And puerarin-6apos; carrying out apos; application of-O-xyloside in preparation of medicine for treating postmenopausal lipid metabolism disorder

Abstract

The invention relates to the field of medicine application of organic compounds, in particular to application of puerarin-6 ''-O-xyloside in preparation of medicine for treating postmenopausal lipid metabolism disorder. Animal experiments of postmenopausal lipid metabolism disorder mice induced by ovariectomy and high fat diet show that the puerarin-6 ''-O-xyloside significantly reduces the TG, TC and LDL-C levels of the mice, and also increases the HDL-C level; the liver function is improved, the ALT and AST levels are reduced, the balloon-like degeneration of liver cells and the accumulation of lipid droplets in the cells are reduced, and the volume of fat cells is reduced. From the aspect of mechanism, the PPARalpha pathway is activated to regulate and control gene expression related to fatty acid oxidation, so that liver beta-oxidation is promoted, and ectopic lipid deposition is inhibited. Therefore, the puerarin-6 ''-O-xyloside can be used for preparing a PPAR alpha agonist, a medicine for treating postmenopausal lipid metabolism disorder and a medicine for treating dyslipidemia.

Description

Technical Field

[0001] This invention relates to the pharmaceutical application of organic compounds, specifically to the application of puerarin-6''-O-xyloside in the preparation of drugs for treating postmenopausal lipid metabolism disorders. Background Technology

[0002] Postmenopausal lipid metabolism disorder refers to a series of lipid metabolism imbalances that occur in women after menopause due to a significant drop in estrogen levels caused by the decline in ovarian function. This condition is often accompanied by changes in body composition, such as reduced skeletal muscle mass, decreased basal metabolic rate, and abnormal accumulation of visceral fat, leading to central obesity and systemic metabolic disorders, which can significantly increase the risk of cardiovascular diseases such as atherosclerosis. Since cardiovascular disease is the leading cause of death among postmenopausal women worldwide, early intervention for this type of lipid metabolism disorder is of great significance.

[0003] Currently, clinical treatments for postmenopausal lipid metabolism disorders mainly include hormone replacement therapy (MHT) and statins. However, MHT has safety concerns, such as increased risk of breast cancer, endometrial cancer, and thrombotic diseases, limiting its long-term use; while statins have adverse effects such as myopathy in some patients, making it difficult to meet the need for long-term safe and effective intervention. Therefore, there is an urgent need to develop an intervention method that combines effectiveness and safety.

[0004] In recent years, phytoestrogens have received widespread attention due to their structural and functional mimicry of endogenous estrogens. Kudzu root has a thousand-year history of medicinal use in my country, widely applied for relieving muscle tension and fever, promoting body fluid production and quenching thirst, and relieving diarrhea. Modern research shows that its main active components are various isoflavone compounds, such as puerarin, daidzein, genistein, and their glycoside derivatives. Studies have confirmed that kudzu root extract or classic formulas containing kudzu root can effectively improve blood lipid levels in patients with metabolic syndrome, lowering TC, TG, and LDL-C while increasing HDL-C, and helping to alleviate menopausal symptoms such as hot flashes and sweating.

[0005] Although the traditional Chinese medicine kudzu root and its isoflavone components show broad application prospects, their active ingredients are complex, the core active substances are unclear, and the targets and mechanisms of action have not been fully elucidated, which restricts their development and application. Therefore, the active ingredients and pharmacological effects of kudzu root need further research. Summary of the Invention

[0006] The purpose of this invention is to provide a new use for puerarin-6''-O-xyloside, namely, for the preparation of a drug for treating postmenopausal lipid metabolism disorders.

[0007] This invention is achieved through the following technical solution: According to a first aspect of the present invention, a novel use of puerarin-6''-O-xyloside is provided, namely, the use of puerarin-6''-O-xyloside in the preparation of PPARα agonists, wherein the structural formula of puerarin-6''-O-xyloside is: .

[0008] In some implementations, PPARα agonists also include pharmaceutically acceptable excipients.

[0009] In some embodiments, the excipients include any one or more of pharmaceutically used carriers, excipients, and diluents.

[0010] In some embodiments, the PPARα agonist is in the form of an oral liquid, injection, enteric solvent, sustained-release preparation, capsule, granule or tablet.

[0011] According to a second aspect of the present invention, a novel use of puerarin-6''-O-xyloside is provided, namely, the use of puerarin-6''-O-xyloside as a PPARα agonist in the preparation of a medicament for treating postmenopausal lipid metabolism disorders, wherein the structural formula of puerarin-6''-O-xyloside is: .

[0012] In some implementations, medications for treating postmenopausal lipid metabolism disorders also include pharmaceutically acceptable excipients.

[0013] In some embodiments, the excipients include any one or more of pharmaceutically used carriers, excipients, and diluents.

[0014] In some embodiments, the dosage form of the drug for treating postmenopausal lipid metabolism disorders is an oral liquid, injection, enteric-coated solution, sustained-release formulation, capsule, granule or tablet.

[0015] According to a third aspect of the present invention, a novel use of puerarin-6''-O-xyloside is provided, namely, the use of puerarin-6''-O-xyloside as a PPARα agonist in the preparation of a medicament for treating dyslipidemia, wherein the structural formula of puerarin-6''-O-xyloside is: .

[0016] In some implementations, the medication for treating dyslipidemia also includes pharmaceutically acceptable excipients.

[0017] In some embodiments, the excipients include any one or more of pharmaceutically used carriers, excipients, and diluents.

[0018] In some embodiments, the dosage form of the drug for treating dyslipidemia is an oral liquid, injection, enteric-coated solution, sustained-release preparation, capsule, granule or tablet.

[0019] The beneficial effects of this invention include: (1) This invention establishes a mouse model of postmenopausal lipid metabolism disorder induced by ovariectomy combined with a high-fat diet and a hepatocyte lipid metabolism disorder model induced by a fatty acid solution composed of OA and PA. For the first time, it systematically reveals that puerarin-6''-O-xyloside can significantly improve serum lipid abnormalities and reduce hepatic lipid deposition by activating the PPARα-mediated fatty acid oxidation pathway, and can effectively promote hepatocyte fatty acid oxidation and reduce lipid accumulation. Its mechanism of action depends on activating the PPARα signaling pathway, thereby upregulating the expression of downstream fatty acid oxidation-related genes such as CPT1A, ACADs, and FABPs, promoting hepatic β-oxidation, inhibiting hepatocyte lipid deposition, and thus restoring lipid homeostasis. This invention clarifies the key role of puerarin-6''-O-xyloside as a PPARα agonist in the regulation of postmenopausal lipid metabolism, thus providing a theoretical basis for the development of safe and effective drugs for postmenopausal metabolic disorders.

[0020] (2) Animal experiments in postmenopausal mice with lipid metabolism disorders induced by ovariectomy combined with a high-fat diet showed that puerarin-6''-O-xyloside significantly reduced TG, TC, and LDL-C levels in mice, while increasing HDL-C levels, improving liver function, reducing ALT and AST levels, alleviating hepatocyte ballooning degeneration and intracellular lipid droplet accumulation, and reducing adipocyte volume. Simultaneously, cell experiments in a hepatocyte lipid metabolism disorder model induced by a fatty acid solution composed of OA and PA showed that puerarin-6''-O-xyloside effectively reduced lipid accumulation in HepG2 cells, accompanied by a decrease in intracellular TG and LDL-C levels. The experimental results indicate that puerarin-6''-O-xyloside can improve obesity and related metabolic disorders caused by estrogen deficiency. Mechanistically, its beneficial effects are achieved by activating the PPARα pathway to regulate gene expression related to fatty acid oxidation, thereby promoting hepatic β-oxidation and inhibiting ectopic lipid deposition. Therefore, puerarin-6''-O-xyloside, as a naturally sourced isoflavone glycoside, exhibits estrogen-like metabolic regulatory properties and can be used to prepare PPARα agonists, drugs for treating postmenopausal lipid metabolism disorders, and drugs for treating dyslipidemia.

[0021] (3) This invention uses puerarin-6''-O-xyloside as the only active ingredient to prepare a drug for treating postmenopausal lipid metabolism disorders. The ingredients are clear, the source is clear, the efficacy is definite, the mechanism is clear, and the safety is high. It is easy to standardize production and quality control, and overcomes the defects of traditional Chinese medicine compound ingredients being complex and having large batch differences. It has good application prospects. Attached Figure Description

[0022] Figure 1 Figure showing how P6OX improves obesity in OVX+HFD mice.

[0023] Figure 2 Figure showing the metabolic status of OVX+HFD mice improved by P6OX.

[0024] Figure 3 The figure shows the transcriptomic, molecular docking, and molecular dynamics simulation results of PPARα and P6OX.

[0025] Figure 4 Figure showing how P6OX improves dyslipidemia in OVX mice through PPARα-mediated fatty acid oxidation.

[0026] Figure 5 Figure showing how P6OX reduces intracellular lipid accumulation in HepG2 cells.

[0027] Figure 6 This diagram illustrates how P6OX reduces intracellular lipid accumulation through the PPARα-mediated fatty acid oxidation pathway.

[0028] Figure 7 This figure illustrates how PPARα antagonists reverse the P6OX-mediated improvement in cellular lipid metabolism.

[0029] Figure 8 This figure shows the reversal of P6OX-induced FAO activation in HepG2 cells by PPARα antagonists. Detailed Implementation

[0030] The present invention will now be described in further detail with reference to the accompanying drawings. Unless otherwise specified, all reagents used in the following experiments are commercially available, and all experimental methods not specifically described are conventional methods known in the art.

[0031] Puerarin-6''-O-xyloside is an active ingredient in kudzu root, belonging to the natural isoflavone class of compounds. Although existing literature reports that puerarin-6''-O-xyloside has certain activities in anti-inflammation, anti-oxidation, and bone protection, its effect on postmenopausal lipid metabolism disorders remains unclear, and the molecular mechanism is still unknown.

[0032] To verify the effect of puerarin-6''-O-xyloside on postmenopausal lipid metabolism disorders, the following experiment was conducted.

[0033] Experiment 1: Animal Experiment I. Chemicals and Reagents Puerarin-6''-O-xyloside (CAS: 114240-18-5) was provided by Guangzhou Jingxin Biotechnology Co., Ltd.

[0034] The high-fat diet (HF60) was purchased from the Guangdong Provincial Medical Laboratory Animal Center.

[0035] Estradiol valerate tablets were purchased from Delpharm Lille SAS (France).

[0036] All enzyme assays were performed using validated commercially available kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) to determine serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), TG, HDL-C, LDL-C, and TC levels.

[0037] II. Laboratory Animals Six-week-old female C57BL / 6J mice were purchased from the Guangdong Provincial Center for Laboratory Animal Science (Guangzhou, China). Mice were acclimatized to SPF conditions for one week with free access to food and water. All experimental protocols were pre-reviewed and formally approved by the Ethics Committee of Guangzhou University of Chinese Medicine on September 23, 2024 (Approval No.: 20240923007).

[0038] After a one-week acclimatization period, mice were randomly divided into five groups: sham-operated group, ovariectomy combined with a high-fat diet model group, low-dose puerarin-6''-O-xyloside group, high-dose puerarin-6''-O-xyloside group, and a positive control group, with six mice in each group. In the sham-operated group, only the adipose tissue around the ovary was removed; ovariectomy was not performed. Mice in the other groups underwent bilateral ovariectomy (OVX). Drug administration began one week post-surgery. During the drug administration period, mice undergoing bilateral ovariectomy were simultaneously fed a high-fat diet (HFD) to establish the model, receiving a high-fat diet (HF60), while mice in the sham-operated group maintained a normal diet.

[0039] Each group was administered medication according to the following protocol: The sham surgery group (SHAM group) was given an equal volume of 0.25wt% sodium carboxymethyl cellulose (CMC-Na) solution by gavage. The ovariectomy combined with high-fat diet model group (referred to as OVX+HFD group) was given an equal volume of 0.25wt% sodium carboxymethyl cellulose (CMC-Na) solution by gavage; Low-dose puerarin-6''-O-xyloside group (PL group): Puerarin-6''-O-xyloside was administered by gavage at a dose of 30 mg / kg / d. High-dose puerarin-6''-O-xyloside group (PH group): Puerarin-6''-O-xyloside was administered by gavage at a dose of 60 mg / kg / d. The positive control group (E2 group) was given estradiol valerate by gavage at a dose of 10 mg / kg / d.

[0040] The administration continued for 12 weeks, once daily, until the sample was collected. The sample was collected after the last administration.

[0041] III. Body Composition Analysis Using the Kubtec / Parameter 3D imaging system (Kubtec, USA), fat mass and lean body mass were measured according to the manufacturer's recommended procedures to assess the distribution of body tissues.

[0042] IV. Tissue Staining Liver tissue was fixed in 4% paraformaldehyde, dehydrated with ethanol, and then embedded in paraffin. Sections were 10 μm thick and stained with hematoxylin and eosin (H&E). For lipid deposition analysis, frozen sections were prepared using OCT embedding and stained with Oil Red O. Images were acquired via whole-section scanning using a digital pathology imaging system (Jiangfeng Biotechnology Co., Ltd., China).

[0043] V. Energy Metabolism Detection Mice were acclimatized overnight in the energy metabolism monitoring system and then continuously monitored for 24 hours under a 12-hour light / dark cycle, with the ambient temperature alternating every 6 hours. The CLAMS-16M system (Columbus, USA) was used to precisely control environmental conditions to assess metabolic status at different temperatures.

[0044] VI. Transcriptome Analysis Total RNA was extracted from mouse liver samples under conditions ensuring high quality and integrity. The RNA was then reverse transcribed into cDNA and ligated with platform-specific adapters to construct sequencing libraries. The libraries were sequenced using the Illumina sequencing platform to generate raw sequence data in FASTQ format. The raw data underwent bioinformatics processing, including quality control and adapter trimming. The processed sequences were mapped to a reference genome using a validated alignment algorithm, and gene expression levels were quantified based on the aligned reads. Statistical methods were used for differential expression analysis, and differentially expressed genes under different experimental conditions were identified after appropriate standardization and significance screening.

[0045] VII. Molecular docking The PPARα structure was downloaded from the RCSB protein database (PDB ID: 7E5I). The protein was pretreated using the Maestro 11.9 platform before molecular docking. The structure of puerarin-6''-O-xyloside (hereinafter referred to as P6OX) was obtained from the PubChem database, converted to a suitable three-dimensional configuration for docking, and energy minimization was performed using Chem3D to obtain a stable conformation. Docking simulations were performed using AutoDockTools (version 1.5.7) to predict the binding conformation and affinity. The docking results were visualized and analyzed using a Python 2.7-based scripting tool to resolve key intermolecular interactions.

[0046] VIII. Molecular Dynamics (MD) Simulation Molecular dynamics simulations of the PPARα-P6OX complex were performed using the GROMACS 2020 software package. The protein force field was obtained using AMBER99SB-ILDN, and the small molecule ligand topology was generated using the sobtop program. The partial charges of the ligand atoms were obtained by fitting the confined electrostatic potential (RESP). The system was placed in a TIP3P water molecule box, and Na was added according to the initial docking structure. + or Cl - Ions balance the total charge.

[0047] The trajectory data were used for structural alignment and root mean square deviation (RMSD) calculations via trjconv in GROMACS. The binding free energy of the PPARα-P6OX complex was calculated using the gmx_MMPBSA module built into GROMACS 2020.

[0048] IX. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) RNA was extracted from liver tissue using TRIzol® reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized using a commercial high-capacity reverse transcription kit (ABclonal Technology Co., Ltd., Wuhan, China), followed by qPCR amplification using PowerUp SYBR™ Green Master Mix. Primer sequences used for qPCR analysis are shown in Table 1.

[0049] Table 1 Primer sequences used for qPCR analysis

[0050] 10. Immunofluorescence Mouse liver tissue samples were fixed with 4% paraformaldehyde (PFA) fixative (15-20 min, room temperature). Permeabilization was performed with PBS containing 0.3% polyethylene glycol tert-octylphenyl ether (Triton X-100) for 15 min, followed by blocking with 5% bovine serum albumin / phosphate-buffered saline (BSA / PBS) for 60 min. Primary antibody was incubated overnight at 4°C in blocking buffer. After washing three times with PBS (5 min each time), the samples were incubated with fluorescently labeled secondary antibody for 1 hour in the dark. Three more washes were performed to remove unbound secondary antibody. DAPI staining was performed for 10 min to label cell nuclei. Images were taken using a Nikon upright fluorescence microscope.

[0051] XI. Statistical Analysis All statistical tests were performed using GraphPad Prism (version 9.5.0). Data are expressed as mean ± standard error. One-way ANOVA was used to assess the significance of differences between groups, with a significance level of 100%. P<0.05.

[0052] XII. Experimental Results 1. Puerarin-6''-O-xyloside (P6OX) has a significant anti-obesity effect in the OVX+HFD mouse model. P6OX improves obesity in OVX+HFD mice, such as Figure 1 As shown in the figure, #### indicates that compared with the SHAM group, P < 0.0001; * indicates that compared with the OVX+HFD group, P < 0.05; ** indicates that compared with the OVX+HFD group, P < 0.01; *** indicates that compared with the OVX+HFD group, P < 0.001; **** indicates that compared with the OVX+HFD group, P < 0.0001.

[0053] from Figure 1 It can be seen that P6OX can significantly improve the obesity phenotype induced in mice by bilateral oophorectomy combined with a high-fat diet (OVX+HFD). Figure 1 A), and significantly reduced body fat percentage (A), Figure 1 B), but it had no significant effect on the percentage of lean body mass. Figure 1 C). Furthermore, P6OX exhibited a dose-dependent weight-reducing effect in OVX+HFD-induced mice ( Figure 1 D and Figure 1 E), and no significant change in food intake was observed. Further analysis showed that P6OX significantly reduced triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels in OVX+HFD mice, while significantly increasing high-density lipoprotein cholesterol (HDL-C) levels. Figure 1 F- Figure 1 I). Furthermore, P6OX administration significantly improved liver function abnormalities induced by bilateral oophorectomy and a high-fat diet (I). Figure 1 J and Figure 1 K). Simultaneously, P6OX can also reduce fasting blood glucose levels in mice in a dose-dependent manner (K). Figure 1 L). Furthermore, representative hematoxylin-eosin (H&E) staining and Oil Red O (ORO) staining of liver tissue showed that P6OX could alleviate hepatocyte ballooning degeneration and reduce lipid droplet accumulation; simultaneously, H&E staining of inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), and brown adipose tissue (BAT) to assess morphological changes indicated that P6OX intervention could reduce the volume of adipocytes in these tissues (L). Figure 1 In summary, these results suggest that puerarin-6''-O-xyloside has a significant anti-obesity effect in the OVX+HFD mouse model.

[0054] 2. Puerarin-6''-O-xyloside (P6OX) improves energy metabolism in ovariectomized mice. P6OX improves the metabolic status of OVX+HFD mice, such as Figure 2 As shown in the figure, #### indicates that compared with the SHAM group, P < 0.0001; ** indicates that compared with the OVX+HFD group, P < 0.01.

[0055] from Figure 2 It can be seen that high-dose P6OX significantly alleviated the decrease in VO2 and VCO2 caused by OVX+HFD modeling. Figure 2 A- Figure 2 D). Furthermore, high-dose P6OX also significantly improved the decreased thermogenesis capacity in OVX+HFD mice ( Figure 2 E, Figure 2 F). The above results indicate that puerarin-6''-O-xyloside can restore the energy metabolism function of OVX+HFD mice to a certain extent.

[0056] 3. PPARα is a potential molecular target for puerarin-6''-O-xyloside (P6OX). Transcriptomic, molecular docking, and molecular dynamics simulation results of PPARα and P6OX are as follows: Figure 3 As shown, P6OX regulates multiple lipid metabolism-related processes and significantly upregulates the transcriptional level of peroxisome proliferator-activated receptor α (PPARα), while also being significantly enriched in metabolic-related pathways. Gene set enrichment analysis (GSEA) further confirmed the activation of the PPAR signaling pathway, suggesting that PPARα-mediated transcriptional regulation plays an important role in P6OX-induced cellular responses. Figure 3 A- Figure 3 C). Molecular docking results showed that P6OX and PPARα could form a stable binding conformation with a binding energy of -8.979 kcal / mol and good spatial complementarity, indicating that they could form a stable complex and had a strong binding potential to the protein target. Figure 3 D). Throughout the molecular dynamics (MD) simulation, the root mean square deviation (RMSD) of the complex remained below 0.35 nm, indicating minimal overall structural fluctuations and a tendency for the system to stabilize. Figure 3 E). Residue flexibility analysis (RMSF) showed limited flexibility only in the N-terminus, C-terminus, and near the 260th amino acid residue, while the ligand-binding domain maintained low motility. Figure 3 F). The radius of gyration (Rg) decreased slightly during the simulation, suggesting enhanced compactness and improved stability of the complex. Furthermore, the trend of the solvent-accessible surface area (SASA) was consistent with that of Rg, further supporting the improved conformational stability of the complex. Figure 3 G, Figure 3 H). P6OX continuously forms approximately six hydrogen bonds with key amino acid residues within the binding pocket, highlighting the crucial role of the hydrogen bond network in maintaining the stability of the P6OX-PPARα interaction. Figure 3 I). The above results indicate that PPARα is a potential molecular target for P6OX.

[0057] 4. Puerarin-6''-O-xyloside (P6OX) improves lipid metabolism disorders in OVX mice by activating the PPARα-mediated fatty acid oxidation pathway. P6OX improves dyslipidemia in OVX mice through PPARα-mediated fatty acid oxidation, such as... Figure 4 As shown in the figure, # indicates that compared with the SHAM group, P < 0.05; ## indicates that compared with the SHAM group, P < 0.01; ### indicates that compared with the SHAM group, P < 0.001; #### indicates that compared with the SHAM group, P < 0.0001; * indicates that compared with the OVX+HFD group, P < 0.05; ** indicates that compared with the OVX+HFD group, P < 0.01; *** indicates that compared with the OVX+HFD group, P < 0.001; **** indicates that compared with the OVX+HFD group, P < 0.0001.

[0058] from Figure 4 As can be seen, quantitative PCR analysis showed that in mice induced by OVX combined with HFD, P6OX significantly improved the transcriptional levels of a series of key fatty acid oxidation (FAO)-related genes regulated by PPARα. Figure 4 A- Figure 4 G). Simultaneously, P6OX also promotes the expression of genes related to lipid metabolism and thermogenicity (G). Figure 4 H- Figure 4 L). Furthermore, P6OX also exhibits a significant regulatory effect on the expression of inflammation-related genes (L). Figure 4 M- Figure 4 Immunofluorescence staining results were consistent with qPCR data, showing that P6OX increased the protein expression levels of PPARα and carnitine palmitoyltransferase 1A (CPT1A) in liver tissue in a dose-dependent manner. Figure 4 Q). In summary, P6OX improves lipid metabolism disorders in OVX mice by activating the PPARα-mediated fatty acid oxidation (FAO) pathway.

[0059] Experiment 2: Cell Experiment I. Chemicals and Reagents Puerarin-6''-O-xyloside (CAS: 114240-18-5) was provided by Guangzhou Jingxin Biotechnology Co., Ltd.

[0060] Estradiol valerate tablets were purchased from Delpharm Lille SAS (France).

[0061] All enzymatic assays were performed using validated commercially available kits (Nanjing Jiancheng Biotechnology Institute, Nanjing, China) to determine the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), TG, HDL-C, LDL-C, and TC.

[0062] BODIPY 493 / 503 were purchased from Beyotime Biotechnology (Shanghai, China). The antibodies included rabbit anti-PPARα (#AF5301, Affinity Biosciences Ltd., China) and rabbit anti-CPT1A (#15184-1-AP, Proteintech Group, Inc., China). The PPARα antagonist GW6471 (CAS: 880635-03-0) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd. (Shanghai, China).

[0063] The purity of the oleic acid (OA) used is ≥99%, CAS No.: 112-80-1; the purity of the palmitic acid (PA) used is ≥99%, CAS No.: 57-10-3.

[0064] II. Cell Culture HepG2 cells were divided into seven groups: blank control group (CON group), model group (OAPA group), low-dose puerarin-6''-O-xyloside group (PL group), medium-dose puerarin-6''-O-xyloside group (PM group), high-dose puerarin-6''-O-xyloside group (PH group), positive control group (E2 group), and PPARα antagonist group (PH-GW6471 group).

[0065] To establish a lipid metabolism disorder model, cells in the model group and the drug intervention group (i.e., low-dose puerarin-6''-O-xyloside group, medium-dose puerarin-6''-O-xyloside group, high-dose puerarin-6''-O-xyloside group, positive control group, and PPARα antagonist group) were treated with a fatty acid solution composed of 40 mM oleic acid (OA) solution and 20 mM palmitic acid (PA) solution in a volume ratio of 4:1. The blank control group was treated with only an equal amount of dimethyl sulfoxide (DMSO). The induction lasted for 24 hours.

[0066] To evaluate the protective effect of puerarin-6''-O-xyloside, the low-dose, medium-dose, and high-dose groups of puerarin-6''-O-xyloside were treated with fatty acid solution supplemented with 50 μM, 100 μM, and 200 μM of puerarin-6''-O-xyloside, respectively. The positive control group was treated with fatty acid solution supplemented with 100 nM estradiol valerate. The PPARα antagonist group was treated with fatty acid solution supplemented with 200 μM puerarin-6''-O-xyloside and 10 μM GW6471. Cells were collected 24 hours after treatment for subsequent experiments.

[0067] III. Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) RNA was extracted from HepG2 cells using TRIzol® reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized using a commercial high-capacity reverse transcription kit (ABclonal Technology Co., Ltd., Wuhan, China), followed by qPCR amplification using PowerUp SYBR™ Green Master Mix. Primer sequences used for qPCR analysis are shown in Table 2.

[0068] Table 2 Primer sequences used for qPCR analysis

[0069] IV. Immunofluorescence HepG2 cell samples were fixed with 4% paraformaldehyde (PFA) fixative (15-20 min, room temperature). Permeabilization was performed with PBS containing 0.3% polyethylene glycol tert-octylphenyl ether (Triton X-100) for 15 min, followed by blocking with 5% bovine serum albumin / phosphate-buffered saline (BSA / PBS) for 60 min. Primary antibody was incubated overnight at 4°C in blocking buffer. After washing three times with PBS (5 min each time), the cells were incubated with fluorescently labeled secondary antibody for 1 hour in the dark. Three more washes were performed to remove unbound secondary antibody. DAPI staining was performed for 10 min to label cell nuclei. Images were taken using a Nikon upright fluorescence microscope.

[0070] V. Statistical Analysis All statistical tests were performed using GraphPad Prism (version 9.5.0). Data are expressed as mean ± standard error. One-way ANOVA was used to assess the significance of differences between groups, with a significance level of 100%. P <0.05.

[0071] VI. Experimental Results 1. Puerarin-6''-O-xyloside (P6OX) improves lipid content in HepG2 cells. P6OX reduces intracellular lipid accumulation in HepG2 cells, such as Figure 5 As shown, Figure 5 In A, *** indicates that P < 0.001 compared to a concentration of 0 μM; Figure 5 In BD, #### indicates that compared with the CON group, P < 0.0001; ** indicates that compared with the OAPA group, P < 0.01; *** indicates that compared with the OAPA group, P < 0.001; **** indicates that compared with the OAPA group, P < 0.0001.

[0072] from Figure 5 As can be seen, the CCK-8 assay was used to eliminate the interference of drug toxicity on the experimental results. The results showed that P6OX did not exhibit significant cytotoxicity at concentrations below 200 μM. Figure 5 A). P6OX dose-dependently reduced elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) levels induced by a fatty acid solution composed of OA and PA, but had no significant effect on total cholesterol (TC) levels. Figure 5 B- Figure 5 D). BODIPY staining results showed that in the HepG2 cell model induced by a fatty acid solution composed of OA and PA, intracellular lipid content was significantly reduced after P6OX intervention. Figure 5 E). In summary, the above results indicate that puerarin-6''-O-xyloside has a positive regulatory effect on cellular lipid metabolism.

[0073] 2. Puerarin-6''-O-xyloside (P6OX) reduces intracellular lipid accumulation through the PPARα-mediated fatty acid oxidation pathway. P6OX reduces intracellular lipid accumulation through the PPARα-mediated fatty acid oxidation pathway, such as... Figure 6 As shown in the figure, ## indicates that compared with group CON, P < 0.01; ### indicates that compared with group CON, P < 0.001; #### indicates that compared with group CON, P < 0.0001; * indicates that compared with group OAPA, P < 0.05; ** indicates that compared with group OAPA, P < 0.01; *** indicates that compared with group OAPA, P < 0.001; **** indicates that compared with group OAPA, P < 0.0001.

[0074] from Figure 6 It can be seen that qPCR results show that P6OX significantly reversed the downregulation of genes related to the fatty acid oxidation (FAO) pathway induced by fatty acid solution composed of OA and PA. Figure 6 A- Figure 6 G). Furthermore, P6OX significantly suppressed the expression of adipogenesis-related genes while upregulating the transcriptional levels of thermogenesis-related markers (G). Figure 6H- Figure 6 N). Immunofluorescence analysis further confirmed that P6OX could effectively restore the protein expression of PPARα and CPT1A in HepG2 cells after treatment with fatty acid solution composed of OA and PA. Figure 6 O, Figure 6 Therefore, the ability of P6OX to reduce intracellular lipid accumulation can be attributed to its regulatory role in the PPARα-driven fatty acid oxidation (FAO) signaling pathway.

[0075] 3. PPARα antagonists reverse the effects of puerarin-6''-O-xyloside (P6OX)-mediated improvement in cellular lipid metabolism. The effects of PPARα antagonists on reversing P6OX-mediated improvements in cellular lipid metabolism are as follows: Figure 7 As shown in the figure, #### indicates that compared with the CON group, P < 0.0001; **** indicates that compared with the OAPA group, P < 0.0001; ++ indicates that compared with the PH group, P < 0.01.

[0076] from Figure 7 It can be seen that the PPARα antagonist GW6471 significantly reversed the decrease in intracellular triglyceride (TG) and total cholesterol (TC) levels induced by P6OX treatment. Figure 7 A, Figure 7 B). Furthermore, Bodipy staining results showed that GW6471 could reverse the reduction in fatty acid accumulation induced by P6OX (B). Figure 7 C). The above results indicate that the PPARα antagonist GW6471 weakened the ameliorative effect of P6OX on lipid metabolism disorders, suggesting that PPARα mediates the regulatory effect of P6OX on lipid metabolism.

[0077] 4. PPARα antagonists eliminate the activation of the fatty acid oxidation (FAO) pathway by puerarin-6''-O-xyloside (P6OX) in HepG2 cells. PPARα antagonists reversed P6OX-induced FAO activation in HepG2 cells, as shown in the following figure. Figure 8As shown in the figure, # indicates that compared with group CON, P < 0.05; ## indicates that compared with group CON, P < 0.01; ### indicates that compared with group CON, P < 0.001; #### indicates that compared with group CON, P < 0.0001; * indicates that compared with group OAPA, P < 0.05; ** indicates that compared with group OAPA, P < 0.01; *** indicates that compared with group OAPA, P < 0.001; **** indicates that compared with group OAPA, P < 0.0001; + indicates that compared with group PH, P < 0.05; ++ indicates that compared with group PH, P < 0.01; +++ indicates that compared with group PH, P < 0.001; ++++ indicates that compared with group PH, P < 0.0001.

[0078] from Figure 8 As can be seen from the qPCR analysis, GW6471 significantly inhibited the upregulation of key FAO genes induced by P6OX treatment, indicating that this antagonist can effectively block the activation of the PPARα pathway by P6OX. Figure 8 A- Figure 8 E). Furthermore, GW6471 can effectively reverse the regulatory effects of P6OX on lipid metabolism, thermogenic and inflammation-related pathways (E). Figure 8 F- Figure 8 L). Furthermore, to further validate these results at the protein level, immunofluorescence experiments were conducted. The results showed that GW6471 inhibited PPARα and CPT1A protein expression induced by P6OX treatment (L). Figure 8 M, Figure 8 N). The above results indicate that PPARα is a key molecular target for P6OX to exert its metabolic regulatory role.

[0079] In summary, animal experiments conducted in postmenopausal mice with lipid metabolism disorders induced by ovariectomy combined with a high-fat diet demonstrated that, compared with the model group, low- and high-dose puerarin-6''-O-xyloside significantly reduced serum TC, TG, and LDL-C, while increasing HDL-C levels (P<0.05); liver oil red staining showed a reduction in lipid droplets and body fat percentage. These effects are close to those of the estradiol valerate control group, indicating that puerarin-6''-O-xyloside has a metabolic improvement capacity comparable to estrogen. Puerarin-6''-O-xyloside is derived from the traditional Chinese medicine kudzu root, has a well-defined structure, and high safety profile. No significant toxic reactions were observed after 12 weeks of continuous administration in animal experiments, demonstrating its therapeutic safety.

[0080] This invention utilizes cell experiments in a hepatocyte lipid metabolism disorder model induced by a fatty acid solution composed of OA and PA to demonstrate that puerarin-6''-O-xyloside effectively reduces lipid accumulation in HepG2 cells, accompanied by a decrease in intracellular TG and LDL-C levels. The experimental results indicate that puerarin-6''-O-xyloside can improve obesity and related metabolic disorders caused by estrogen deficiency.

[0081] Meanwhile, transcriptomics analysis revealed that puerarin-6''-O-xyloside significantly enriches the PPAR signaling pathway and upregulates the expression of fatty acid oxidation-related genes. Molecular docking and kinetic simulations further confirmed that puerarin-6''-O-xyloside stably binds to the PPARα ligand domain (predicted binding energy of -8.979 kcal / mol), and qRT-PCR and immunofluorescence validated its upregulation of PPARα and downstream proteins in the fatty acid oxidation pathway. In an in vitro cell model, the lipid-lowering effect of puerarin-6''-O-xyloside was completely blocked after the addition of the PPARα antagonist GW6471, confirming PPARα as a potential target for puerarin-6''-O-xyloside and providing a reliable basis for subsequent drug development.

[0082] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. Application of puerarin-6''-O-xyloside in the preparation of PPARα agonists, among which, The structural formula of the puerarin-6''-O-xyloside is: 。 2. The application according to claim 1, characterized in that, The PPARα agonist also includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The excipients include any one or more of the pharmaceutically used carriers, excipients, and diluents.

4. The application according to any one of claims 1-3, characterized in that, The dosage forms of the PPARα agonist are oral liquid, injection, enteric-coated, sustained-release, capsule, granule or tablet.

5. The application of puerarin-6''-O-xyloside as a PPARα agonist in the preparation of drugs for treating postmenopausal lipid metabolism disorders, among which, The structural formula of the puerarin-6''-O-xyloside is: 。 6. The application according to claim 5, characterized in that, The medications for treating postmenopausal lipid metabolism disorders also include pharmaceutically acceptable excipients.

7. The application according to claim 6, characterized in that, The excipients include any one or more of the pharmaceutically used carriers, excipients, and diluents.

8. The application according to any one of claims 5-7, characterized in that, The dosage form of the drug for treating postmenopausal lipid metabolism disorders is oral liquid, injection, enteric-coated, sustained-release, capsule, granule or tablet.

9. The application of puerarin-6''-O-xyloside as a PPARα agonist in the preparation of drugs for treating dyslipidemia, among which, The structural formula of the puerarin-6''-O-xyloside is: 。 10. The application according to claim 9, characterized in that, The medication for treating dyslipidemia also includes pharmaceutically acceptable excipients.

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