Use of verbascoside for the preparation of a medicament for the treatment of obesity and related metabolic disorders

Abstract

This invention discloses the application of verbascoside in the preparation of drugs for treating obesity and related metabolic disorders, relating to the field of biopharmaceuticals. The drug upregulates the expression of phosphorylated AMP-activated protein kinase α and carnitine palmitoyltransferase-1α, while downregulating the expression of sterol regulatory element-binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase. This application is the first systematic study of the comprehensive intervention effect of verbascoside on diet-induced obesity and related metabolic disorders. In an in vivo obese mouse model, it was demonstrated that verbascoside can significantly inhibit excessive weight gain induced by a high-fat diet, reduce fasting blood glucose and fasting insulin levels, improve dyslipidemia indicators including triglycerides, total cholesterol, non-esterified fatty acids, and low-density lipoprotein cholesterol, enhance insulin sensitivity, and alleviate macrovesicular steatosis and hepatocellular damage.

Description

Technical Field

[0001] This invention relates to biopharmaceutical technology, specifically to the application of verbascoside in the preparation of drugs for treating obesity and related metabolic disorders. Background Technology

[0002] The incidence of obesity is increasing rapidly worldwide, becoming a major public health problem threatening human health. Obesity is not only manifested as excessive weight gain, but its core harm lies in a series of systemic metabolic disorders caused by the excessive expansion of adipose tissue, including chronic low-grade inflammation, lipid metabolism abnormalities, and insulin resistance (IR). These pathological changes promote each other, forming a vicious cascade reaction of "metabolic inflammation - lipid extravasation - insulin signal attenuation", which significantly increases the risk of type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease.

[0003] In obese states, abnormal hypertrophy of adipocytes leads to increased secretion of chemokines such as monocyte chemoattractant protein-1 (MCP-1), recruiting a large number of macrophages to infiltrate adipose tissue and polarize into the pro-inflammatory M1 phenotype, secreting inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), forming a coronal structure (CLS) surrounding necrotic adipocytes. Simultaneously, abnormal activation of lipid synthesis regulators such as sterol regulatory element-binding protein-1c (SREBP-1c) exacerbates lipid deposition in tissues such as the liver, while continuous stimulation by inflammatory factors interferes with the normal transduction of the insulin receptor substrate-1 (IRS-1) / phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT) signaling pathway, resulting in decreased insulin sensitivity in peripheral tissues.

[0004] Currently, treatments for obesity and related metabolic disorders mainly include lifestyle interventions, bariatric surgery, and drug therapy. Existing clinical drugs often target single pathological mechanisms, such as reducing energy intake by suppressing appetite, reducing fat absorption by inhibiting lipase, or improving insulin sensitivity by activating peroxisome proliferator-activated receptor gamma (PPARγ). However, for the complex pathological network of "inflammation-lipid metabolism disorder-insulin resistance," which involves multiple reinforcing mechanisms, interventions using a single mechanism are often insufficient to comprehensively correct the body's metabolic imbalance. Therefore, finding drugs that can simultaneously target the remodeling of the adipose-immune microenvironment and the repair of the glucose and lipid metabolism signaling axis, exerting a multi-target synergistic intervention effect, is of significant clinical importance.

[0005] Verbascoside is a phenylethyl glycoside natural compound extracted from plants such as Cistanche tubulosa. Previous studies have suggested that it possesses antioxidant, anti-inflammatory, and lipid-regulating biological activities. However, whether verbascoside can simultaneously exert multiple synergistic effects in obesity-induced immunometabolic disorders—including inhibiting adipose tissue inflammation, optimizing lipid metabolism, and enhancing insulin signaling—remains unclear without systematic experimental evidence. Summary of the Invention

[0006] The purpose of this invention is to provide the application of verbascoside in the preparation of drugs for treating obesity and related metabolic disorders, so as to solve the problem of the lack of multi-target synergistic drugs in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the application of verbascoside in the preparation of drugs for treating obesity and related metabolic disorders.

[0009] Furthermore, the obesity-related metabolic disorder includes at least one of dyslipidemia, hepatic steatosis, insulin resistance, and impaired glucose tolerance.

[0010] Furthermore, the drug inhibits the polarization of macrophages in the white adipose tissue of the epididymis towards the pro-inflammatory M1 type and promotes their polarization towards the anti-inflammatory M2 type.

[0011] Furthermore, the drug upregulates the expression of phosphorylated AMP-activated protein kinase α and carnitine palmitoyltransferase-1α, while downregulating the expression of sterol regulatory element-binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase.

[0012] Furthermore, the drug restores insulin signaling by upregulating the expression of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and phosphorylated protein kinase B.

[0013] Furthermore, the targets of the verbascoside include tumor necrosis factor, interleukin-6, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and insulin receptor.

[0014] In a second aspect, the present invention provides a pharmaceutical composition for treating obesity and related metabolic disorders, comprising a therapeutically effective amount of verbascoside and a pharmaceutically acceptable carrier.

[0015] Furthermore, the dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

[0016] Compared with existing technologies, the application of verbascoside provided by this invention in the preparation of drugs for treating obesity and related metabolic disorders is the first systematic study on the comprehensive intervention effect of verbascoside on diet-induced obesity and related metabolic disorders. In an in vivo obese mouse model, it was confirmed that verbascoside can significantly inhibit excessive weight gain induced by a high-fat diet, reduce fasting blood glucose and fasting insulin levels, improve dyslipidemia indicators including triglycerides, total cholesterol, non-esterified fatty acids and low-density lipoprotein cholesterol, improve insulin sensitivity, and reduce macrovesicular steatosis and hepatocyte damage. These effects indicate that verbascoside can provide comprehensive improvement for obesity-related metabolic disorders.

[0017] This invention further reveals that verbascoside can simultaneously act on three interrelated pathological pathways: inflammation control, lipid metabolism optimization, and insulin signal enhancement. Verascoside can reduce immune cell infiltration in adipose tissue, inhibit macrophage polarization towards the pro-inflammatory M1 type, and reduce the secretion of inflammatory factors. It can activate the AMPK signaling axis, enhance fatty acid oxidation, and inhibit SREBP-1c-mediated lipogenesis. It can restore the impaired IRS-1 / PI3K / AKT insulin signaling in obese individuals. The synergistic effect of these three mechanisms provides experimental evidence for multi-target intervention in obesity-related insulin resistance, suggesting that verbascoside can be developed as a candidate drug for the treatment of obesity and related metabolic disorders. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 One of the schematic diagrams illustrating the overall effect of verbascoside in improving metabolic dysfunction in obesity, provided in an embodiment of the present invention;

[0020] Figure 2 This is the second schematic diagram illustrating the overall effect of verbascoside in improving metabolic dysfunction in obesity, as provided in this embodiment of the invention.

[0021] Figure 3 This is one of the target analysis diagrams for the potential effects of verbascoside on obesity based on network pharmacology provided in this embodiment of the invention; wherein, (A) is a Venn diagram of the common target genes of verbascoside and obesity, (B) is a protein-protein interaction (PPI) network of the common target proteins, (C) is a core hub target sub-network identified based on topological parameters, (D) is a schematic diagram of the top ten significantly enriched items in GO functional enrichment analysis, and (E) is a diagram of the KEGG pathway enrichment analysis results.

[0022] Figure 4 The second diagram is an analysis of the potential target of verbascoside for obesity based on network pharmacology, provided for an embodiment of the present invention; wherein (FR) is a molecular docking pattern diagram of verbascoside with thirteen hub proteins.

[0023] Figure 5 The third diagram is an analysis of the potential target of verbascoside for obesity based on network pharmacology, provided for an embodiment of the present invention; wherein, (S) is a schematic diagram of the mechanism model derived from network pharmacology.

[0024] Figure 6 The figures provided in this embodiment of the invention show the experimental results of verbascoside reducing weight gain induced by a high-fat diet; wherein, (A) is a schematic diagram of the animal experimental protocol, (B) is a curve of mouse weight change during the modeling period, (C) is a statistical graph of mouse food intake from week 1 to week 16, (D) is a statistical graph of mouse weight at week 16, (E) is a statistical graph of fasting blood glucose level, (F) is a statistical graph of serum triglyceride content, (G) is a statistical graph of total cholesterol content, (H) is a statistical graph of low-density lipoprotein cholesterol content, (I) is a curve of mouse weight change during drug intervention, (J) is a statistical graph of final weight at week 22, and (K) is a statistical graph of weight gain at week 22.

[0025] Figure 7 One of the experimental results provided in this embodiment of the invention is shown in Figure 1, which shows the effect of verbascoside on improving glucose intolerance and regulating lipid homeostasis. Among them, (A) is the oral glucose tolerance test and the area under the curve (AUC) statistical graph, and (B) is the insulin tolerance test and the AUC statistical graph.

[0026] Figure 8 The second figure shows the experimental results of verbascoside improving glucose intolerance and regulating lipid homeostasis provided in the embodiments of the present invention; wherein, (C) is a statistical chart of fasting blood glucose level, (D) is a statistical chart of fasting insulin level, (E) is a statistical chart of insulin resistance index, (F) is a statistical chart of insulin sensitivity index, (G) is a statistical chart of triglyceride content, (H) is a statistical chart of total cholesterol content, (I) is a statistical chart of non-esterified fatty acid content, and (J) is a statistical chart of low-density lipoprotein cholesterol content;

[0027] Figure 9 One of the experimental results provided in this embodiment of the invention is shown in Figure 1, which shows the effect of verbascoside on reducing hepatic steatosis and promoting epididymal white adipose tissue remodeling. Among them, (A) is a statistical chart of liver weight, (B) is a representative chart of liver hematoxylin-eosin staining, (C) is a statistical chart of serum alanine aminotransferase level, and (D) is a statistical chart of aspartate aminotransferase level.

[0028] Figure 10Figure 2 shows the experimental results of verbascoside reducing hepatic steatosis and promoting epididymal white adipose tissue remodeling provided in the embodiments of the present invention; wherein, (E) is a representative H&E staining image of epididymal white adipose tissue, and (F) is a quantitative statistical image of adipocyte area.

[0029] Figure 11 One of the experimental results of verbascoside restoring the immune homeostasis of epididymal white adipose tissue provided in the embodiments of the present invention; wherein, (A) is a statistical graph of serum mononuclear cell chemokine-1 level, (B) is a statistical graph of serum resistin level, (C) is a statistical graph of serum tumor necrosis factor-α level, and (D) is a statistical graph of serum leptin level.

[0030] Figure 12 The second figure shows the experimental results of verbascoside in restoring the immune homeostasis of epididymal white adipose tissue provided in the embodiments of the present invention; wherein, (E) is a representative immunohistochemical staining image of IL-6, IL-10, F4 / 80, CD11c and CD206 in epididymal white adipose tissue, and (F) is a representative result of flow cytometry detection of M1 and M2 macrophages.

[0031] Figure 13 The experimental results provided in this embodiment of the invention demonstrate the effect of verbascoside on restoring immune homeostasis in epididymal white adipose tissue. Figure 3 Among them, (G), (H), and (I) are bar charts showing the flow cytometry analysis results of the proportion of macrophage-related marker positive cells in mice of different treatment groups;

[0032] Figure 14 Figure 1 shows the experimental results of verbascoside inhibiting lipid accumulation and inflammatory response in 3T3-L1 adipocytes provided in this embodiment of the invention; wherein, (A) is a representative figure of Oil Red O staining of differentiated adipocytes, (B) is a quantitative statistical figure of Oil Red O staining, (C) is a figure of cell viability determination of preadipocytes by verbascoside, (D) is a figure of extracellular triglyceride content, (E) is a figure of extracellular non-esterified fatty acid content, (F) is a figure of extracellular glycerol content, (G) is a figure of intracellular non-esterified fatty acid content, (H) is a figure of intracellular triglyceride content, (I) is a figure of IL-6 secretion level, and (J) is a figure of MCP-1 secretion level.

[0033] Figure 15The figure shows the experimental results of verbascoside regulating glucose and lipid metabolism and insulin signaling in 3T3-L1 adipocytes provided in the embodiments of the present invention; wherein, (AD) is a schematic diagram of the mRNA expression levels of adipokine-1, CCAAT / enhancer-binding protein α, fatty acid synthase and acetyl-CoA carboxylase before qRT-PCR analysis, and (E) is a schematic diagram of the immunoblotting analysis results of IRS-1, CPT-1α, PI3K, SREBP-1c, AMPKα, p-AMPKα, AKT, p-AKT, PPARγ, C / EBPα and PKA. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0035] As attached Figure 1 To be continued Figure 15 As shown:

[0036] Example 1: Prediction and validation of anti-obesity target of verbascoside based on network pharmacology and molecular docking.

[0037] This embodiment uses network pharmacology and molecular docking technology to systematically predict and verify the potential target and molecular mechanism of verbascoside in anti-obesity.

[0038] 1. Identification of verbascoside's putative target and obesity-related targets

[0039] First, a list of probable targets for verbascoside was obtained from the Swiss Target Prediction, SEA, and CTD databases. These predicted targets were mapped to the standard official gene symbols in the Uniprot database, and duplicates were removed, ultimately resulting in 174 verbascoside-related targets. Simultaneously, searches were conducted in the GeneCards, DrugBank, and OMIM databases using "Obesity" as a keyword. After removing entries with low evidence strength, 3021 obesity-related targets were obtained. The intersection of these two sets of targets was analyzed using Venny 2.1 software, ultimately identifying 114 overlapping targets as candidate therapeutic targets for verbascoside for subsequent network and enrichment analyses, as shown in the attached figure. Figure 3 As shown in Figure A.

[0040] 2. Construction and analysis of protein-protein interaction (PPI) networks

[0041] The 114 common targets were imported into the STRING database. With an interaction score median > 0.700 (high confidence), protein-protein interaction data were extracted and visualized and topological parameters calculated using Cytoscape 3.10.3 software. The constructed PPI network contains 102 nodes and 556 edges, with an average connection count of 10.902, as shown in the attached figure. Figure 3 As shown in B. The topological parameters of the network nodes were calculated, and the core hub proteins and their topological parameters were identified, as shown in Table 1.

[0042] Table 1: Topological parameters of key targets in the verbascoside-related obesity PPI network

[0043] Common name Degree Betweenness Closeness TNF 38 0.1023 0.5611 IL-6 37 0.1024 0.5872 ESR1 25 0.0736 0.5372 CCL2 (MCP-1) 24 0.0112 0.4879 CXCL8 24 0.0135 0.4856 PPARγ 23 0.0734 0.5000 MAPK1 20 0.0118 0.5000 IFN-γ 18 0.0034 0.4654 FASN 5 0.0029 0.3755 CPT1α 4 0.0000 0.3378 INSR 4 <0.0001 0.3915 AKT 2 0.0000 0.3459 CAT 1 0.0000 0.2544

[0044] As shown in Table 1, among the top-ranked nodes, TNF (degree value 38) and IL-6 (degree value 37) exhibited the highest degree values ​​and were significantly ranked high in terms of centrality and near-centrality, suggesting that they are potential key hub proteins driving obesity-related low-grade inflammation. ESR1 also showed high connectivity and centrality, suggesting its bridging role between inflammation and metabolic regulation. MCP-1 (CCL2) was closely associated with these hub proteins, suggesting that immune cell recruitment and inflammation amplification are potential processes regulated by verbascoside. Furthermore, nodes related to lipid metabolism included PPARγ, FASN, and CPT-1α, which are associated with lipidogenesis, fatty acid synthesis, and β-oxidation, respectively. Based on topological parameters, a core hub-target subnetwork was further identified, as shown in the appendix. Figure 3 As shown in C.

[0045] 3. Functional annotation and pathway enrichment analysis

[0046] To elucidate the multi-pathway regulatory mechanism of verbascoside, GO functional enrichment and KEGG pathway analysis were performed on 114 candidate targets. GO enrichment analysis identified 1727 terms, including 1508 biological processes (BP), 149 molecular functions (MF), and 70 cellular components (CC). (See attached...) Figure 3 D summarized the top 10 significantly enriched terms in BP, MF, and CC. The rich BP terms mainly involve the regulation of cell migration / movement and responses to external or exogenous stimuli; MF terms focus on cytokines, receptor signaling, and kinase regulatory activities; and CC terms are enriched in membrane microdomains, lipid rafts, and extracellular matrix-related structures.

[0047] KEGG enrichment analysis identified 183 significantly enriched signaling pathways, as shown in the attached figure. Figure 3E is shown. Enrichment analysis highlighted pathways closely associated with metabolic inflammation and obesity-related complications, including the AGE-RAGE signaling pathway, the PI3K-AKT signaling pathway, and the FoxO signaling pathway, as well as signaling pathways reflecting vascular and lipid pathology, stress response, and inflammation.

[0048] 4. Molecular docking to verify binding stability

[0049] Using AutoDock Vina software, verbascoside was molecularly docked with 13 preferred hub proteins in the core network (including SREBP-1c, TNF, IL-6, INSR, AKT, PPARγ, etc.). The results showed that the binding energy of all verbascoside-protein complexes was less than -7.0 kcal / mol, and the specific docking scores are shown in Table 2.

[0050] Table 2: Molecular docking binding free energy between verbascoside and core target protein

[0051] Target Docking score (kcal / mol) SREBP-1c -10.74 TNF -9.76 IL6 -9.48 IFNγ -9.00 CAT -8.52 CPT-1 -8.39 CCL2 (MCP-1) -8.34 MAPK1 -8.12 PPARγ -7.83 INSR -7.67 AKT1 -7.50 FASN -7.49

[0052] Note: The lower the binding energy, the more stable the binding between the ligand and the receptor.

[0053] Among them, verbascoside showed the lowest binding energy (-10.74 kcal / mol) with SREBP-1c, exhibiting strong binding activity; the binding energies with TNF and IL-6 were -9.76 and -9.48 kcal / mol, respectively. The binding modes of each docking complex are shown in the attached figure. Figure 4 As shown in FR. Molecular docking results, from a structural biology perspective, confirmed the stable ligand-receptor affinity between verbascoside and the predicted core target, validating the reliability of the network pharmacology prediction. The integrated mechanism model is shown in the attached figure. Figure 5 As shown in S.

[0054] Example 2: In vivo efficacy evaluation of verbascoside in a diet-induced obese mouse model.

[0055] This embodiment uses a high-fat diet (HFD) induced diet-induced obesity (DIO) mouse model to evaluate the in vivo intervention effect of verbascoside on obesity and related metabolic disorders.

[0056] 1. Experimental materials and establishment of the DIO mouse model

[0057] The test drug, verbascoside (purity ≥98%), was purchased from Shanghai Yuanye Biotechnology Co., Ltd., China. Simvastatin (SIM) was used as the positive control at a dose of 60 mg / kg. Male C57BL / 6 mice (8-10 weeks old) were purchased from the Animal Experiment Center of Xinjiang Medical University and housed in a controlled environment with a temperature of 21±2°C, relative humidity of 50±5%, and a 12-hour light / dark cycle, with free access to food and water.

[0058] The experimental protocol is attached. Figure 6 As shown in Figure A, mice were randomly divided into a normal fat diet group (NFD group) and a high-fat diet group (HFD group, with 60% of their calories coming from fat) after acclimatization, and underwent a 16-week dietary intervention. After induction, fasting blood glucose (FBG), fasting insulin (FINS), and lipid markers (TG, TC, LDL-C) were measured. Mice in the HFD group whose body weight exceeded the average body weight of the NFD group by more than 20% were considered to have successfully established an obesity model.

[0059] Mice that successfully modeled the disease were randomly divided into three groups: the HFD model group (continued high-fat diet and gavage with an equal volume of solvent), the SIM treatment group (high-fat diet combined with simvastatin 60 mg / kg gavage), and the Act treatment group (high-fat diet combined with verbascoside 60 mg / kg gavage). Drug intervention was administered once daily for 6 weeks. Body weight and food intake were recorded weekly during the experiment.

[0060] 2. Effects of verbascoside on weight gain and lipid metabolism

[0061] During the 16-week dietary induction period, the body weight of mice in the HFD group steadily increased, gradually deviating from that of the NFD group, as shown in the attached figure. Figure 6 As shown in B. By week 16, the final body weight of mice in the HFD group (40.55±5.83 g) was significantly higher than that in the NFD group (30.10±2.55 g), an increase of approximately 34.72% (see attached figure). Figure 6 D). Along with increased body weight, mice in the HFD group exhibited significant systemic metabolic impairment; compared to the NFD group, fasting blood glucose (see attached image) was significantly lower. Figure 6 E), serum triglycerides (attached) Figure 6 F), Total cholesterol (see appendix) Figure 6 G) and low-density lipoprotein cholesterol (LDL-C) Figure 6 H) levels were significantly elevated.

[0062] During a 6-week drug intervention, verbascoside effectively attenuated HFD-induced sustained weight gain, as shown in the appendix. Figure 6 As shown in Figure I. By the study endpoint, compared to the HFD model group, the Act treatment group mice had a weight reduction of approximately 8.61% (see Appendix). Figure 6 J), the overall weight increase decreased by approximately 60.96% (see appendix). Figure 6 (K). During the intervention, verbascoside was more effective than the positive control drug simvastatin in limiting weight gain. The above data indicate that verbascoside can effectively reduce excessive weight gain induced by long-term HFD.

[0063] 3. The effect of verbascoside on improving glucose tolerance and insulin resistance.

[0064] The effects of verbascoside on systemic metabolic homeostasis were evaluated using an oral glucose tolerance test (OGTT) and an insulin tolerance test (ITT). OGTT results are attached. Figure 7 As shown in Figure A, HFD-fed mice exhibited significant glucose intolerance, with blood glucose levels rising sharply after glucose gavage, peaking at 15 minutes. Verbascoside intervention significantly reduced circulating glucose levels at all time points and decreased the area under the OGTT curve (AUC), suggesting that verbascoside can improve glucose tolerance and glucose clearance rate in HFD-fed mice.

[0065] The ITT results are attached. Figure 7 As shown in Figure B, blood glucose levels decreased in all groups after insulin injection, reaching their lowest level at 30 minutes. However, the HFD model group mice remained in a state of relative hyperglycemia, reflecting impaired peripheral tissue responsiveness to insulin. Verbascoside intervention effectively masked this hyperglycemic characteristic and reduced the AUC value of ITT, suggesting that it significantly improved insulin tolerance and peripheral insulin sensitivity.

[0066] 4. The regulatory effect of verbascoside on systemic lipid homeostasis

[0067] Consistent with the development of insulin resistance and dyslipidemia, chronic HFD feeding induces a typical metabolic syndrome-like phenotype, characterized by fasting blood glucose (see appendix). Figure 8 C) Fasting insulin (attached) Figure 8 D) HOMA-IR index (attached) Figure 8 E) and serum TG, TC, NEFA, LDL-C (with appendix) Figure 8 A significant increase in GJ, while the HOMA-IS index (with...) Figure 8 F) Significantly reduced. Verbascoside treatment significantly reversed the above abnormalities. Compared with the HFD model group, the Act treatment group showed varying degrees of reduction in FBG, FINS, HOMA-IR, TG, TC, NEFA, and LDL-C levels, while the HOMA-IS index increased.

[0068] 5. The effect of verbascoside on improving hepatic steatosis and epididymal white adipose tissue hypertrophy.

[0069] In the DIO mouse model, HFD feeding led to significant hepatomegaly, with liver weight increasing to approximately 1.64 times that of the NFD control group, consistent with severe lipid droplet accumulation in the liver tissue. Verbascoside intervention significantly reversed this phenotype; compared to the HFD model group, the Act treatment group showed a liver weight reduction of approximately 26.35%, demonstrating superior efficacy compared to the simvastatin group (see appendix). Figure 9 A). Biochemical markers of liver injury further support the hepatoprotective effect of verbascoside: compared with the HFD model group, serum AST and ALT levels in the Act treatment group decreased by approximately 21.99% and 45.15%, respectively (see appendix). Figure 9 The CD (Cellular Diagnostic and Circulatory System) indicates that hepatocellular damage has been reduced and liver function has been partially restored.

[0070] The results of liver H&E staining are attached. Figure 9 As shown in Figure B, the liver lobule structure in the NFD group was clear and the hepatocyte morphology was normal; the hepatocytes in the HFD group showed typical macrovesicular steatosis and ballooning degeneration, with round lipid droplets of varying sizes visible in the cytoplasm; after Act intervention, the lipid droplet burden in the hepatocytes was significantly reduced, the hepatic cord structure became more regular, and the histological morphology was basically restored to a level close to that of the NFD group.

[0071] Pathological analysis of the epididymal white adipose tissue (eWAT) showed that adipocytes in the NFD group were smaller, more uniform in morphology, and more densely packed. HFD feeding induced significant adipocyte hypertrophy, decreased adipocyte density within the field of view, irregular cell outlines, widened intercellular spaces, and the presence of corona structures (CLS) formed by the aggregation of immune cells around necrotic adipocytes, consistent with the pathological characteristics of chronic inflammation of adipose tissue. The average area of ​​eWAT adipocytes in the HFD group was approximately 11031±4706 μm², which significantly decreased to approximately 3713±1551 μm² after verbascoside treatment. Figure 10 The EF indicates that adipocyte hypertrophy was effectively inhibited and tissue structure was improved.

[0072] 6. The effect of verbascoside on the remodeling of the lipid immune microenvironment

[0073] In obese individuals, adipose tissue dysfunction is closely associated with chronic low-grade inflammation. Results of the detection of circulating inflammatory mediators are attached. Figure 11 As shown in AD, compared with the NFD group, HFD feeding significantly exacerbated the systemic inflammatory response, while verbascoside treatment could partially alleviate this response. Compared with the HFD model group, the serum levels of monocyte chemoattractant protein-1 (MCP-1), resistin (RETN), tumor necrosis factor-α (TNF-α), and leptin in the Act treatment group were reduced to varying degrees, suggesting that verbascoside can inhibit the HFD-induced chronic low-grade inflammatory state.

[0074] The immunohistochemical evaluation results of the local immune microenvironment of eWAT are attached. Figure 12 As shown in Figure E, NFD-fed mice showed less immune cell infiltration in their eWAT, while HFD-fed mice exhibited significantly increased macrophage infiltration. Infiltrating adipose tissue macrophages (ATM) were predominantly pro-inflammatory M1 subsets (F4 / 80⁺CD11c⁺), with a relative decrease in alternately activated anti-inflammatory M2 macrophages (CD206⁺). Simultaneously, the CLS structure surrounding necrotic adipocytes was more abundant, accompanied by upregulation of pro-inflammatory mediator (IL-6) and downregulation of anti-inflammatory factor (IL-10). Verbascoside intervention effectively reduced the overall ATM infiltration abundance in eWAT, inhibited M1 polarization, relatively increased the proportion of M2 macrophages, and significantly reduced CLS density.

[0075] Flow cytometry further validated the above findings, as shown in the attached figure. Figure 12 As shown in Figure F, compared with the NFD control group, HFD feeding increased the total number of ATMs in eWAT and caused a significant M1 shift. Verbascoside intervention reduced the abundance of total ATMs, prevented excessive M1 infiltration, and promoted a restorative trend in the proportion of M2 macrophages. These results indicate that verbascoside has the effect of reshaping the adipose-mediated immune microenvironment and restoring local immune homeostasis.

[0076] Example 3: Verification of the lipid-lowering and anti-inflammatory effects of verbascoside in a 3T3-L1 adipocyte model.

[0077] This embodiment uses a 3T3-L1 adipocyte model to verify in vitro the effects of verbascoside on lipid accumulation and inflammatory response in adipocytes.

[0078] 1. Cell viability assay

[0079] The cytotoxicity of verbascoside on 3T3-L1 preadipocytes was determined using the MTT assay. Results showed that cell viability did not significantly decrease after 24 hours of treatment with verbascoside at concentrations of 0, 25, 50, 75, and 100 μM. (See attached image) Figure 14 As shown in C, this indicates that verbascoside has no significant cytotoxic effect on preadipocytes within the experimental concentration range.

[0080] 2. Effects on lipid accumulation in adipocytes

[0081] After 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes using the MDI cocktail method, they were treated with different concentrations (50, 100 μM) of verbascoside for 72 hours. Oil Red O staining results are attached. Figure 14 As shown in AB, verbascoside reduced lipid droplet aggregation in adipocytes in a dose-dependent manner, and the total lipid content was significantly reduced at a concentration of 100 μM.

[0082] Biochemical assays of intracellular and extracellular lipid metabolites further support this conclusion, as shown in the attached figure. Figure 14 As shown in DH. Compared with the MDI-induced group, treatment with a specific concentration of verbascoside resulted in varying degrees of decrease in TG, NEFA, and glycerol levels in the cell supernatant; simultaneously, intracellular TG and NEFA levels also showed a decreasing trend. Accompanied by reduced lipid droplet accumulation, glucose consumption in differentiated 3T3-L1 adipocytes increased. This phenotype is consistent with the subsequently recorded recovery of insulin signaling and downregulation of SREBP-1c-driven adipogenesis.

[0083] 3. Effects on the secretion of inflammatory factors in adipocytes

[0084] Pathological lipid over-accumulation in hypertrophic adipocytes is often accompanied by amplified inflammatory signals. Detection results are attached. Figure 14 As shown in Figure IJ, the levels of MCP-1 and IL-6 secreted by mature adipocytes differentiated under MDI induction were significantly increased, suggesting that they were in a pro-inflammatory state. Verbascoside treatment could weaken this response, with varying degrees of reduction in the secretion levels of MCP-1 and IL-6 compared to the MDI-induced group.

[0085] Example 4: Validation of the mechanism by which verbascoside regulates metabolic function through the IRS-1 / AKT / SREBP-1c signaling axis.

[0086] This embodiment further explores the molecular mechanism by which verbascoside regulates glucose and lipid metabolism and insulin signaling in 3T3-L1 adipocytes at the protein and gene levels.

[0087] To determine the mechanism by which verbascoside regulates glucose and lipid metabolism during 3T3-L1 differentiation, we analyzed the expression changes of adipogenesis and metabolism-related biomarkers at the mRNA and protein levels. The results showed that, compared with the control group, MDI-induced downregulation of preadipocyte-1 (Pref-1) mRNA levels, while upregulating the mRNA levels of CCAAT / enhancer-binding protein α (C / EBPα), fatty acid synthase (FASN), and acetyl-CoA carboxylase (ACC), as shown in the attached figure. Figure 15 As shown in AD, verbascoside treatment significantly counteracted the aforementioned changes induced by MDI, manifested by inhibiting the expression of FASN and ACC, which is consistent with blocking terminal adipogenesis, inhibiting programmed adipogenesis, and promoting fatty acid oxidation, thereby helping to limit excessive lipid deposition.

[0088] The results of immunoblotting analysis further revealed the regulatory mechanism centered on AMPK, as shown in the appendix. Figure 15As shown in Figure E, MDI induction decreased the ratio of phosphorylated AMPKα to total AMPKα (p-AMPKα / AMPKα), while simultaneously decreasing the protein levels of CPT-1α and protein kinase A (PKA), and increasing the protein levels of adipogenesis regulators SREBP-1c, PPARγ, and C / EBPα. Verbascoside treatment reversed this pattern of changes, characterized by upregulation of the p-AMPKα / AMPKα ratio, CPT-1α, and PKA protein levels, while downregulating the protein levels of SREBP-1c, PPARγ, and C / EBPα. These changes indicate enhanced fatty acid β-oxidation and lipolysis, while inhibiting lipogenesis and TG deposition, consistent with the aforementioned transcriptional data.

[0089] Regarding the insulin signaling pathway, MDI induction reduced the protein levels of IRS-1 and PI3K, as well as the phosphorylation level of AKT (p-AKT / AKT ratio). Verbascoside treatment restored the expression of IRS-1 and PI3K and enhanced the phosphorylation level of AKT, indicating that the function of the IRS-1 / PI3K / AKT insulin signaling pathway was restored.

[0090] Based on the above mechanistic studies, verbascoside promotes fatty acid oxidation by activating the AMPK signaling axis, while inhibiting the expression of key regulatory factors of adipogenesis such as SREBP-1c to reduce lipogenesis and reconstructing the insulin signaling function of IRS-1 / PI3K / AKT, thereby synergistically inhibiting terminal differentiation of adipocytes and alleviating lipid accumulation and insulin resistance during differentiation.

[0091] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of verbascoside in the preparation of drugs for treating obesity and related metabolic disorders.

2. The application of verbascoside according to claim 1 in the preparation of drugs for treating obesity and related metabolic disorders, characterized in that, The obesity-related metabolic disorders include at least one of dyslipidemia, hepatic steatosis, insulin resistance, and impaired glucose tolerance.

3. The application of verbascoside according to claim 2 in the preparation of drugs for treating obesity and related metabolic disorders, characterized in that, The drug inhibits the polarization of macrophages in the white adipose tissue of the epididymis towards the pro-inflammatory M1 type and promotes their polarization towards the anti-inflammatory M2 type.

4. The application of verbascoside according to claim 2 in the preparation of drugs for treating obesity and related metabolic disorders, characterized in that, The drug upregulates the expression of phosphorylated AMP-activated protein kinase α and carnitine palmitoyltransferase-1α, while downregulating the expression of sterol regulatory element-binding protein-1c, fatty acid synthase, and acetyl-CoA carboxylase.

5. The application of verbascoside according to claim 2 in the preparation of drugs for treating obesity and related metabolic disorders, characterized in that, The drug restores insulin signaling by upregulating the expression of insulin receptor substrate-1, phosphatidylinositol 3-kinase, and phosphorylated protein kinase B.

6. The application of verbascoside according to claim 2 in the preparation of drugs for treating obesity and related metabolic disorders, characterized in that, The targets of the verbascoside include tumor necrosis factor, interleukin-6, sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor γ, and insulin receptor.

7. A pharmaceutical composition for treating obesity and related metabolic disorders, characterized in that, It contains a therapeutically effective amount of verbascoside and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition is an oral formulation or an injectable formulation.

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