effects of different components on the expression of cellular inflammatory factors (Figure a: IL-6; Figure b: TNF-α; Figure c: IL-1β, n=3).

[0025] Figure 5 The effects of different components on intracellular ROS levels.

[0026] Figure 6 The effects of different components on intracellular lipid accumulation levels.

[0027] Figure 7 The total ion chromatograms of ScL in positive and negative ion modes are shown in Figure a (positive ion, negative ion).

[0028] Figure 8 The number distribution of ScL component classifications.

[0029] Figure 9 This refers to the animal experiment procedure.

[0030] Figure 10 The effect of ScL on mouse body weight (Figure a shows the mouse body weight change curve, Figure b shows the final weight gain of the mouse, n=8).

[0031] Figure 11 HE-stained sections showing the effects of ScL on the morphology of mouse liver and kidney, and serum levels of ALT, ALT, BUN, and Cr (Figure a: liver; Figure b: kidney, 400×, n=3; Figure c: alanine aminotransferase; Figure d: aspartate aminotransferase; Figure e: blood urea nitrogen; Figure f: creatinine, n=8).

[0032] Figure 12The effect of ScL on serum oxidative stress indicators in mice (Figure a: SOD; Figure b: GSH; Figure c: CAT; Figure d: MDA, n=8).

[0033] Figure 13 The inhibitory effect of ScL on inflammatory factors in mouse serum (Figure a: IL-6; Figure b: TNF-α; Figure c: IL-1β, n=8).

[0034] Figure 14 The effect of ScL on blood lipid levels in mice (Figure a: TC level; Figure b: TG level; Figure c: LDL-C level; Figure d: HDL-C level, n=8).

[0035] Figure 15 Representative images and quantitative analysis results of Oil Red O staining of the mouse aorta (Figure a is a representative image of Oil Red O staining; Figure b is the quantitative analysis result, n=4.).

[0036] Figure 16 The images and quantitative analysis results of the HE of mouse arterial valves are shown in Figure a (representative image of HE; Figure b shows the quantitative analysis results, n=4).

[0037] Figure 17 Representative images and quantitative analysis results of Oil Red O in mouse arterial valves (Figure a shows a representative image of Oil Red O; Figure b shows the quantitative analysis results of necrotic cores, n=4).

[0038] Figure 18 The images and quantitative analysis results of Masson staining of mouse arterial valves are shown in Figure a (a representative image of Masson staining; b is the collagen fiber count, n=4).

[0039] Figure 19 This is a flowchart of serum metabolomics.

[0040] Figure 20 This is a flowchart of liver proteomics.

[0041] Figure 21 The PCA dispersion plots are for the Con group, HFD group, and ScL-H group samples.

[0042] Figure 22OPLS-DA score plots for samples from the Con, HFD, and ScL-H groups (Figure a: HFD group vs. Con group; Figure b: ScL-H group vs. HFD group); OPLS-DA permutation test plots for samples from the Con, HFD, and ScL-H groups (Figure c: HFD group vs. Con group; Figure d: ScL-H group vs. HFD group); OPLS-DA S-plots for samples from the Con, HFD, and ScL-H groups (Figure e: HFD group vs. Con group; Figure f: ScL-H group vs. HFD group).

[0043] Figure 23 Volcano diagram of differential metabolites.

[0044] Figure 24 Cluster analysis of metabolites.

[0045] Figure 25 For KEGG enrichment analysis of differential metabolites.

[0046] Figure 26 The PCA dispersion plots are for the Con group, HFD group, and ScL-H group samples.

[0047] Figure 27 Volcano plot for differentially expressed proteins.

[0048] Figure 28 Cluster analysis for differentially expressed proteins.

[0049] Figure 29 KEGG enrichment analysis for differentially expressed proteins (Figure a shows the HFD and Con groups; Figure b shows the ScL-H and HFD groups).

[0050] Figure 30 The expression of related proteins in each group was detected by Western blotting (Figure a shows the Western blotting chemical imaging, Figure be shows the statistical results, n=3). Detailed Implementation

[0051] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0052] The reagents used in this application were as follows: ethanol, ethyl acetate, petroleum ether, n-butanol, dichloromethanol, and methanol were purchased from Shanghai Maclean Biotechnology Co., Ltd.; interleukin-6 (IL-6) ELISA kit, tumor necrosis factor (TNF-α) ELISA kit, and interleukin-1β (IL-1β) ELISA kit were purchased from Wuhan Elite Biotechnology Co., Ltd.; chromatographic acetonitrile, chromatographic methanol, and formic acid were purchased from Thermo Fisher Scientific. Malondialdehyde (MDA) detection kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.; glutathione peroxidase (GSH) detection kit, superoxide dismutase (SOD) detection kit, and catalase (CAT) detection kit were purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd. Methanol (≥ 99.0%) and acetonitrile (≥ 99.9%) were purchased from Thermo Fisher Scientific; formic acid (LC-MS grade) was purchased from TCI (Shanghai) Chemical Industry Development Co., Ltd.; 20X PBS buffer was purchased from Shanghai Sangon Biotech; NH4HCO3 was purchased from Shanghai Yuanye Biotechnology; chromatographic grade acetone was purchased from Wokai; iRT standard peptides were purchased from Biognosys; ammonium formate (≥ 99.9%) was purchased from Sigma; succinic acid-D4 (mixed internal standard, 2 μg / mL), L-valine-D8 (mixed internal standard, 2 μg / mL), cholic acid-D4 (mixed internal standard, 2 μg / mL), D-fluorescein free acid (mixed internal standard, 2 μg / mL), and L-carnitine-D3 (mixed internal standard, 2 μg / mL) were purchased from Shanghai Yuanye Biotechnology; the BCA kit was purchased from ThermoScientific.

[0053] Instruments used: FLB-500A universal high-speed grinder, Shanghai Haozhuang Instrument Co., Ltd.; SB-5200 DTD ultrasonic cleaner, Ningbo Xinzhi Biotechnology Co., Ltd.; DZKW-D-2 constant temperature water bath, Beijing Yongguangming Medical Instrument Co., Ltd.; SHA-A water bath constant temperature shaker, Tianjin Saidelis Laboratory Analytical Instrument Manufacturing Plant; FA2004B electronic balance, Shanghai Youke Instrument Co., Ltd.; ALPHA1-2LDplus freeze dryer, Decai Instrument Equipment (Beijing) Co., Ltd.; TDL-5M medical refrigerated centrifuge, Sichuan Shuke Instrument Co., Ltd.; ZNCL-BS magnetic stirrer, Beijing Shentai Weiye Instrument Equipment Co., Ltd.; EPOCH2TS enzyme-linked immunosorbent assay (ELISA) analyzer, BioTek; benchtop high-speed refrigerated centrifuge, Eppendorf (Shanghai) International Trade Co., Ltd.; ACQUITY UPLC I-Class Plus high-performance liquid chromatograph, Waters; ACQUITY UPLC HSS column, Thermo-Orbitrap-QE. HF high-resolution liquid chromatography-mass spectrometry (LC-MS / MS), Thermo Fisher.

[0054] The raw material used in this experiment was yellow willow catkins, which originated from the Xinjiang Uygur Autonomous Region.

[0055] Each experiment in this application was performed in triplicate, and the results are expressed as mean ± standard deviation. One-way ANOVA analysis was performed on the experimental data using GraphPad Prism 8.0.2 software. p < 0.05 was considered statistically significant, and p < 0.01 was considered highly statistically significant.

[0056] Unless otherwise specified, the technical means used in the following implementation examples are conventional means well known to those skilled in the art. Example 1: Uses of the effective fraction of ethyl acetate from yellow willow buds

[0057] This application provides the use of the effective fraction of ethyl acetate from *Gynostemma pentaphyllum* in the preparation of drugs for the prevention or treatment of atherosclerosis.

[0058] The atherosclerosis mentioned includes those induced by high fat content.

[0059] The effective component of ethyl acetate from *Gynostemma pentaphyllum* can prevent or treat atherosclerosis by improving oxidative stress levels, reducing inflammation levels, lowering blood lipid levels, reducing the area of ​​atherosclerotic plaques, reducing cholesterol accumulation, and enhancing the ability of intracellular cholesterol to be excreted.

[0060] The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* also improves oxidative stress by increasing the activity levels of antioxidant enzymes SOD, GSH, and CAT; reduces inflammation by decreasing the levels of inflammatory factors IL-6, TNF-α, and IL-1β; and lowers blood lipid levels by decreasing the levels of TC, TG, and LDL-C; thereby preventing or treating atherosclerosis.

[0061] The effective component of the ethyl acetate of *Gynostemma pentaphyllum* also promotes the conversion of cholesterol into bile acids and steroid hormones by upregulating the expression of CYP11A1, the rate-limiting enzyme in steroid hormone synthesis, and CYP7A1, the rate-limiting enzyme in bile acid synthesis, thereby reducing cholesterol accumulation in the body and preventing or treating atherosclerosis.

[0062] The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* may further enhance the efflux of intracellular cholesterol by activating the expression of membrane transport proteins ABCG1 and ABCA1, thereby preventing or treating atherosclerosis.

[0063] Furthermore, the applied drugs include single-component or compound preparations.

[0064] The dosage form of the drug includes one of the following: tablets, capsules, oral liquids, syrups, pills, injections, or lyophilized powder for injection. Example 2: Separation, screening, and structural identification of effective components from the ethyl acetate fraction of *Gynostemma pentaphyllum*.

[0065] 1. Experimental Methods (1) Preparation of alcohol extract of yellow willow flower and extracts of different extract parts The medicinal material *Houttuynia cordata* was extracted using ethanol extraction at a material ratio of 1:50 to obtain a total extract. After extraction, the extract was filtered and the filtrate was collected. The ethanol was removed by vacuum concentration in a rotary evaporator. The concentrated extract was then dried in a 50°C oven to obtain a dry extract. The extract was dissolved in an appropriate amount of double-distilled water and then sequentially extracted with equal volumes of PE, EtOAc, and n-BuOH to obtain PE extract, EtOAc extract, n-BuOH extract, and an aqueous (Aq) residue, respectively. Each extract was concentrated under reduced pressure in a rotary evaporator and then dried in a 50°C oven to obtain the corresponding dry extract.

[0066] (2) Determination of total polyphenol and flavonoid content in extracts from different parts of *Hydrocotyle vulgaris* flowers Following the instructions provided by the supplier, the total polyphenol and flavonoid content of the extracts from four different extraction fractions was determined using the corresponding kit.

[0067] (3) Separation of different components from the ethyl acetate fraction of yellow willow buds An appropriate amount of the ethyl acetate fraction extract was separated by silica gel column chromatography using a gradient elution system of dichloromethane-methanol mixed solvent. The elution gradient was successively pure dichloromethane, dichloromethane-methanol (v / v ratios of 80:1, 60:1, 40:1, 20:1, 10:1, 5:1, 1:1), and pure methanol, and each eluent fraction was collected separately. Based on the results of thin-layer chromatography, fractions with the same polarity were combined, concentrated under reduced pressure, and then the fractions with different polarities were obtained. All obtained extracts were stored at 4°C for later use.

[0068] (4) Effects of different components of the ethyl acetate fraction of yellow willow buds on cell viability RAW 264.7 cells and HUVEC cells in logarithmic growth phase were harvested and treated with 1×10⁻⁶ cells. 5 Cells were seeded at a density of 1 / mL in 96-well plates and cultured for 24 h. A control group (Con), experimental groups with different concentrations of each component from section 2.2.4, and a blank control group were set up, with three replicates for each group. The corresponding concentration of extract was added to each treatment group, and after a certain period of culture, CCK-8 solution was quickly added to each well, and the cells were incubated in the dark for 30 min. The absorbance (OD value) at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula shown in Figure 1.

[0069]

[0070] (5) Screening of the most active components By evaluating in vitro anti-inflammatory activity, lipid deposition improvement effect and antioxidant damage activity, we systematically screened different polar components. Based on the comprehensive results of various activity tests, we determined the component with the best activity and measured its flavonoid and polyphenol content. We plan to use it as the research object for further in-depth research.

[0071] (6) UPLC-HRMS analysis of the composition of ScL ① Sample pretreatment Take about 100 mg of the active component (ScL) of the ethyl acetate fraction of *Salix viminalis* obtained by screening and put it into a 1.5 mL centrifuge tube. Add 1 mL of 70% methanol-water containing mixed internal standard, 2 μg / mL. Vortex for 1 min to fully dissolve it. Centrifuge at 12000 rpm for 10 min at 4℃. After passing through a 0.45 μm membrane, take 200 μL for analysis.

[0072] ②Acquisition conditions for liquid chromatography-mass spectrometry LC-MS analysis was performed using an ACQUITY UPLC I-Class high-performance liquid chromatograph and a Thermo-Orbitrap-QE HF high-resolution liquid chromatography-mass spectrometer. The LC separation conditions were as follows: the column was an ACQUITY UPLC HSS T3 (100 mm × 2.1 mm, 1.8 μm); mobile phase A was 0.1% formic acid in water; and mobile phase B was acetonitrile. The flow rate was constant at 0.35 μL / min, and the injection volume was 2 μL.

[0073] ③Mass spectrometry conditions The sample was first ionized using an electrospray ionization source, and scanning and data acquisition were performed in both positive and negative ion modes, with a mass spectrometry scanning range covering 100 to 1500 m / z. The main parameter settings were as follows: spray voltage 3800 V in positive ion mode, spray voltage -3200 V in negative ion mode, sheath gas and auxiliary gas flow rates set to 35 arb and 8 arb respectively, heater and capillary temperatures controlled at 350℃ and 320℃ respectively, S-Lens voltage set to 50 V, collision energies set to 10%, 20%, and 40% respectively, full scan mass spectrometry (Full MS) resolution of 60000, and MS2 resolution of 15000.

[0074] ④ Data processing Before pattern recognition, raw data underwent baseline filtering, peak identification, integration, and retention time correction using the metabolomics software XCMS v4.5.1. Compound identification was based on precise mass number, secondary fragmentation, and isotopic distribution, using the LuMet-TCM database and the HERB public database for qualitative analysis. Search parameters included retention times within ±0.3 min of the retention times of standards in the database, and a mass tolerance of 5 ppm for primary mass spectrometry.

[0075] 2. Experimental Results (1) Total polyphenol content and flavonoid content of extracts from different parts of yellow willow flower The total polyphenol and total flavonoid contents of different extraction fractions are shown in the attached figure. Figure 1 As shown in the figure, among the three extraction fractions and the aqueous layer, the ethyl acetate fraction had the highest total polyphenol and total flavonoid content, with the total polyphenol content reaching 43.99 mg / g and the total flavonoid content reaching 28.94 mg / g.

[0076] (2) Effects of different components of the ethyl acetate fraction of yellow willow buds on cell viability ① Effects of different components from the ethyl acetate fraction of *Gynostemma pentaphyllum* on the activity of HUVECs The effects of each component on the activity of HUVECs were detected using the CCK-8 assay. The results are shown in the appendix. Figure 2 As shown in the figure, when the concentration of each extract reached 200 μg / mL, the activity of HUVECs cells was not significantly affected compared with the normal control group (p>0.05), indicating that at this concentration, each extract had no significant cytotoxicity to HUVECs cells. Therefore, this study selected 200 μg / mL as the experimental concentration for subsequent in vitro activity evaluation to ensure that the subsequent experimental results could truly reflect the pharmacological effects of the samples rather than cytotoxicity.

[0077] ② Effects of different components of the ethyl acetate fraction of *Gynostemma pentaphyllum* on the viability of RAW264.7 cells. The effects of each component on the viability of RAW264.7 cells were detected using the CCK-8 assay. Results are shown in the appendix. Figure 3 As shown in the figure, when the concentration of each extract reached 200 μg / mL, the activity of RAW264.7 cells was not significantly affected compared with the normal control group (p>0.05), indicating that at this concentration, each extract had no significant cytotoxicity to RAW264.7 cells. Therefore, this study selected 200 μg / mL as the experimental concentration for subsequent in vitro activity evaluation to ensure that the subsequent experimental results could truly reflect the pharmacological effects of the samples rather than cytotoxicity.

[0078] (3) Effects of each component on the expression of cellular inflammatory factors The results of the effects of different components on the expression of cellular inflammatory factors are shown in the appendix. Figure 4 As shown in the figure, among the tested components, component 6 with a ratio of 10:1 significantly inhibited the expression of TNF-α, IL-6 and IL-1β in RAW264.7 cells induced by lipopolysaccharide (LPS) (p<0.01), indicating that this component has certain anti-inflammatory activity.

[0079] (4) Effects of each component on intracellular ROS levels The effects of different components on intracellular ROS levels are shown in the appendix. Figure 5 As shown in the figure, all nine components reduced the ROS level in HUVEC cells induced by H2O2 to varying degrees. Compared with the model group (LPS), the inhibitory effects of the 80:1 component to the pure methanol component on ROS were all statistically significant (p<0.01), with the 5:1 component showing the most prominent inhibitory effect and exhibiting the best antioxidant activity.

[0080] (5) Effects of each component on intracellular lipid accumulation An in vitro model of abnormal fat deposition was established in RAW264.7 cells by inducing ox-LDL. Oil Red O staining was used to detect the anti-fat deposition effects of nine components in vitro. Results are shown in the appendix. Figure 6 As shown in the figure. The results showed that 20:1, 10:1 and 5:1 all significantly inhibited lipid accumulation in RAW264.7 cells (p<0.01), with 10:1 showing the best effect, indicating that this component has the potential to inhibit macrophage foaming.

[0081] In addition, the total polyphenol and flavonoid content of the three ScL fractions with the best in vitro activity was determined. The results showed that the total polyphenol content of ScL was 127.43 mg / g and the flavonoid content was 97.12 mg / g. Compared with the ethyl acetate extract, the total polyphenols and flavonoids were significantly enriched.

[0082] (6) Compositional analysis and structural identification of ScL Compositional analysis and structural identification of ScL were performed using UPLC-HRMS. The total ion chromatogram of ScL is attached. Figure 7 As shown in the figure. A total of 1841 compounds from ScL were identified by comparing the LuMet-TCM database and the Herb public database, covering 20 major classes. Among them, compounds with a peak area exceeding 1% of the total area include apigenin, (+)-abscisic acid, cyclohexanoic acid, enanthocyanidin-16B,17-diol, and 6-gingerol. The identified compounds were sorted by peak area percentage from largest to smallest, and the top 50 compounds are presented (see Table 1).

[0083] Table 1: Composition and Structural Identification Results of ScL

[0084] (7) Number distribution of ScL component classification Compositional analysis of ScL by UPLC-HRMS revealed that the compounds contained in ScL were mainly concentrated in terpenes, phenylpropanoids, organic heterocyclic compounds, flavonoids, phenols, fatty acyls, and steroids, as shown in the attached figure. Figure 8 As shown in the figure, terpenoids were the most numerous, with 299 identified, accounting for 19.93% of the total number of compounds with clear classifications; phenylpropanoids numbered 174, accounting for 17.53%; organic heterocyclic compounds numbered 263, accounting for 11.60%; flavonoids numbered 168, accounting for 11.20%; and phenolic compounds numbered 123, accounting for 8.20%. Example 3: Study on the activity of effective components from the ethyl acetate fraction of *Gynostemma pentaphyllum* in improving atherosclerosis in ApoE- / - mice.

[0085] 1. Test Methods (1) Laboratory animals Eight male C57BL / 6J mice (6-8 weeks old) and forty male ApoE knockout (ApoE- / -) C57BL / 6J mice, weighing between 17.0 ± 2.0 g, were purchased from Nanjing Shuborn Biotechnology Co., Ltd. They were housed in an SPF-grade animal facility (Experimental Animal Use License: SYXK(New)2023-0001). Animal testing ethics were reviewed and approved by the Experimental Animal Center of Xinjiang University, license number: XJUAE-2025-034. The mice were housed at a temperature of 23-25℃ and humidity of 55%-65%, with a 12 / 12 h light / dark cycle. After one week of acclimatization, relevant experiments were conducted. All mice had free access to water and food.

[0086] (2) Animal experiment procedure See the attached document for animal experiment procedures. Figure 9 As shown in the figure. This experiment used 8 male C57BL / 6J mice and 40 male ApoE gene knockout (ApoE- / -) C57BL / 6J mice to study the activity of improving atherosclerosis. After one week of adaptive feeding, the ApoE- / - mice were switched to a high-fat diet (20% fat, 1.25% cholesterol, 0.5% sodium cholate), while the C57BL / 6J mice continued to be given a normal maintenance diet. The feeding was carried out for 12 consecutive weeks to induce an atherosclerosis model.

[0087] (3) Grouping of experimental animals and gavage strategy Table 2: Animal Experiment Grouping and Gavage Strategy

[0088] Note: Con: Control group, normal control group; HFD: High-fat diet group, high-fat diet feeding group; Simva: simvastatin control group; ScL-L: Low dose group; ScL-M: Medium dose group; ScL-H: High dose group, the same below.

[0089] After the modeling was completed, ApoE- / - mice were randomly divided into 5 groups of 8 mice each according to their body weight. C57BL / 6J mice were set up as a blank control group. The grouping is shown in Table 2. Each group was intervened for 4 consecutive weeks.

[0090] (4) Sample collection After drug administration, all mice were anesthetized and euthanized, and relevant tissue samples were collected for subsequent indicator detection. All animal experimental procedures in this study followed relevant ethical guidelines for laboratory animals. The following samples were collected: ① Serum samples: Collect 0.5-1 mL of whole blood from each mouse, centrifuge, and collect the supernatant serum, which is then stored at -80℃.

[0091] ② Intact aorta: Four mice were taken from each group, and their aortas were removed and placed in 4% formaldehyde solution and stored at room temperature; the remaining four mice had their aortas removed, rapidly frozen in liquid nitrogen, and then stored at -80℃.

[0092] ③ Heart and other tissues: The tissues were divided into two groups. One group was placed in cryovials, flash-frozen in liquid nitrogen, and stored at -80°C. The other group of tissues was placed in a 4% formaldehyde solution and stored at room temperature.

[0093] (5) Mouse body weight measurement and evaluation of liver and kidney toxicity Formaldehyde-fixed mouse kidney and liver tissues were collected from each group, routinely dehydrated, cleared, paraffin-embedded, embedded, sectioned, and stained with hematoxylin and eosin (HE) for observation and description of kidney and liver pathology. In addition, mouse serum was collected to detect the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr).

[0094] (6) Determination of biochemical indicators and enzyme-linked immunosorbent assay in mice The levels of SOD, CAT, GSH, and MDA, as well as the levels of TC, TG, LDL-C, and HDL-C, and the levels of TNF-α, IL-6, and TNF-α in mouse serum were measured according to the instructions of the test kit.

[0095] (7) Aortic Oil Red O staining The isolated aortic sample was carefully dissected under a dissecting microscope, removing the adventitia and surrounding excess tissue. The aorta was then longitudinally dissected to fully expose the intima, facilitating the infiltration of subsequent staining solution. The treated aorta was fixed in 4% paraformaldehyde solution for 2 hours, and then rinsed three times with phosphate-buffered saline (PBS) for 5 minutes each time to thoroughly remove any residual 4% paraformaldehyde solution.

[0096] After fixation, the samples were rinsed with 60% isopropanol for 2 min, then transferred to freshly prepared Oil Red O staining solution and stained at room temperature in the dark for 15-20 min. After staining, the samples were separated in 60% isopropanol for 10 s to remove non-specifically bound dyes, and immediately rinsed three times with PBS. Following this treatment, atherosclerotic plaque areas of the aortic intima were stained orange-red, while normal vessel areas appeared white or light pink, suitable for subsequent image acquisition and quantitative analysis of plaque area. Oil Red O staining was performed according to the manufacturer's instructions.

[0097] (8) Hematoxylin-eosin (HE) staining of mouse arterial valve sections Mouse hearts were fixed overnight in 4% paraformaldehyde solution. After fixation, the tissues were rinsed three times with PBS. The tissues were dehydrated with graded ethanol and then embedded in paraffin. During embedding, liquid paraffin was injected into the mold, the tissue orientation was adjusted, and after the bottom paraffin solidified, more paraffin was added. The mold was then placed in an embedding cassette and allowed to cool and solidify.

[0098] After trimming the embedded paraffin blocks, they are placed in a paraffin microtome for continuous sectioning, with a section thickness of 4-5 μm. Intact tissue sections are cut and flattened in a 40℃ water bath to prevent detachment from the slide. After being removed from the slide, they are placed in a 60℃ constant temperature oven to bake until the moisture evaporates and the paraffin melts. Then, they are stored at room temperature for later use.

[0099] (9) Mouse arterial valve sections stained with Oil Red O To perform histopathological evaluation of the aortic root, tissue specimens attached to the heart were fixed in 4% paraformaldehyde, dehydrated and protected with a gradient sucrose solution, and then embedded in OCT embedding medium. Serial frozen sections were prepared using a cryostat, and stained with Oil Red O to assess lipid deposition in the lesion area.

[0100] (10) Masson staining of mouse arterial valve sections Paraffin sections were prepared as described above. After melting the wax by baking at 72°C for 20 min, the sections were dewaxed and hydrated sequentially with xylene and graded ethanol, rinsed with running water, and stained. After staining, the sections were dehydrated with graded ethanol, cleared with xylene, mounted with neutral resin, and observed and photographed under a microscope.

[0101] (11) Data statistics The images were digitized using ImageJ software. The resulting data were then subjected to a one-way ANOVA using Graphpad Pism 9.0. The results are expressed as mean ± standard deviation (mean ± SD). p < 0.05 was considered statistically significant, and p < 0.01 was considered statistically significant. The software was then used to create graphs.

[0102] 2. Experimental Results (1) Effect of ScL on mouse body weight The results of the effect of ScL on mouse body weight are shown in the appendix. Figure 10 As shown in the figure, during the experiment, the body weight of mice in the control group (Con), model control group (HFD), positive control group (Simva), and the test groups of effective components of *Salix viminalis* extract (ScL-L, ScL-M, ScL-H) gradually increased over time. At the end of the intervention, compared with the control group (Con), the body weight increase of the model control group (HFD) was significant (p<0.01); compared with the model control group (HFD), there was no significant difference in body weight increase between the low-dose group (ScL-L) and the medium-dose group (ScL-M) (p>0.05), while the body weight increase of the high-dose group (ScL-H) and the positive control group (Simva) was significantly increased (p<0.01).

[0103] (2) Effects of ScL on the liver and kidneys of mice Mouse liver and kidney tissues were stained with hematoxylin and eosin (HE) and observed under a 400x microscope. The results are shown in the attached figure. Figure 11 As shown in the figure, compared with the control group (Con) mice, the liver and kidney tissues of the model control group (HFD) and mice in different doses of ScL groups showed fat vacuoles, indicating that the liver and kidney tissues of the mice were structurally intact, but fat accumulation was observed in the tissues, indicating that the high-fat diet had a significant impact on the liver and kidney tissues of the mice. This phenomenon was significantly reversed in the ScL-H and Simva groups, and the liver and kidney function indicators of the mice were normal, indicating that ScL has no significant toxic effect on mice.

[0104] (3) Effects of ScL on mouse serum markers ① Oxidative stress markers in mouse serum The effects of ScL on serum oxidative stress levels in mice with high-fat diet-induced atherosclerosis were investigated by measuring serum levels of superoxide dismutase (SOD), glutathione peroxidase (GSH), catalase (CAT), and malondialdehyde (MDA). The results are attached. Figure 12 As shown. (From the appendix) Figure 11(a) It can be seen that, compared with the Con group mice, the SOD activity in the serum of the HFD group mice was significantly decreased (p<0.01); compared with the HFD group mice, the SOD activity in the serum of the ScL-H group mice was significantly increased (p<0.01). (See attached...) Figure 11 (b) It can be seen that, compared with the Con group mice, the serum GSH content of the HFD group mice was significantly decreased (p<0.01); compared with the HFD group mice, the serum GSH activity level of the ScL-H group mice was significantly increased (p<0.01). (See attached...) Figure 11 (c) It can be seen that, compared with the Con group mice, the serum CAT activity in the HFD group mice was significantly decreased (p<0.05); compared with the HFD group mice, the serum CAT activity in both the ScL-M and ScL-H groups mice was significantly increased (p<0.01). (See attached...) Figure 11 (d) It was found that compared with the Con group mice, the serum MDA content of the HFD group mice was significantly increased (p<0.05); compared with the HFD group mice, the serum MDA content of the ScL-M and ScL-H groups mice was significantly decreased (p<0.01). These results demonstrate that *Salix viminalis* flower extract can increase the activity of SOD and CAT and the content of GSH in the serum of atherosclerotic mice, and decrease the serum MDA level. This indicates that ScL can significantly reduce the level of oxidative stress in atherosclerotic model mice, thereby exerting a protective effect in delaying the progression of atherosclerosis. It suggests that ScL may effectively intervene in the pathological process of atherosclerosis by reducing vascular oxidative damage and inhibiting the inflammatory cascade mediated by oxidative stress.

[0105] ②Inflammatory factor markers in mouse serum The effects of ScL on serum inflammatory factors in mice with high-fat diet-induced atherosclerosis were investigated by measuring serum levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). The results are attached. Figure 13 As shown. (From the appendix) Figure 13 (a) It can be seen that, compared with the Con group mice, the serum IL-6 level in the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the serum IL-6 level in the ScL-M group and the ScL-H group mice was significantly decreased (p<0.01). (See attached diagram) Figure 13 (b) It was found that compared with the Con group mice, the serum TNF-α level in the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the serum TNF-α levels in the ScL-L, ScL-M, and ScL-H groups mice were significantly decreased (p<0.01). (See attached...) Figure 13(c) It was found that compared with the Con group mice, the serum IL-1β level in the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the serum IL-1β level in the ScL-L group mice was significantly decreased (p<0.05), and the serum IL-1β levels in the ScL-M and ScL-H groups mice were both significantly decreased (p<0.01). These results indicate that ScL can alleviate the inflammatory response in mice by reducing the serum levels of IL-6, TNF-α, and IL-1β, thereby alleviating high-fat diet-induced atherosclerosis.

[0106] ③ Mouse blood lipid levels Investigating the effects of ScL on serum TC, TG, LDL-C, and HDL-C in mice is significant for studying its activity in improving atherosclerosis. By measuring serum TC, TG, LDL-C, and HDL-C levels in mice, the effects of ScL on serum lipids in mice with high-fat diet-induced atherosclerosis were examined. The results are attached. Figure 14 As shown. (From the appendix) Figure 14 (a) It can be seen that, compared with the Con group mice, the serum TC content of HFD mice was significantly increased (p<0.01); compared with the HFD group mice, the serum TC level of ScL-H mice was significantly decreased (p<0.01). (See attached diagram) Figure 14 (b) It can be seen that, compared with the Con group mice, the serum TG content of the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the serum TG content of the ScL-L group mice was significantly decreased, and the serum TG levels of the ScL-M and ScL-H groups mice were significantly decreased (p<0.01). (See attached...) Figure 14 (c) It can be seen that, compared with the Con group mice, the serum LDL-C content of the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the serum LDL-C content of the ScL-L group mice was significantly decreased (p<0.05), and the serum LDL-C content of the ScL-M and ScL-H groups mice was significantly decreased (p<0.01). (See attached...) Figure 14 (d) It can be seen that, compared with the Con group mice, the serum HDL-C content of the HFD group mice was significantly decreased (p<0.01); compared with the HFD group mice, the serum LDL-C content of the ScL-H group mice was significantly increased (p<0.01).

[0107] (4) Effects of ScL on atherosclerotic plaques in mice ① Whole aorta of mice stained with Oil Red O From the appendix Figure 15It was found that, compared with the Con group mice, the aortic plaque area of ​​the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the aortic plaque area of ​​the ScL-M group mice was significantly decreased (p<0.05), and the aortic plaque area of ​​the ScL-H group mice was significantly decreased (p<0.01).

[0108] ② Hematoxylin-eosin (HE) staining of mouse arterial valve sections HE staining analysis was performed on arterial valve sections from each group of mice to analyze the area of ​​necrotic core and plaque at the arterial valve sites, and the progression of atherosclerosis in each group was statistically analyzed. The results are shown in the attached figure. Figure 16 As shown in the figure. Compared with the Con group mice, the HFD group mice had a significantly increased necrotic core area at the arterial valve lesion site (p<0.01); compared with the HFD group mice, the ScL-M group mice and the ScL-H group mice had a significantly decreased necrotic core area at the arterial valve lesion site (p<0.01). Compared with the Con group mice, the HFD group mice had a significantly increased fibrous cap area at the lesion site (p<0.05); compared with the HFD group mice, the ScL-H group mice had a significantly decreased fibrous cap area at the lesion site (p<0.05).

[0109] ③ Mouse arterial valve sections stained with Oil Red O Oil Red O staining was performed on frozen sections of mouse arterial valves to examine the development of atherosclerosis in each group of mice. The results are attached. Figure 17 As shown in the figure, compared with the Con group mice, the plaque area at the aortic valve of the HFD group mice was significantly increased (p<0.01); compared with the HFD group mice, the plaque area at the aortic valve of the ScL-L group mice was significantly decreased (p<0.05), and the plaque area at the aortic valve of the ScL-M group mice and the ScL-H group mice was significantly decreased (p<0.01). The results of this application indicate that after ScL intervention, the Oil Red O staining positive area in the aortic valve region of atherosclerotic model mice was significantly reduced, suggesting that ScL can effectively inhibit local lipid accumulation and exert a protective effect in delaying the progression of atherosclerosis.

[0110] ④ Masson staining of mouse arterial valve sections Masson staining was performed on frozen sections of mouse arterial valves to examine the stability of atherosclerotic plaques at the arterial valve sites in each group of mice. The results are attached. Figure 18As shown in the figure, compared with the Con group mice, the collagen fiber thickness at the atherosclerotic plaque site was significantly reduced in the HFD group mice (p<0.01); compared with the HFD group, the collagen fiber thickness at the plaque site was significantly increased in the ScL-M and ScL-H groups mice (p<0.01). This indicates that ScL can effectively increase the collagen fiber thickness at the aortic valve plaque site in mice and the collagen fiber deposition at the aortic valve plaque site in atherosclerotic mice, suggesting that it may improve plaque stability by enhancing the fibrous cap thickness, thereby reducing the risk of serious cardiovascular events such as plaque rupture and secondary thrombosis. Example 4: Exploring the mechanism of action of ScL in improving atherosclerosis in ApoE- / - mice based on metabolomics, proteomics, and experimental validation.

[0111] 1. Experimental Methods (1) Serum metabolomics analysis Based on the results of the study on the anti-atherosclerotic activity of ScL in Example 3, serum from mice in the Con group, HFD group, and ScL-H group was collected for metabolomics analysis. Six biological samples were randomly selected from each group (n=6). The specific method is as described in Appendix. Figure 19 The process is shown in the diagram.

[0112] (2) Liver proteomics analysis Liver tissues were collected from mice in the Con, HFD, and ScL-H groups for proteomics analysis. Three biological samples were randomly selected from each group (n=3). Specific methods are detailed in the appendix. Figure 20 The process shown (3) Western Blot verification of the expression of related proteins in liver tissue Three biological samples (n=3) were randomly selected from each group. Total protein was extracted from 50 mg of liver tissue, and protein concentration was determined using the BCA method (Bio-Rad Laboratories, Inc.). Equal amounts of protein (20-30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were blocked with 5% skim milk at room temperature for 1 hour, followed by overnight incubation at 4°C with primary antibodies against ABCG1, ABCA1, CYP11A1, CYP7A1, and GAPHD. After primary antibody incubation, the membranes were washed three times with Tris-buffered saline (TBST) containing 1% Tween-20 for 10 minutes each time. Secondary antibody incubation was performed at 26°C for 1 hour, followed by three washes with TBST. Protein bands were visualized using an enhanced chemiluminescence reagent, with consistent exposure times for all samples. Semi-quantitative analysis of band intensity was performed using ImageJ software, with GAPHD used as an internal control.

[0113] 2. Experimental Results (1) Serum metabolomics ① Principal component analysis of samples Principal component analysis (PCA) provides a direct visual observation of the overall distribution of the sample set and the dispersion between groups. (Appendix) Figure 21 The PCA scatter plots for the Con, HFD, and ScL-H groups are shown. In the plot, PC1 on the horizontal axis and PC2 and PC3 on the vertical axis represent the scores of the first, second, and third principal components, respectively. The plots show that the samples from the Con, HFD, and ScL-H groups are all within the 95% confidence interval.

[0114] ② Sample Orthogonal-Partial Least Squares-Discriminant Analysis The samples were analyzed using Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), and OPLS-DA score plots were generated. The results are attached. Figure 22 As shown. (Attached) Figure 22 (a) and (b) show the OPLS-DA score plots among the Con, HFD, and ScL-H groups. The plots show a clear separation trend among the Con, HFD, and ScL-H groups, indicating significant pairwise differences in serum metabolites among these groups. The reliability and predictive ability of the OPLS-DA model were tested using the corresponding ordination test (n=200), as shown in the attached figure. Figure 22 As shown in (c) and (d), the permutation test results meet the requirements in pairwise comparisons, indicating that the model does not have the risk of overfitting and has high interpretability and predictability, allowing for further analysis of the data results. The S-plot can visually demonstrate the correlation between sample metabolites and principal components; the results are shown in the appendix. Figure 22 As shown in (e) and (f), there are differentially expressed metabolites with large fold differences among the samples in each group.

[0115] ③ Volcano plot analysis of differential metabolites From the appendix Figure 23 It was found that 946 differentially expressed metabolites were present in the Con and HFD groups, of which 271 were upregulated and 675 were downregulated, reflecting the differences in metabolites in atherosclerotic mice. In the ScL-H and HFD groups, 971 differentially expressed metabolites were present, of which 757 were upregulated and 214 were downregulated, indicating that ScL has a significant effect on metabolites in atherosclerotic mice.

[0116] ④ Hierarchical cluster analysis of differential metabolites The hierarchical clustering results of overall metabolites in the Con group, HFD group, and ScL-H group are shown in the attached figure. Figure 24 As shown, the heatmap visually presents the differences in metabolite expression through color gradients, with red indicating high expression and blue indicating low expression. The graph clearly shows the overall distribution of metabolite differences among the Con group, HFD group, and ScL-H group.

[0117] ⑤ KEGG enrichment analysis of differentially metabolized metabolites Using the MetaboAnalyst online platform, KEGG pathway enrichment analysis was performed on differentially expressed metabolites between the ScL-H group and the HFD group, as shown in the attached figure. Figure 25 As shown, 10 metabolic pathways involved in the action of ScL on the serum of mice with high-fat diet-induced atherosclerosis were screened according to p-value from smallest to largest and p<0.05. These pathways included steroid hormone biosynthesis, ovarian steroid hormone production, α-linolenic acid metabolism, primary bile acid biosynthesis, arachidonic acid metabolism, linoleic acid metabolism, choline metabolism in cancer, sphingolipid metabolism, neuroactive ligand-receptor interaction, and phenylalanine metabolism. Through KEGG pathway enrichment analysis and combined with existing research reports, 7 metabolic pathways were found to be related to improving atherosclerosis, including steroid hormone biosynthesis, primary bile acid biosynthesis, arachidonic acid metabolism, α-linolenic acid metabolism, linoleic acid metabolism, sphingolipid metabolism, and phenylalanine metabolism.

[0118] (2) Liver tissue proteomics ① Principal component analysis of samples Principal component analysis (PCA) was used to observe the overall distribution and inter-group dispersion of samples. (Appendix) Figure 26 The figure shows the PCA scatter plots for the Con, HFD, and ScL-H group samples. The horizontal axis PC1 and the vertical axes PC2 and PC3 represent the scores of the first, second, and third principal components, respectively. The figure shows that the samples from the Con, HFD, and ScL-H groups are all within the 95% confidence interval.

[0119] ② Volcano plot analysis of differentially expressed proteins From the appendix Figure 27 It was found that 265 differentially expressed proteins were present in the Con and HFD groups, of which 176 were upregulated and 89 were downregulated, reflecting the differences in protein expression in atherosclerotic mice. In the ScL-H and HFD groups, 338 differentially expressed proteins were present, of which 144 were upregulated and 194 were downregulated, indicating that ScL has a significant effect on protein expression in atherosclerotic mice.

[0120] ③ Hierarchical cluster analysis of differentially expressed proteins The hierarchical clustering results of overall metabolites in the Con group, HFD group, and ScL-H group are shown in the attached figure. Figure 28 As shown, the clustering heatmap provides a clear visual representation of the differences in protein expression among samples in each group. (See attached image.) Figure 28 It can intuitively present the overall distribution of protein expression differences among the Con group, HFD group and ScL-H group, as well as the reversal of protein expression after ScL intervention.

[0121] ④ Screening and identification of differentially expressed proteins After log transformation, the data were used to assess the differences in protein expression between groups using two parameters: Foldchange (FC) and p-value obtained by t-test. In this study, p-value < 0.05, FC ≥ 1.5, or FC ≤ 1 / 1.5 were used as heterogeneous screening criteria to screen differentially expressed proteins among the samples in each group. The main differentially expressed proteins of ScL in ApoE- / - mice fed a high-fat diet are shown in Tables 3 and 4.

[0122] Table 3: Differentially upregulated proteins between the ScL group and the HFD group

[0123] Table 4: Differentially downregulated proteins between the ScL group and the HFD group

[0124] The differentially regulated and downregulated proteins that met the p<0.05 condition were sorted in ascending order of their upregulation and downregulation folds, and the sorting results are shown in Tables 3 and 4.

[0125] ⑤ KEGG enrichment analysis of differentially expressed proteins KEGG pathway enrichment analysis was performed on differentially expressed proteins between the HFD group and the Con group, and between the ScL-H group and the HFD group. (See appendix) Figure 29 Ten metabolic pathways were selected based on p-values ​​from smallest to largest (p < 0.05). The steroid biosynthesis pathway was enriched in both groups using the KEEGG enrichment method. Steroid biosynthesis is a core metabolic pathway for the body to produce cholesterol and all steroid hormones, and it is related to the development of atherosclerosis.

[0126] (3) Western Blot was used to verify the expression of related proteins in liver tissue. Combined analysis of serum metabolomics and liver tissue proteomics suggests that ScL may accelerate cholesterol consumption and efflux by promoting cholesterol metabolism and enhancing the synthesis of bile acids and other steroid hormones. Based on this, this study hypothesizes that ScL may promote the conversion of cholesterol into bile acids and steroid hormones by upregulating the expression of steroid hormone synthesis rate-limiting enzyme CYP11A1 and bile acid synthesis rate-limiting enzyme CYP7A1, thereby reducing cholesterol accumulation in the body. Simultaneously, ScL may activate the expression of membrane transport proteins ABCG1 and ABCA1, further enhancing the intracellular cholesterol efflux capacity. These mechanisms work together to help maintain cholesterol metabolic homeostasis, thus exerting a protective effect against atherosclerosis.

[0127] The expression of relevant proteins in each group was detected by Western blotting and is shown in the figure below. Figure 30 As shown in the figure, compared with the Con group, the levels of ABCA1 and CYP11A1 in the liver tissue of mice in the HFD group were significantly decreased (p<0.05), and the levels of ABCG1 and CYP7A1 decreased even more significantly (p<0.01). Compared with the HFD group, the levels of ABCA1, ABCG1, and CYP11A1 in the liver tissue of mice in the ScL-H group were significantly increased (p<0.01), and CYP7A1 was also significantly increased, but the increase was not as great as the former three (p<0.05). ABCA1 and ABCG1 are important targets for mediating cholesterol excretion by cells. Increased expression of these two can effectively improve the excretion of intracellular cholesterol from the body, prevent fat accumulation, and thus improve atherosclerosis. CYP11A1 and CYP7A1 are key rate-limiting enzymes in the synthesis of steroid hormones and bile acids, respectively. Increased expression of these two can accelerate cholesterol metabolism and thus improve atherosclerosis.

[0128] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Their purpose is to enable those who need this technology to understand the content of the present invention and implement it. They do not limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of the effective component of ethyl acetate from *Gynostemma pentaphyllum* in the preparation of drugs for the prevention or treatment of atherosclerosis.

2. The application according to claim 1, characterized in that, The atherosclerosis mentioned includes those induced by high fat content.

3. The application according to claim 1 or 2, characterized in that, The effective component of ethyl acetate from *Gynostemma pentaphyllum* can prevent or treat atherosclerosis by improving oxidative stress levels, reducing inflammation levels, lowering blood lipid levels, reducing the area of ​​atherosclerotic plaques, reducing cholesterol accumulation, and enhancing the ability of intracellular cholesterol to be excreted.

4. The application according to claim 1 or 2, characterized in that, The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* also improves oxidative stress by increasing the activity levels of antioxidant enzymes SOD, GSH, and CAT; reduces inflammation by decreasing the levels of inflammatory factors IL-6, TNF-α, and IL-1β; and lowers blood lipid levels by decreasing the levels of TC, TG, and LDL-C; thereby preventing or treating atherosclerosis.

5. The application according to claim 1 or 2, characterized in that, The effective component of the ethyl acetate of *Gynostemma pentaphyllum* also promotes the conversion of cholesterol into bile acids and steroid hormones by upregulating the expression of CYP11A1, the rate-limiting enzyme in steroid hormone synthesis, and CYP7A1, the rate-limiting enzyme in bile acid synthesis, thereby reducing cholesterol accumulation in the body and preventing or treating atherosclerosis.

6. The application according to claim 1 or 2, characterized in that, The effective fraction of ethyl acetate from *Gynostemma pentaphyllum* may further enhance the efflux of intracellular cholesterol by activating the expression of membrane transport proteins ABCG1 and ABCA1, thereby preventing or treating atherosclerosis.

7. The application according to claim 1 or 2, characterized in that, The drug includes single-component or compound preparations.

8. The application according to claim 1 or 2, characterized in that, The dosage form of the drug includes one of the following: tablets, capsules, oral liquids, syrups, pills, injections, or lyophilized powder for injection.

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