Use of a fine wind wheel herb extract in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease

Abstract

The application belongs to the technical field of medicine, and particularly relates to application of a fine wind wheel herb extract in preparation of a medicine for preventing or treating non-alcoholic fatty liver disease. The fine wind wheel herb is crushed, alcohol-extracted, and subjected to reduced-pressure recovery to obtain a concentrate, the concentrate is subjected to petroleum ether and ethyl acetate extraction to obtain an ethyl acetate extract, and the ethyl acetate extract is subjected to macroporous resin purification to obtain the fine wind wheel herb extract. The extract significantly inhibits palmitic acid-induced lipid accumulation of HepG2 cells, increases PPARa, CPT1A, and reduces SREBP-1c, FASN, and CD36 protein expression, and inhibits liver cell oxidative stress. The extract can also significantly alleviate lipid accumulation of NAFLD mice induced by a high-fat diet, increase antioxidant capacity, and reduce liver damage. The extract inhibits alpha-glucosidase activity and increases adiponectin receptor 2 expression. It is shown that the fine wind wheel herb extract can effectively reduce blood lipids, reduce liver lipid accumulation, inhibit oxidative stress, has a sugar absorption inhibition and liver cell protection effect, and can be used for alleviating NAFLD progression.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of *Gnaphalium affine* extract in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome caused by various factors without a history of excessive alcohol consumption. The main characteristic of NAFLD is hepatocellular steatosis, often accompanied by obesity and hyperlipidemia. Based on disease progression, it can be classified into simple fatty liver, steatohepatitis, liver fibrosis and cirrhosis, and hepatocellular carcinoma. NAFLD can be caused by a variety of factors and diseases, with the key to its development being an imbalance in lipid metabolism in the liver, including the uptake, oxidation, synthesis, esterification, and triglyceride transport of free fatty acids. Abnormalities in one or more of these processes can induce the accumulation of triglycerides (TG) in the liver, leading to fatty liver. In recent years, the widely accepted pathogenesis of NAFLD is the "multiple-hit hypothesis," which points out that multiple factors (insulin resistance, hormones secreted by adipose tissue, oxidative stress, endoplasmic reticulum stress, nutritional factors, gut microbiota, and genetic and epigenetic factors) induce NAFLD. Oxidative stress remains a major factor in liver damage and disease progression in cases of steatosis, serving as the starting point for both hepatic and extrahepatic injury. The prevalence of NAFLD in the general population is as high as 20-30%, and 2-3% of NAFLD patients will develop non-alcoholic steatohepatitis (NASH). Therefore, the prevention and treatment of NAFLD are of paramount importance.

[0003] Currently, treatment for NAFLD, besides dietary and lifestyle modifications, commonly used medications are mainly divided into three categories: insulin sensitizers (biguanides), lipid-lowering drugs (statins, etc.), and hepatoprotective drugs (antioxidants). Although these drugs have shown efficacy in improving metabolic syndrome in animal studies and clinical practice, they also have numerous side effects, such as myopathy, liver damage, and gastrointestinal reactions. In recent years, Rezdiffra, approved by the FDA, can be used in conjunction with diet and exercise to improve liver fibrosis and NASH symptoms, but its long-term clinical benefits are still under investigation. Traditional Chinese medicine possesses pharmacological characteristics such as multi-component, multi-target, and multi-pathway synergistic effects, low toxicity, and the ability to be used long-term. Therefore, finding traditional Chinese medicines that can prevent and treat NAFLD is crucial.

[0004] Clinopodium gracile (Benth.) Matsum, also known as slender windmill vegetable, is a plant belonging to the genus Clinopodium in the Lamiaceae family. It is distributed throughout areas south of the Yangtze River and is rich in medicinal resources, possessing properties of dispelling wind and clearing heat, promoting blood circulation, detoxifying, and reducing swelling. Medicinal plants in the Clinopodium genus contain various chemical components, including flavonoids and their glycosides, triterpenoid saponins, iridoid glycosides, diterpenoid glycosides, and free triterpenoids, exhibiting hemostatic, antibacterial, anti-inflammatory, endothelial cell protective, immunomodulatory, antioxidant, and antitumor effects. In Fujian, Clinopodium gracile is used in traditional medicine to treat diabetes, improving glucose and lipid metabolism disorders in diabetic patients.

[0005] This invention aims to investigate the ameliorative effect of C. gracile extract (CGE) on NAFLD. Summary of the Invention

[0006] Purpose of the invention: This invention provides a method for preparing CGE and its application in drugs for the prevention and treatment of NAFLD.

[0007] Technical solution:

[0008] This invention provides the application of *Gnaphalium affine* extract in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease.

[0009] The *Gnaphalium affine* extract was prepared according to the following steps:

[0010] The dried whole herb of *Syngonium sibiricum* was pulverized and extracted by reflux with ethanol and its aqueous components, methanol and its aqueous components, or propanol and its aqueous components, or by maceration extraction. The extract was concentrated under reduced pressure, and the concentrated extract was dispersed in water and extracted four times sequentially with petroleum ether and ethyl acetate. The ethyl acetate extract was subjected to column chromatography with macroporous resin. After washing away impurities with water, it was eluted with a gradient of ethanol (range 10-90%). The ethanol eluent (30-70%) was collected, concentrated and dried under reduced pressure to obtain *Syngonium sibiricum* extract CGE.

[0011] The CGE described in this invention can be mixed with conventional excipients and / or excipients to prepare various dosage forms for prevention or treatment. Injections, capsules, tablets, granules, sugar-coated pills, and solutions are all possible drug dosage forms. Capsules, tablets, granules, and sugar-coated pills may contain one or more common excipients—carriers or excipients: such as starch, microcrystalline fibers, etc.; binders: such as carboxymethyl cellulose, polyvinylpyrrolidone, etc.; humectants: such as glycerin; disintegrants: such as calcium carbonate, etc.; adsorbents: such as kaolin, etc.; lubricants: such as talc, etc. Pharmaceutical excipients can be in various forms, such as solids, semi-solids, and liquids, and formulation excipients can be of various types. Solutions may contain common solvents, solubilizers, emulsifiers, preservatives, etc., such as water, ethanol, glycerin, polyethylene glycol, benzyl benzoate, etc. Dosage forms such as capsules, tablets, granules, sugar-coated pills, and solutions can be prepared using known conventional methods. The effective fraction extract of *Gnaphalium affine* can be mixed with one or more excipients to prepare the dosage form.

[0012] The NAFLD cell model described in this invention consists of palmitic acid (PA)-induced HepG2 cells treated with CGE.

[0013] The CGE described in this invention has the effect of reducing lipid accumulation in HepG2 cells.

[0014] The CGE described in this invention has the effect of increasing the expression of PPARα, CPT1A, and AdipoR2 and decreasing the expression of SREBP-1c, FASN, and CD36 proteins.

[0015] The CGE described in this invention can alleviate PA-induced intracellular oxidative stress in HepG2 cells, and has the effects of increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and reducing the content of malondialdehyde (MDA) and reactive oxygen species (ROS).

[0016] The NAFLD model mice described in this invention were induced by a high-fat diet and treated with CGE.

[0017] The CGE described in this invention can improve lipid accumulation and pathological damage in the liver of NAFLD mice.

[0018] The CGE described in this invention can alleviate oxidative stress in the liver of NAFLD mice and has the functions of reducing serum MDA content and increasing SOD and GSH-Px activity.

[0019] The CGE described in this invention has a liver-protective effect and can reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum.

[0020] The CGE active ingredient described in this invention has an affinity for PPARα protein.

[0021] The CGE described in this invention has a significant inhibitory effect on α-glucosidase activity.

[0022] Peroxisome proliferator-activated receptors (PPARs) regulate fatty acid homeostasis in various tissues, including the liver, adipose tissue, muscle, and other organs. PPARα is primarily expressed in the liver, controlling fatty acid synthesis, translocation, and β-oxidation. First, carnitine palmitoyltransferase 1A (CPT1A) is a crucial regulator of fatty acid oxidation (FAO), and its regulation is controlled by PPARα. Mitochondria are the primary organelle for fatty acid oxidation. In the cytoplasm, fatty acids are activated to form acyl-CoA, which is then transported to the mitochondrial matrix via CAT1A-mediated mitochondrial membrane translocation for FAO. Second, activation of PPARα can effectively reduce hepatic fatty acid synthesis and triglyceride (TG) accumulation by decreasing the expression of sterol-regulatory element-binding protein (SREBP-1c). PPARα regulates the expression of fatty acid synthesis genes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase 1 (ACC1), which are closely associated with SREBP-1c. Cluster of differentiation 36 (CD36) is a highly glycosylated single-chain transmembrane protein involved in free fatty acid uptake. PPARα specifically regulates CD36 to maintain intracellular fatty acid homeostasis. Therefore, PPARα is a potential target for the treatment of NAFLD. ROS are mainly produced in mitochondria and control oxidative stress. Lipid metabolism disorders lead to disruption of the mitochondrial electron transport chain, resulting in excessive ROS production. Under normal circumstances, oxygen free radicals generated in the mitochondrial electron transport chain are cleared by GSH-Px and SOD. However, in NAFLD mice, excessive ROS production by hepatocytes exceeds the clearance capacity of antioxidant enzymes, leading to persistent oxidative stress. Excessive ROS production decreases mitochondrial membrane potential, increases mitochondrial membrane permeability, and increases cytochrome C release, leading to oxidative damage to cells. Therefore, reducing ROS production and inhibiting hepatocyte oxidative damage may be an effective means to alleviate NAFLD.

[0023] Adiponectin is an adipokine secreted by adipocytes that is negatively correlated with obesity symptoms. It participates in glucose metabolism, maintains energy balance, and has antioxidant, anti-inflammatory, anti-atherosclerotic, and insulin-antagonistic effects. Increased adiponectin levels in mammals promote fatty acid oxidation. Current research indicates that adiponectin levels are decreased in NAFLD patients. The effects of adiponectin are primarily mediated by the adiponectin receptor (AdipoR). AdipoR1 is mainly expressed in skeletal muscle, while AdipoR2 is mainly expressed in the liver. AdipoR2 can also activate PPARα. It regulates lipid metabolism. By activating the AdipoR2 receptor, it can effectively regulate glucose and lipid metabolism, including promoting glucose uptake and utilization, thereby lowering blood glucose levels.

[0024] Alpha-glucosidase is a glycosidic enzyme found on the brush border of small intestinal mucosal cells. This enzyme hydrolyzes disaccharides, ultimately converting carbohydrates entering the small intestine into monosaccharides for absorption. Inhibiting alpha-glucosidase activity slows the rate of carbohydrate hydrolysis and glucose production, and delays glucose absorption, thus effectively inhibiting the timing and progression of postprandial blood glucose elevation. Used alone or in combination with other hypoglycemic agents, it can lower blood glucose levels in diabetic patients. Type 2 diabetes is a common complication of non-alcoholic fatty liver disease (NAFLD); therefore, inhibiting alpha-glucosidase activity can lower blood glucose levels in NAFLD patients with abnormal glucose metabolism.

[0025] Beneficial effects

[0026] Fenofibrate (FNB) is used to treat hyperlipidemia in adults where diet control therapy is ineffective. Its effect on lowering triglycerides and mixed hyperlipidemia is more significant than its effect on cholesterol. FNB can lower serum cholesterol and triglycerides. The main adverse reactions of fenofibrate include gastrointestinal irritation, such as diarrhea, nausea, and constipation. It may also cause myositis, myopathy, and rhabdomyolysis. Therefore, this invention uses FNB as a positive control drug.

[0027] This invention provides a novel application of CGE in the treatment of NAFLD. Results show that CGE exhibits good effects in reducing lipid accumulation, alleviating oxidative damage, protecting the liver, and inhibiting α-glucosidase activity. In vitro experiments show that CGE significantly reduces the levels of TG and TC in PA-induced HepG2 cells, decreases the Oil Red O staining area, significantly promotes the expression of PPARα, CPT1A, and AdipoR2 proteins, and inhibits the expression of SREBP-1c, FASN, and CD36 proteins; significantly enhances SOD and GSH-Px activities, and inhibits MDA and ROS production. In vivo experiments show that CGE significantly reduces the Oil Red O staining area in the liver of NAFLD mice and alleviates liver pathological damage; significantly reduces serum TG, TC, NEFA, ALT, and AST levels, and reduces liver TG and TC content; significantly increases serum SOD and GSH-Px activities, and reduces MDA production. These findings suggest that CGE has the effects of alleviating steatosis, inhibiting oxidative stress, and protecting the liver, and can be used for the prevention and treatment of NAFLD. Furthermore, CGE's inhibitory effect on lipid accumulation is comparable to that of FNB, and its ability to reduce oxidative damage and protect the liver is superior to that of the positive control drug FNB. FNB also lacks hepatoprotective effects and has significant side effects, such as causing liver dysfunction with long-term use. CGE reduces NAFLD by regulating PPARα, and compared to FNB, the active components of CGE (rutin, rosmarinic acid, apigenin, hesperidin, or lemon balm glycoside) have a stronger affinity for PPARα protein. The IC50 of CGE inhibiting α-glucosidase is 74.1 μg / mL. This indicates that CGE, as an extract of *Gnaphalium affine*, has better therapeutic effects than FNB, with hepatoprotective effects, fewer side effects, and a mechanism of action that inhibits α-glucosidase activity. Attached Figure Description

[0028] Figure 1 The effect of CGE on PA-induced lipid accumulation in HepG2 cells; including A. TG content in HepG2 cells; B. TC content in HepG2 cells; C. Representative images stained with Oil Red O. Results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group.

[0029] Figure 2 The effects of CGE on the expression of PPARα, CPT1A, SREBP-1c, FASN, CD36, and AdipoR2, which are related to glucose and lipid metabolism, induced by PA in HepG2 cells; A. Protein expression and statistical graphs of PPARα, CPT1A, SREBP-1c, FASN, and CD36; B. Protein expression and statistical graphs of AdipoR2. Results are expressed as mean ± SEM.# P < 0.05, ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group.

[0030] Figure 3 The effect of CGE on PA-induced oxidative stress in HepG2 cells; including: A. SOD activity in HepG2 cells; B. GSH-Px activity in HepG2 cells; C. MDA content in HepG2 cells; D. Representative image of intracellular ROS (DCFH-DA staining); E. Relative fluorescence intensity of DCFH-DA staining. Results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group. & P < 0.05 vs. FNB group.

[0031] Figure 4 Effects of CGE on body weight and liver weight in high-fat diet-induced NAFLD mice; where A. mouse body weight; B. mouse liver weight; C. mouse liver weight coefficient; results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group. & P < 0.05 vs. FNB group.

[0032] Figure 5 Effects of CGE on liver histopathology in high-fat diet-induced NAFLD mice; A. Liver appearance; B. Representative images of hematoxylin and eosin (H&E) staining.

[0033] Figure 6 Effects of CGE on lipid accumulation in high-fat diet-induced NAFLD mice; A. Serum TC content in mice; B. Serum TG content in mice; C. Liver TC content in mice; D. Liver TG content in mice; E. Serum NEFA content in mice; F. Representative image of mouse liver stained with Oil Red O. Results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group.

[0034] Figure 7Effects of CGE on serum ALT and AST activities in high-fat diet-induced NAFLD mice; A. Serum ALT activity in mice. B. Serum AST activity in mice. Results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, *P < 0.05, **P < 0.01 vs. model group. & P < 0.05, && P < 0.01 vs. FNB group.

[0035] Figure 8 Effects of CGE on oxidative stress induced by a high-fat diet in NAFLD mice; A. Serum SOD activity; B. Serum GSH-Px activity; C. Serum MDA content. Results are expressed as mean ± SEM. ## P < 0.01 vs. normal group, **P < 0.01 vs. model group, && P < 0.01 vs. FNB group.

[0036] Figure 9 The affinity of CGE's active ingredient for PPARα protein. Detailed Implementation

[0037] The present invention will be described in detail through the following embodiments, but the present invention is not limited thereto. All technologies implemented based on the above content of the present invention are within the scope of the present invention.

[0038] Experimental materials: HepG2 human liver cancer cells were purchased from the Type Culture Collection, USA. PA was purchased from China Pharmaceutical Chemical Reagent Co., Ltd. Fenofibrate was purchased from Solarbio Biotechnology Co., Ltd. PPARα antibody was purchased from Shenyang Wanlei Biotechnology Co., Ltd., China. SREBP-1c antibody was purchased from Santa Cruz Biotechnology Co., Ltd. Anti-GAPDH antibody was purchased from Bioworld Technology Co., Ltd. CPT1A and FASN antibodies were purchased from Wuhan Proteintech Co., Ltd., China. Total cholesterol (TC) and TG biochemical indicators were purchased from Zhejiang Dongou Diagnostic Products Co., Ltd. Non-esterified fatty acids (NEFA), SOD, GSH-Px, MDA, ALT, and AST detection kits were purchased from Nanjing Jiancheng Bioengineering Institute. Reactive oxygen species detection kits were purchased from Beyotime Biotechnology Co., Ltd.

[0039] Laboratory animals: SPF-grade male C57BL / 6 mice (6-8 weeks old, weighing 18-22 g) were provided by the Animal Medical Center of Yangzhou University. High-fat diet was purchased from Nantong Trofi Feed Technology Co., Ltd.

[0040] Example 1: Preparation of extracts from effective parts of *Gnaphalium affine*

[0041] The extraction steps are as follows: The dried whole herb of *Gnaphalium affine* (1.5 kg) was pulverized and extracted by reflux with 80% ethanol. The concentrated extract was then extracted four times with petroleum ether and ethyl acetate, respectively. The ethyl acetate fraction was separated using macroporous resin D101, eluted sequentially with 10%, 30%, 50%, 70%, and 90% ethanol solutions. Finally, the 30%–70% ethanol eluate was collected, concentrated under reduced pressure, and dried to obtain CGE (14.1 g). Six components were identified in the *Gnaphalium affine* extract: rutin, hesperidin, rosmarinic acid, lemon balm glycoside, apigenin, and isochorin. The contents of rutin, hesperidin, lemon balm glycoside, and isochorin in CGE were 48.35, 80.36, 115.67, and 11.58 mg / g, respectively.

[0042] Example 2: The effect of CGE on improving PA-induced lipid accumulation in hepatocytes

[0043] 1. Detect the levels of TG and TC in HepG2 cells.

[0044] Selected HepG2 cells in good logarithmic growth phase were digested and the HepG2 cell suspension was seeded into 6-well cell culture plates (1×10⁻⁶ cells / well). 5 Cells were cultured in wells (number per well). When cells reached 80% confluence, the experimental groups were as follows: the normal group consisted of naturally cultured HepG2 cells; the model group consisted of HepG2 cells co-cultured with 250 μM PA; and the remaining groups consisted of HepG2 cells co-cultured with CGE (7.5, 15, and 30 μg / mL) and FNB (100 μM) and PA. After 24 h of culture, the culture medium was discarded, the cells were washed three times with PBS, and 100 μL of lysis buffer was added to each well. Cells were lysed on ice for 15 min, and scraped off. The cells were centrifuged at 12000 g / min for 10 min at 4 ℃, and the supernatant was collected. The cellular protein content was detected according to the bovine serum albumin (BCA) kit, and the intracellular TG and TC content was detected according to the TG and TC kit instructions. The experiment was performed in triplicate.

[0045] The results are as follows Figure 1As shown in Figure A, compared with the normal group, PA significantly increased the intracellular TG content in HepG2 cells. Compared with the model group, both the CGE (15, 30 μg / mL) group and the FNB (100 μM) (i.e., FNB 36 μg / mL) group significantly reduced the intracellular TG content in HepG2 cells, with inhibition rates of 73.6%, 76.6%, and 65.8%, respectively. Figure 1 As shown in Figure B, compared with the normal group, PA significantly increased the intracellular TC content in HepG2 cells. Compared with the model group, both the CGE (15, 30 μg / mL) group and the FNB (100 μM) group significantly reduced the intracellular TC content in HepG2 cells.

[0046] 2. Oil Red O staining to observe lipid droplets in HepG2 cells

[0047] Cell grouping and culture were performed according to the steps in Example 2.1. Cells were removed from the incubator, washed three times with phosphate buffer solution (PBS), fixed with 4% paraformaldehyde for 15 min, stained with Oil Red O for 1 h, and counterstained with hematoxylin for 15 s. Finally, the Oil Red O-stained cells were observed using an Olympus microscope.

[0048] The results are as follows Figure 1 As shown in Figure C, PA significantly increased the number of lipid droplets in HepG2 cells; compared with the model group, the CGE (15, 30 μg / mL) groups significantly reduced the Oil Red O staining area of ​​HepG2 cells. This indicates that CGE can effectively inhibit lipid accumulation in the NAFLD cell model.

[0049] Example 3: Regulation of PPARα, CPT1A, SREBP-1c, FASN, CD36, and AdipoR2 proteins in PA-induced hepatocytes by CGE

[0050] Cell grouping and culture were performed according to step 1 of the example. After cell processing, proteins were extracted, quantified using the BCA method, and the expression of PPARα, CPT1A, SREBP-1c, FASN, CD36, and AdipoR2 was detected by Western blot. The steps were as follows: HepG2 cell samples were lysed with lysis buffer, and protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. Then, the membrane was blocked with TBST buffer containing 6% skim milk or 5% BSA at room temperature for 2 h. Primary antibody (1:1000 dilution) was added, and the membrane was incubated at 4 °C for 12 h. Then, the membrane was washed three times with TBST, and secondary antibody (1:5000 dilution) was added, and the membrane was incubated at room temperature for 2 h. Finally, the membrane was fixed and developed using an electrochemiluminescence staining system, and then photographed using a gel imaging system. Relative quantitative analysis was performed using ImageJ software.

[0051] PPARα plays a crucial role in maintaining lipid homeostasis, participating in multiple processes including fatty acid synthesis, uptake, and β-oxidation. PPARα can inhibit CD36 expression to reduce intracellular fatty acid uptake. It can also increase CPT1A gene expression, thereby promoting fatty acid oxidation. Furthermore, FASN and ACC1 are downstream target genes of SREBP-1c; activating PPARα can reduce SREBP-1c expression and inhibit the transcription of FASN and ACC1 target genes, thus weakening hepatic fatty acid synthesis. Results are as follows... Figure 2 As shown in Figures AB, compared with the normal group, PA-induced PPARα and CPT1A protein expression levels in HepG2 cells were significantly decreased, while SREBP-1c, FASN, and CD36 protein expression levels were significantly increased. Compared with the model group, CGE (15, 30 μg / mL) groups significantly upregulated PPARα and CPT1A protein expression levels and significantly downregulated SREBP-1c, FASN, and CD36 expression levels. Figure 2 As shown in CD, compared with the normal group, the protein expression level of AdipoR2 in HepG2 cells induced by PA was significantly reduced. Compared with the model group, the protein expression level of AdipoR2 in the CGE (30 μg / mL) group was significantly upregulated. This indicates that CGE can improve glucose and lipid metabolism disorders in the NAFLD cell model by inhibiting fatty acid synthesis and uptake and promoting fatty acid β-oxidation, and its mechanism of action is related to the upregulation of PPARα protein expression.

[0052] Example 4: CGE inhibits PA-induced oxidative stress in hepatocytes

[0053] 1. Effects of CGE on PA-induced SOD, GSH-Px, and MDA levels in hepatocytes

[0054] Cells were grouped and cultured according to the steps in Example 2.1, and then lysed. Cellular protein levels were detected using a BCA kit, and intracellular SOD, GSH-Px activities, and MDA levels were detected according to the kit instructions.

[0055] The results are as follows Figure 3 As shown in Figures AB, compared with the normal group, PA-induced SOD and GSH-Px activities in HepG2 cells were significantly reduced. Compared with the model group, SOD activity in hepatocytes in the CGE (15, 30 μg / mL) and FNB (100 μM) groups was significantly increased, with promotion rates of 87.8%, 152.7%, and 93.9%, respectively. GSH-Px activity in hepatocytes in the CGE (30 μg / mL) and FNB (100 μM) groups was significantly upregulated, with promotion rates of 112.4% and 49.5%, respectively, and the upregulation of PA-induced GSH-Px activity in hepatocytes in the CGE (30 μg / mL) group was significantly stronger than that in the FNB (100 μM) group. Figure 3 As shown in Figure C, compared with the normal group, the MDA content in HepG2 cells induced by PA was significantly increased. Compared with the model group, the MDA content in hepatocytes in both the CGE (15, 30 μg / mL) group and the FNB (100 μM) group was significantly decreased, and the effect of CGE (15, 30 μg / mL) group in reducing PA-induced MDA content in hepatocytes was significantly stronger than that in the FNB (100 μM) group. This indicates that the antioxidant effect of CGE is superior to that of FNB.

[0056] 2. Effects of CGE on PA-induced ROS production in hepatocytes

[0057] Cell grouping was performed according to the steps in Example 2.1, with HepG2 cells (1×10⁻⁶) grouped together. 5 Cells were seeded per well in 6-well plates and incubated at 37°C in a 5% CO2 incubator. Following the kit instructions, 1 mL of serum-free medium containing the DCFH-DA probe (2 μM) was added to each well of cells, and the plates were incubated at 37°C for 30 min. After washing three times with serum-free medium to remove excess probe, the cells were observed using an inverted fluorescence microscope.

[0058] The results are as follows Figure 3 As shown in the DE, compared with the normal group, PA significantly increased intracellular ROS production in HepG2 cells. Compared with the model group, CGE (15, 30 μg / mL) groups significantly reduced ROS content. This indicates that CGE can effectively inhibit oxidative stress in the NAFLD cell model.

[0059] Example 5: The preventive and therapeutic effects of CGE on high-fat diet-induced NAFLD in mice.

[0060] Establishment and administration of the NAFLD animal model: C57BL / 6 mice were acclimatized for 3 days in an environment (23±2 ℃, 12 h / 12 ​​h light-dark cycle). Fifty mice were divided into five groups: normal diet (ND), high-fat diet (HFD), HFD + CGE 40 mg / kg, HFD + CGE 80 mg / kg, and HFD + FNB 80 mg / kg, with 10 mice in each group. The normal control group was fed a standard diet, while the HFD groups were fed a diet consisting of 60% high-fat (60% fat, 19.4% protein, 20.6% carbohydrates) + 1% cholesterol + 0.25% bile salts (TP23300-X, purchased from Nantong Trofi Biotechnology Co., Ltd.), with free access to water. Mice were fed a high-fat diet for 10 weeks, with CGE and FNB administered concurrently from weeks 3 to 10. Animals were treated at the end of the 10th week. They were fasted for 12 hours before treatment, but water was allowed. Blood was collected from the abdominal aorta and centrifuged at 12,000 g / min for 10 minutes at 4 ℃ to collect serum.

[0061] 1. Effects of CGE on body weight and liver weight in high-fat diet-induced NAFLD mice

[0062] The livers of mice were harvested and weighed. Liver weight coefficient = liver (g) / body weight (100 g)

[0063] NAFLD is often accompanied by obesity and increased liver weight. Results are as follows: Figure 4 As shown, compared with the normal group, the body weight, liver weight, and liver index of NAFLD mice induced by a high-fat diet were significantly increased. Compared with the model group, the body weight of mice in the CGE (40, 80 mg / kg) and FNB (80 mg / kg) groups was significantly reduced, with inhibition rates of 63.2%, 75.4%, and 108.8%, respectively; liver weight was significantly reduced, with inhibition rates of 71.4%, 85.7%, and 57.1%, respectively. Compared with the model group, the liver index of the CGE (40, 80 mg / kg) groups was significantly reduced, with inhibition rates of 100% and 128.57%, respectively. Although the body weight and liver weight of the FNB (80 mg / kg) group decreased, the liver index increased to some extent. Compared with the positive control FNB group, the liver index of the CGE 80 mg / kg group was significantly reduced. The above results indicate that CGE can effectively inhibit the increase in body weight and liver weight in HFD-induced NAFLD mice and has a hepatoprotective effect, while FNB has no hepatoprotective effect.

[0064] 2. Effects of CGE on liver histopathology in high-fat diet-induced NAFLD mice

[0065] A portion of the liver from the experimental mice was fixed in 10% formalin solution, embedded in OCT, and paraffin sections were prepared. Hematoxylin and eosin (H&E) staining was then performed, and the staining of the liver tissue was observed under a microscope.

[0066] The results are as follows Figure 5 As shown, compared with the ND group, the livers of NAFLD mice induced by a high-fat diet were significantly enlarged and yellowish in color. Compared with the model group, CGE at 40 and 80 mg / kg alleviated this phenomenon to varying degrees. H&E staining of liver tissue showed that hepatocytes in the normal group were arranged in a cord-like structure with normal structure. The model group showed diffuse lipid vacuoles with fusion, and some inflammatory infiltration around the portal areas. Treatment with CGE at 40 and 80 mg / kg significantly improved the degree of hepatic steatosis and inflammatory response.

[0067] 3. Effects of CGE on lipid accumulation in high-fat diet-induced NAFLD mice

[0068] The levels of total cholesterol (TC), triglycerides (TG), and NEFA in mouse serum were detected using a kit. 10 mg of liver tissue from each mouse was rinsed twice in physiological saline, then homogenized with 10% isopropanol. After homogenization, the liver tissue was centrifuged at 12000 g / min for 10 min at 4 °C. The supernatant was collected, and the TG and TC levels in the liver tissue were detected according to the kit instructions. Frozen sections of liver tissue from the experimental mice were stained with Oil Red O, and the staining was observed under a microscope.

[0069] Serum lipid and liver lipid levels can reflect the degree of lipid accumulation in mice. Oil Red O staining of liver tissue can further observe the number, size, and distribution of lipid droplets in the mouse liver. Results are as follows... Figure 6 As shown in the AE, compared with the normal group, the serum and liver tissue contents of NAFLD mice induced by a high-fat diet were significantly increased, and the serum NEFA content was significantly increased. Compared with the HFD group, the serum TC and TG contents in the CGE (40, 80 mg / kg) and FNB (80 mg / kg) groups were significantly decreased (TC inhibition rates were 35.0%, 91.5%, and 80.5%, respectively; TG inhibition rates were 68.0%, 84.0%, and 96.0%, respectively), and the liver tissue contents of TC and TG were significantly reduced (TC inhibition rates were 40.6%, 77.5%, and 94.8%, respectively; TG inhibition rates were 59.4%, 88.9%, and 104.0%, respectively). The serum NEFA content was also significantly reduced (inhibition rates were 40.1%, 90.8%, and 101.3%, respectively). Figure 6As shown in Figure F, a large number of lipid droplets accumulated in the liver tissue of the model group mice. After CGE treatment, the number of lipid droplets in the mouse liver was significantly reduced. These results indicate that CGE can significantly alleviate lipid accumulation in NAFLD mice induced by HFD.

[0070] 4. Effects of CGE on serum ALT and AST activities in high-fat diet-induced NAFLD mice

[0071] Abnormally elevated ALT and AST enzyme activities are important markers of liver injury. Results as follows: Figure 7 As shown in Figures AB, a high-fat diet significantly increased serum ALT and AST activities in mice. Compared with the model group, the CGE (80 mg / kg) group showed a significant decrease in serum ALT activity (inhibition rate of 101.8%), and the CGE (40, 80 mg / kg) groups showed a significant decrease in serum AST activity (inhibition rates of 92.4% and 100.0%, respectively). The FNB (80 mg / kg) group showed a trend of increasing serum ALT, while AST activity showed no significant difference compared to the model group. Compared with the positive control FNB group, the CGE 80 mg / kg group showed a significant decrease in serum ALT and AST enzyme activities. This indicates that CGE alleviated high-fat diet-induced liver damage in NAFLD mice and had a hepatoprotective effect, while FNB had no hepatoprotective effect.

[0072] 5. Effects of CGE on oxidative stress in high-fat diet-induced NAFLD mice

[0073] Oxidative stress damage, a key factor in the progression of NAFLD to NASH, forms a "parallel attack" with lipid accumulation, accelerating NAFLD development. The activity of antioxidant enzymes and lipid peroxidation products in mice reflect their oxidative stress status. Results are as follows... Figure 8 As shown in AC, compared with the normal group, the serum SOD and GSH-Px activities of NAFLD mice induced by a high-fat diet were significantly reduced, while the MDA content was significantly increased. Compared with the model group, CGE (40, 80 mg / kg) significantly increased the serum SOD and GSH-Px activities of mice (SOD promotion rates were 80.3% and 91.6%, respectively; GSH-Px promotion rates were 62.7% and 70.4%, respectively). Meanwhile, compared with the model group, the CGE (40, 80 mg / kg) group and the FNB (80 mg / kg) group significantly reduced the MDA content, with inhibition rates of 20.7%, 79.0%, and 70.7%, respectively. Compared with the FNB (80 mg / kg) group, the serum GSH-Px activity of mice in the CGE (40, 80 mg / kg) group was significantly upregulated. These results indicate that CGE can effectively improve the oxidative stress state of NAFLD mice, and its antioxidant effect in vivo is superior to that of FNB.

[0074] Example 6: The active ingredient CGE has an affinity for PPARα protein.

[0075] The binding energies and interaction modes between CGE active compounds and the PPARα target were analyzed using the protein-ligand docking software AutoDockTools-1.5.7. The molecular structures of naringin, hesperidin, rosmarinic acid, lemon balm glycoside, apigenin, isochorin, and FNB were retrieved from the PubChem website (https: / / pubchem.ncbi.nlm.nih.gov / ). The PPARα protein structure (PDB ID, 1I7G; resolution, 2.20 Å) was downloaded from the PDB website (http: / / www.rcsb.org / pdb / home / home.do). Molecular docking was performed using AutoDockTools-1.5.7.

[0076] The affinity of CGE's active compounds for the PPARα target was assessed using molecular docking analysis. For example... Figure 9 As shown in Table 1, each active compound binds to the PPARα protein via visible hydrogen bonds. The binding energies of PPARα to naringin, rosmarinic acid, apigenin, hesperidin, or lemon balm glycoside are all lower than those of FNB (−7.6 kcal / mol). This indicates that the active components of CGE (naringin, rosmarinic acid, apigenin, hesperidin, or lemon balm glycoside) have a stronger affinity for the PPARα protein. This further supports the view that CGE alleviates NAFLD by modulating PPARα.

[0077]

[0078] Example 7: Inhibitory effect of CGE on α-glucosidase

[0079] The activity of α-glucosidase was determined using 4-nitrophenol-α-D-glucanopyranoside (PNP-G) as a substrate. 40 μl of CGE (final concentrations of 7.5, 15, 30, 60, and 120 μg / mL) and 30 μL of α-glucosidase (0.35 mg / mL) were added to 500 μL of KH₂PO₄ buffer (67 mM, pH=6.8). After incubation at 37 °C for 10 min, 30 μL of PNP-G (0.01 M) was added to initiate the reaction. After reacting at 37 °C for 10 min, 1 mL of 0.1 M Na₂CO₃ was added to terminate the reaction. The OD value of p-nitrobenzene (PNP) released from PNP-G under enzymatic action was measured at 405 nm. Enzyme activity units are defined as the amount of enzyme required to release 1 μmol of PNP from PNP-G within 1 min at 37℃ and pH=6.8. The inhibition rate is calculated. The enzyme inhibition assay is repeated three times, with three replicates each time. The inhibition rate against α-glucosidase is calculated using the following formula: Inhibition rate = (OD value of control group - OD value of drug group) / OD value of control group × 100%.

[0080] The results are shown in Table 2: Compared with the control group, CGE (7.5-120 μg / mL) showed a highly significant inhibitory effect on α-glucosidase in a dose-dependent manner. The IC50 of CGE inhibiting α-glucosidase was... 50 It was 74.1 μg / mL.

[0081] Table 2. Inhibitory effect of CGE on α-glucosidase ( ±s, n=9)

[0082]

[0083] Note: ** P<0.01 vs control group.

Claims

1. The application of *Gnaphalium affine* extract in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that... The extract of *Symplocos buergeriana* was prepared by the following steps: The dried whole herb of *Symplocos buergeriana* was pulverized and extracted by reflux with an ethanol-water solution or by maceration. The extract was concentrated under reduced pressure. The concentrated extract was dispersed in water and extracted sequentially with petroleum ether and ethyl acetate. The ethyl acetate fraction was subjected to column chromatography with macroporous resin. After washing away impurities with water, the extract was eluted with an ethanol gradient. The ethanol eluent was collected, concentrated under reduced pressure, and dried to obtain the *Symplocos buergeriana* extract. The ethanol gradient elution conditions were elution with 10-90% ethanol. The ethanol eluent was collected as 30-70% ethanol.

2. The application according to claim 1, characterized in that, The main components of the *Gnaphalium affine* extract are rutin, hesperidin, rosmarinic acid, lemon balm glycoside, apigenin, and isochorin.

3. The application according to claim 1 or 2, characterized in that, The drug is a pharmaceutical composition formed from *Gnaphalium affine* extract and pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The pharmaceutical composition is prepared as a solution, capsule, tablet, granule or sugar-coated pill.

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