Application of Lonicera Confusa in Nonalcoholic Fatty Liver Disease (NAFLD) and Obesity

Abstract

The application discloses application of lonicera cornuta in non-alcoholic fatty liver disease (NAFLD) and obesity, and use of the lonicera cornuta provided by the application can effectively reduce the increase of body weight and body fat rate of a model mouse induced by a high-fat diet, can effectively relieve and / or treat obesity; in addition, the lonicera cornuta provided by the application can also significantly improve biochemical indexes TC, TG, LDL-C and HDL-C, ALT and AST of the model mouse, play a role in liver protection, can not only improve hepatocyte damage by improving liver antioxidant stress capacity and inhibiting expression of inflammatory factors, but also improve liver steatosis by reducing expression of lipogenic genes, activate AMPK alpha, inhibit activity of an important regulator SREBP-1c of lipid synthesis, thereby reduce expression of lipid synthesis genes FASN, SCD1 and protein ACC, improve hepatocyte antioxidant stress capacity and improve liver steatosis, and can effectively relieve and / or treat NAFLD.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of Gynostemma pentaphyllum in non-alcoholic fatty liver disease and obesity, and more specifically to the application of Gynostemma pentaphyllum as the main component in the preparation of products for treating non-alcoholic fatty liver disease (NAFLD) and obesity. Background Technology

[0002] Non-alcoholic fatty liver disease (NAFLD) encompasses a series of disease processes. NAFLD can progress to non-alcoholic steatohepatitis, gradually leading to liver fibrosis, and may eventually develop into cirrhosis, liver failure, and even liver cancer. Currently, clinical management of NAFLD is limited to lifestyle interventions (including low-calorie diets, exercise, and weight loss). There are no approved drug therapies specifically for NAFLD.

[0003] Currently, there are three main categories of drugs used for nonalcoholic fatty liver disease (NAFLD) / nonalcoholic steatohepatitis (NASH). The first category consists of drugs targeting oxidative stress, inflammation, and fibrosis, including antioxidants (vitamin E), tumor necrosis factor α pathway modulators (enlicasin, pentoxifylline, ZSP1601), and immunomodulators (cenicriviroc, belapectin). The second category comprises antidiabetic drugs, including incretins, thiazolidinediones (TZDs), and sodium-glucose cotransporter 2 (CGG) inhibitors. The third category targets hepatic fat accumulation and the resulting metabolic stress, including peroxisome proliferator-activated receptor agonists (e.g., pioglitazone, erafibrano, saroglieza), bile acid-farnesol X receptor axis modulators (obeticholic acid), lipogenesis inhibitors (aramchol, NDI-010976), and fibroblast growth factor 21 / 19 analogs. However, most of these drugs are still in phase 2 and 3 clinical trials. While antidiabetic drugs help improve some NAFLD / NASH disease parameters, their application in NAFLD / NASH treatment still needs further validation through phase 3 clinical trials. The biggest challenge in drug treatment targeting metabolic disorders is overcoming the high proportion of adverse reactions. In the clinical management of NAFLD, Western medicine lacks drugs specifically for treating fatty liver, limiting interventions to lifestyle changes (including low-calorie diets, exercise, and weight loss), even including natural vitamin E supplementation, or administering hepatoprotective and enzyme-lowering drugs when transaminase levels are elevated; even if transaminase levels return to normal in the short term after medication, they may rebound quickly upon discontinuation, failing to address the root cause. However, global liver cancer incidence statistics show that the incidence of liver cancer caused by NAFLD is increasing the fastest. Therefore, effective NAFLD drugs play a crucial role in reducing the incidence of liver cancer.

[0004] Obesity is a chronic metabolic disease characterized by excessive accumulation of body fat and excessive weight. It often induces and increases the risk of many chronic diseases, such as hypertension, type II diabetes, and cardiovascular and cerebrovascular diseases, severely reducing life expectancy and quality of life. Current treatments for obesity include lifestyle interventions, surgical treatment, and drug therapy. However, the first two methods have poor adherence and high risks; therefore, the development of anti-obesity drugs shows great promise.

[0005] Currently, the main drugs approved in China for the treatment of obesity are orlistat, liraglutide, benaglutide, smegglutide, and telpoglutide. These drugs are chemical drugs and biological drugs, respectively. Compared with chemical drugs and biological drugs, traditional Chinese medicine has unique advantages in the treatment of metabolic diseases such as obesity, such as fewer toxic side effects and less likelihood of drug resistance. In recent years, it has been favored by researchers of anti-obesity drugs.

[0006] Clematis filamentosa Dunn, a plant belonging to the genus Clematis in the Ranunculaceae family, has medicinal uses for its stems and leaves. It is particularly effective in treating angina pectoris and hypertension. Currently, there are Guanxinkang tablets and capsules on the market with Clematis filamentosa as the main active ingredient, used to treat hypertension and coronary heart disease. However, to date, there are no studies or reports on the use of Clematis filamentosa for treating NAFLD and obesity. Summary of the Invention

[0007] The purpose of this invention is to provide an application of Aristolochia debilis in NAFLD.

[0008] Another object of the present invention is to provide an application of Aristolochia debilis in obesity.

[0009] To address the aforementioned problems, this invention provides an application of Aristolochia debilis in NAFLD and obesity.

[0010] Furthermore, this invention provides an application of Aristolochia debilis as the main component in the preparation of a treatment for NAFLD and obesity.

[0011] Furthermore, the products mentioned include pharmaceuticals or health supplements.

[0012] Furthermore, the aforementioned medicine or health product is a preparation made with Aristolochia debilis as the main active ingredient, plus commonly used pharmaceutical excipients or auxiliary ingredients.

[0013] Furthermore, the formulation includes liquid formulations, solid formulations, or semi-solid formulations.

[0014] The beneficial effects of the application of Gynostemma pentaphyllum in NAFLD and obesity provided by this invention are as follows:

[0015] 1. After intervention with Gynostemma pentaphyllum, the body weight, body fat percentage and liver weight of mice were significantly reduced, the regularity of hepatocyte arrangement was effectively improved, and the fat accumulation in liver tissue was significantly reduced.

[0016] 2. The biochemical indicators of TC, TG, LDL-C and HDL-C, ALT and AST in mice were effectively improved, proving that Gynostemma pentaphyllum has a protective effect on the liver.

[0017] 3. Ganmutong can not only improve hepatocyte damage by enhancing the liver's antioxidant stress capacity and inhibiting the expression of inflammatory factors, but also improve hepatic steatosis by reducing the expression of adipogenic genes.

[0018] 4. Ganmutong can activate AMPKα and inhibit the activity of SREBP-1c, an important regulator of lipid synthesis, thereby reducing the expression of lipid synthesis genes FASN and SCD1 and protein ACC, improving the antioxidant stress capacity of hepatocytes, and improving hepatic steatosis. Attached Figure Description

[0019] Figure 1 Results of the analysis of Aristolochia debilis; A: Photograph of Aristolochia debilis; B: Mass spectrometry analysis results of chemical components of Aristolochia debilis;

[0020] Figure 2 Effects of Aristolochia debilis on the morphology and organs of mice in the treatment of non-alcoholic fatty liver disease and obesity; A: Oil Red staining (magnification: 100X); B: Body weight, liver mass, and liver index; C: H&E staining (magnification: 100X); D: Subcutaneous white adipose tissue (sWAT) mass and subcutaneous white adipose tissue index; E: Mouse morphology photographs; F: Body fat percentage; G: Mouse liver photographs; n = 10 / group; ## P<0.01, ### P<0.001 vs. control group C; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group M;

[0021] Figure 3 The effects of Gynostemma pentaphyllum on biochemical indicators in mice with non-alcoholic fatty liver disease; A: total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL-C) and high-density lipoprotein (HDL-C) in plasma; B: alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in plasma and liver; n = 10 / group; # P<0.05, ## P<0.01, ### P<0.001 vs. control group C; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group M;

[0022] Figure 4The effect of Gynostemma pentaphyllum on liver function in mice with non-alcoholic fatty liver disease; A: the ratio of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione S-transferase (GST), and reduced glutathione (GSH) to oxidized glutathione (GSSG) in plasma; B: results of inflammatory factor gene expression detected by real-time PCR; C: results of adipogenic gene expression detected by real-time PCR; n = 10 / group; # P<0.05, ## P<0.01, ### P<0.001 vs. control group C; * P<0.05, ** P<0.01, *** P < 0.001 vs. model group M;

[0023] Figure 5 The effect of Gynostemma pentaphyllum on the AMPKα / SREBP-1c signaling pathway related to hepatic lipid synthesis in the treatment of non-alcoholic fatty liver disease; A: Western blotting results; B: pAMPKα / AMPKα expression ratio; C: ImageJ quantitative analysis of the relative expression levels of SREBP-1c, ACC protein, and the reference internal reference protein β-actin. ### P<0.001 vs. control group C;

[0024] *** P<0.001 vs model group M. Detailed Implementation

[0025] To make the purpose, technical solution and effects of this application clearer and more explicit, the following describes this application in further detail with reference to the accompanying drawings and embodiments.

[0026] Materials Methods

[0027] 1.1 Material Preparation

[0028] The *Aristolochia debilis* was collected from Shixing County, Shaoguan City, Guangdong Province. Phytomorphological identification confirmed that it has opposite, trifoliate, thick, leathery, broadly ovate leaves with a blunt, pointed apex, glabrous on both sides, with five basal veins, slightly raised above and prominently raised below, and inconspicuous lateral veins. It also has large flowers, 3 cm in diameter, with four sepals, glabrous inside and densely pubescent outside; stamens in the outer whorl nearly equal in length to the sepals, the inner whorl shorter, with linear filaments and a prominent connective; and narrowly ovate achenes, about 6 mm long, with short, soft hairs, and a persistent style 5–8 cm long, filiform, with spreading, long, soft hairs. Figure 1A). Chemical composition analysis of the collected wild *Aristolochia debilis* was performed using ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The analysis revealed monoterpenes, sesquiterpenes, aldehydes, ketones, alkenes, phenols, and alcohols, with a rich content of flavonoids including noriheptacortin, rutin, baicalin, euryptoside, quercetin, and morin D. Figure 1 B). After collecting the leaves, air-dry them, then grind them in a high-speed pulverizer at 35,000 revolutions per minute for 5 minutes. Pass the powder through a 40-mesh sieve and collect it for later use.

[0029] High-fat feed (HFD, product number HF60, 60kcal% fat high-fat rat food) and growth feed (product number: LF10C, 10kcal% fat low-fat rat food) were purchased from Detz Biotechnology (Wuxi) Co., Ltd.

[0030] Fatty acid-free bovine serum albumin (BSA, batch number ST023) was purchased from Beyotime.

[0031] Sodium oleate (OA, lot number: O1383) and sodium palmitate (PA, catalog number: P5585) were purchased from Sigma.

[0032] Hematoxylin (batch number: G1080), eosin (batch number: G1100), fuchsin (batch number: G1165) and aniline blue (batch number: G1350) staining agents were purchased from Solarbio.

[0033] Saturated Oil Red O staining solution (batch number: G1260) was purchased from Servicebio.

[0034] Alanine aminotransferase (ALT, batch number: C009-2-1), aspartate aminotransferase (AST, batch number: C010-2-1), high-density lipoprotein cholesterol (HDL-C, batch number: A112-1-1), low-density lipoprotein cholesterol (LDL-C, batch number: A113-1-1), triglycerides (TG, batch number: A110-1-1), total cholesterol (TC, batch number: A111-1-1), malondialdehyde (MDA, batch number: A003-1), and superoxide dismutase (SOD, product number: A001-3) were purchased from Nanjing Jiancheng Biotechnology Institute.

[0035] The inventors designed primers for TNFα, MCP1, IL6, IL10, SCD1, FASN, SREBP1, and PPARγ using NCBI and Primer Blast, respectively. The primer sequences are shown in Table 1 below. The primers in Table 1 were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0036] Table 1 Primer sequences of the target gene

[0037]

[0038] Antibodies for phosphorylated adenosine monophosphate activated protein kinase α (pAMPKα, batch number: 2535T) and adenosine monophosphate activated protein kinase α (AMPKα, batch number: 2532S) were ordered from Cell Signaling Technology; sterol regulatory element binding protein 1c (SREBP-1c, batch number: ab28481) was ordered from Abcam; antibodies for acetyl-CoA carboxylase (ACC, batch number: A19627) and β-actin (β-actin, batch number: AC026) were ordered from ABclonal Company in Wuhan, China; and goat anti-rabbit IgG (H+L)-HRP conjugate (batch number: abs20040ss) was ordered from Absin Biotech (Shanghai) Co., Ltd.

[0039] 1.2 Animal Research

[0040] Forty male C57BL / 6 mice (18±2g) aged 6–7 weeks were purchased from Zhuhai Beston Biotechnology Co., Ltd. and housed in an SPF-grade animal experimental center under the following conditions: room temperature 22–25℃, 12h day-night cycle, 55% relative humidity, and free access to water and food.

[0041] After one week of acclimatization, purchased mice were randomly divided into four groups (n=10 per group): control group (C, growth diet); model group (M, high-fat diet); high-fat diet + low-dose Aristolochia debilis group (L, high-fat diet + 1% Aristolochia debilis powder); and high-fat diet + high-dose Aristolochia debilis group (H, high-fat diet + 5% Aristolochia debilis powder). Treatment continued for 16 weeks, with mouse weight recorded weekly. After treatment, mice were fasted overnight and weighed. Blood, liver, kidney, spleen, and subcutaneous white adipose tissue (sWAT) samples were collected and weighed, and stored at -80°C. Organ index = organ weight / body weight × 100%.

[0042] 1.3 Biochemical Parameter Detection Methods

[0043] Plasma or liver tissue biochemical parameters, including ALT, AST, HDL-C, LDL-C, TG, TC, MDA, SOD, GST, GSH, and GSSG, should be tested using commercially available kits according to the manufacturer's instructions.

[0044] 1.4 Histological analysis and Oil Red O staining detection method

[0045] Mouse liver tissue (8 μm thick) was fixed in 4% neutral paraformaldehyde solution and stained with hematoxylin and eosin (H&E) staining solution. Histopathological scores were calculated based on NAFLD activity scores. Frozen sections of mouse liver tissue (8 μm thick) were fixed in 4% neutral paraformaldehyde solution, stained with Oil Red O, and mounted with glycerol to obtain information on lipid droplet accumulation in liver tissue sections and cells. Results were identified using ImageJ. The tissue was observed and photographed under an optical microscope.

[0046] 1.5 Immunoblotting

[0047] Total protein was extracted from cells and liver tissue using a lysis buffer containing RIPA, protease inhibitors, and phosphatase inhibitors. Protein concentrations in liver tissue and cells were determined using the BCA Protein Assay Kit (A55865, Thermo Scientific).

[0048] Equal volumes of protein sample were mixed with RIPA solution and loading dye in a specific ratio and loaded onto a 10% SDS-PAGE gel to separate the target. The sample was then transferred to a polyvinylidene fluoride (PVDF) membrane according to standard procedures. After blocking with 5% skim milk for 1 hour, the target protein was incubated overnight at 4°C with primary antibodies (SREBP-1c (1:2000), ACC (1:2000), AMPKα (1:5000), p-AMPKα (1:5000), and β-actin (1:10000) were used as internal control proteins). Additionally, HRP-labeled goat anti-rabbit IgG (1:10000) was used for further incubation at 37°C for 1 hour. Protein bands were then captured using an ECL Advanced kit (GE Biosciences), and grayscale analysis was performed using ImageJ software.

[0049] 1.6 Quantitative Real-Time PCR Experimental Method

[0050] 40 mg of mouse liver tissue was ground and total RNA was extracted using an RNA extraction kit (LS1040, Promega). Reverse transcription was then performed using a cDNA reverse transcription kit (11141ES60, Yeasen) according to the manufacturer's instructions. The reverse transcription conditions were 25℃ for 5 min, 55℃ for 15 min, and 85℃ for 5 min. qPCR was then performed using a qPCR kit (11201ES08, Yeasen). The qPCR program was 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 60℃ annealing for 20 s, and 72℃ extension for 35 s, for 40 cycles, with a 2... -ΔΔCtmRNA was calculated using a relative quantification method. Primer sequences for the target genes (TNFα, MCP1, IL6, IL10, SCD1, FASN, SREBP1, PPARγ) are shown in Table 1 and were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0051] 1.7 Statistical Analysis

[0052] Statistical analysis was performed using GraphPad Prism 8.0 and SPSS 26.0 software. All trials were repeated at least three times, and results are expressed as mean ± standard deviation (X ± SD). A p-value < 0.05 was considered statistically significant.

[0053] Example 1: Effects of Aristolochia debilis on body weight, liver weight, liver structure, and fat accumulation in HFD-induced mice.

[0054] After 16 weeks of HFD feeding, the livers of the model group mice showed lipid accumulation and a dull surface, while the abdomen and subcutaneous tissues of the mice also showed significant fat accumulation. Figure 2 As shown in Figure A: Oil Red O staining results of liver tissue showed that a large number of red lipid droplets appeared in the liver tissue of the model group, indicating significant fat accumulation; while all treatment groups were able to significantly reduce fat deposition in liver tissue, indicating that Gynostemma pentaphyllum can improve liver lipid accumulation caused by fat.

[0055] like Figure 2 As shown in B, the body weight, liver weight, and liver index of mice in the model group were significantly higher than those in the control group, while the body weight, liver weight, and liver index of mice in each drug administration group were significantly lower than those in the model group.

[0056] like Figure 3 As shown in Figure B, plasma ALT and AST levels in the model group were significantly higher than those in the control group, while ALT and AST levels in each treatment group were significantly lower than those in the model group.

[0057] Figure 2 B and Figure 3 B and others jointly proved that the NAFLD model was successfully constructed.

[0058] like Figure 2 As shown in Figure C: The H&E staining results show that the liver cells in the model group were arranged irregularly, while the regularity of the liver cell arrangement in each drug-treated group was effectively improved compared to the model group.

[0059] like Figure 2 As shown in D: Compared with the model group, subcutaneous white fat accumulation was significantly reduced in each treatment group, and the high-dose group was superior to the low-dose group.

[0060] like Figure 2As shown in E, the mice in the model group were significantly fatter than those in the control group, while the body size of each drug-treated group was significantly improved compared to the model group.

[0061] like Figure 2 As shown in F, the body fat percentage of mice in the model group was significantly higher than that in the control group, while the body fat percentage of each drug-treated group was significantly improved compared to the model group.

[0062] like Figure 2 As shown in G: The livers of mice in the model group were significantly enlarged compared to the control group, while the livers of mice in the drug-treated group were significantly smaller compared to the model group.

[0063] Figure 2 E and 2F together prove that the obesity model was successfully established.

[0064] Figure 2 The experimental data corresponding to B, 2D, and 2F are shown in Table 2.

[0065] Table 2 Summary of experimental data on mouse body weight, body fat percentage, liver weight and liver index, and subcutaneous white fat weight and subcutaneous white fat index.

[0066] control group Model group low-dose group High-dose group body weight / g 27.40±0.89 41.60±0.89 33.20±1.30 30.00±2.55 Body fat percentage 2.9520±0.7504 5.2886±0.9431 4.1921±0.1111 3.8558±0.2996 Liver mass / g 1.1257±0.3116 1.7118±0.1930 1.0465±0.3116 0.8754±0.0729 Liver index 0.0413±0.0114 0.0411±0.0042 0.0317±0.0099 0.02945±0.0046 Subcutaneous white fat mass / g 0.3917±0.1205 2.0875±0.2812 1.2441±0.2562 0.8477±0.1339 Subcutaneous white fat index 0.0143±0.0044 0.0502±0.0068 0.0374±0.0071 0.0283±0.0037

[0067] As shown in Table 2, in terms of mouse body weight, the low-dose group was 20.2% lower than the model group, and the high-dose group was 27.9% lower than the model group; in terms of body fat percentage, the low-dose group was 20.7% lower than the model group, and the high-dose group was 27.1% lower than the model group; in terms of mouse liver weight, the low-dose group was 38.9% lower than the model group, and the high-dose group was 48.9% lower than the model group; and in terms of subcutaneous white fat weight, the low-dose group was 40.4% lower than the model group, and the high-dose group was 59.4% lower than the model group.

[0068] This demonstrates that after intervention with Gynostemma pentaphyllum, the body weight, body fat percentage, subcutaneous white fat, and liver weight of mice were significantly reduced, the regularity of hepatocyte arrangement was effectively improved, and fat accumulation in liver tissue was significantly reduced. This indicates that Gynostemma pentaphyllum can effectively reduce the weight gain induced by a high-fat diet in model mice, achieving the effect of alleviating and / or treating obesity induced by a high-fat diet in model animals. Furthermore, no discomfort, death, deformity, or carcinogenicity was observed in the model mice during this process, which also confirms the safety of Gynostemma pentaphyllum. This provides the possibility for the use of Gynostemma pentaphyllum in the preparation of foods, health products, and pharmaceuticals with weight loss / weight reduction functions.

[0069] Example 2: Effects of Aristolochia debilis on Biochemical Indicators in NAFLD Mice

[0070] like Figure 3 As shown in Figure A: Compared with the model group, all drug-treated groups significantly reduced the levels of TC, TG, and LDL-C in plasma, while increasing the level of HDL-C.

[0071] like Figure 3 As shown in B, each treatment group significantly reduced the activity of ALT and AST enzymes in mouse plasma and liver, indicating that Gynostemma pentaphyllum has a good protective effect on liver function in mice with non-alcoholic fatty liver disease.

[0072] Figure 3 The experimental data corresponding to A and 3B are shown in Table 3.

[0073] Table 3 Summary of experimental data on plasma TC, TG, LDL-C, HDL-C, ALT, AST, and liver ALT, AST.

[0074]

[0075]

[0076] Table 3 shows that, regarding mouse plasma TC, the low-dose group was 50.9% lower than the model group, and the high-dose group was 72.6% lower than the model group; regarding mouse plasma TG, the low-dose group was 79% lower than the model group, and the high-dose group was 85.1% lower than the model group; regarding mouse plasma LDL, the low-dose group was 43.3% lower than the model group, and the high-dose group was 66.3% lower than the model group; and regarding mouse plasma HDL, the low-dose group was 73.83% higher than the model group, and the high-dose group was 89% higher than the model group. 1%; Regarding mouse plasma ALT, the low-dose group was 73.2% lower than the model group, and the high-dose group was 82.3% lower than the model group; Regarding mouse plasma AST, the low-dose group was 49.2% lower than the model group, and the high-dose group was 57.8% lower than the model group; Regarding mouse liver ALT, the low-dose group was 39.8% lower than the model group, and the high-dose group was 56.5% lower than the model group; Regarding mouse liver AST, the low-dose group was 14.1% lower than the model group, and the high-dose group was 36.7% lower than the model group.

[0077] This shows that after intervention with Ganmutong, the biochemical indicators TC, TG, LDL-C, HDL-C, ALT, and AST in mice were effectively improved, proving that Ganmutong has a protective effect on the liver.

[0078] Example 3: Mechanism study of the improvement of liver function in NAFLD mice by Aristolochic acid.

[0079] To further verify the mechanism of Ganmutong's protective effect on liver function, the inventors tested the oxidative stress, inflammatory markers, and expression of adipogenic genes in mice.

[0080] like Figure 4As shown in Figure A: Compared with the model group, the MDA level, SOD and GST activities, and GSH / GSSG ratio of each treatment group were significantly increased, demonstrating that Ganmutong can improve antioxidant stress capacity, thereby playing a protective role in the liver.

[0081] like Figure 4 As shown in Figure B: Compared with the model group, the expression levels of TNFα and MCP1 in each treatment group were significantly reduced, proving that Ganmutong can significantly inhibit the expression of pro-inflammatory factors TNFα and MCP1 in liver tissue; compared with the model group, the expression levels of IL6 and IL10 in each treatment group were significantly increased, proving that Ganmutong can promote the expression of anti-inflammatory factors IL6 and IL10. Therefore, it can be concluded that the protective effect of Ganmutong on the liver is related to the inhibition of inflammatory response.

[0082] like Figure 4 As shown in Figure C: Real-time PCR results showed that the expression levels of adipogenic genes SCD1, FASN, SREBP1, and PPARγ in the liver of mice in the model group were higher than those in the control group; while the expression levels of each adipogenic gene in the liver tissue of mice in the drug-treated group were significantly downregulated, and the high-dose group was better than the low-dose group, proving that Gynostemma pentaphyllum can reduce the expression of adipogenic genes.

[0083] Figure 4 The experimental data corresponding to A, 4B, and 4C are shown in Table 4.

[0084] Table 4. Summary of experimental data for MDA, SOD, GST, GSH, GSSG, GSH / GSSG, TNFα, MCP1, IL6, IL10, SCD1, FASN, SREBP1, and PPARγ.

[0085] control group Model group low-dose group High-dose group <![CDATA[MDA / mmol·g -1 ]]> 377.7461±49.6821 774.5555±41.6783 760.8710±10.6129 445.8293±53.5269 <![CDATA[SOD / mmol·g -1 ]]> 2986.5467±24.7293 1690.0800±55.6223 2344.6947±258.5622 2932.1212±149.3180 <![CDATA[GST / mmol·g -1 ]]> 186.8217±50.1851 81.9329±8.2641 159.4026±12.8757 248.6702±18.7942 GSH / GSSG 7.8301±0.1271 3.4198±0.0397 5.5694±0.1882 13.0697±1.6804 TNFα 1.0243±0.2850 9.5500±3.3835 6.4248±0.7579 1.5678±0.8621 MCP1 1.0322±0.3277 12.3727±2.4585 7.0010±0.7068 2.4253±0.7927 IL6 1.1526±0.0969 0.6719±0.1560 1.2334±0.3213 2.6437±0.3013 IL10 1.1405±0.1585 0.7502±0.1081 1.6186±0.2159 3.1170±0.8544 SCD1 1.0000±0.0114 8.3368±0.0429 5.8830±0.6884 0.3663±0.0245 FASN 1.0198±0.2533 2.7918±0.3915 1.1217±0.3686 1.0278±0.0498 SREBP1 1.0003±0.0318 1.5256±0.0914 1.0189±0.1010 0.6087±0.0891 PPARγ 1.0089±0.1593 2.7920±0.4060 0.7509±0.1732 0.6225±0.1970

[0086] Table 4 shows that, regarding mouse MDA, the low-dose group was 17.7% lower than the model group, and the high-dose group was 42.4% lower; regarding mouse SOD, the low-dose group was 38.7% higher than the model group, and the high-dose group was 73.5% higher; regarding mouse GST, the low-dose group was 94.6% higher than the model group, and the high-dose group was 203.5% higher; regarding mouse GSH / GSSG, the low-dose group was 62.9% higher than the model group, and the high-dose group was 282.2% higher; regarding mouse TNFα mRNA expression, the low-dose group was 32.7% lower than the model group, and the high-dose group was 83.6% lower; regarding mouse MCP1... Regarding mRNA expression levels, the low-dose group was 43.4% lower than the model group, and the high-dose group was 80.4% lower than the model group; regarding mouse IL6 mRNA expression levels, the low-dose group was 83.6% higher than the model group, and the high-dose group was 293.5% higher than the model group; regarding mouse IL10 mRNA expression levels, the low-dose group was 115.8% higher than the model group, and the high-dose group was 315.5% higher than the model group; regarding mouse adipogenic gene SCD1 mRNA expression levels, the low-dose group was 29.4% lower than the model group, and the high-dose group was 95.6% lower than the model group; regarding mouse adipogenic gene FASN mRNA expression levels, the low-dose group was 59.8% lower than the model group, and the high-dose group was 63.2% lower than the model group; regarding mouse adipogenic gene SREBP1… In terms of mRNA expression levels, the low-dose group was 33.2% lower than the model group, and the high-dose group was 60.1% lower than the model group; in terms of mouse adipogenic gene PPARγ mRNA expression levels, the low-dose group was 73.1% lower than the model group, and the high-dose group was 77.7% lower than the model group.

[0087] Therefore, it can be seen that Ganmutong can not only improve hepatocyte damage by enhancing the liver's antioxidant stress capacity and inhibiting the expression of inflammatory factors, but also improve hepatic steatosis by reducing the expression of adipogenic genes.

[0088] Example 4: Effects of Aristolochia debilis on key proteins and genes involved in lipid synthesis in NAFLD mice

[0089] To further elucidate the protective effect of Gynostemma pentaphyllum against non-alcoholic fatty liver disease induced by a high-fat diet, the inventors used Western blotting to detect the expression of key proteins and genes involved in lipid synthesis in the AMPK / SREBP-1c signaling pathway.

[0090] like Figure 5As shown, the expression ratio of pAMPKα / AMPKα proteins involved in energy regulation was significantly inhibited in the liver tissue of model group mice, preventing energy transfer and thus significantly upregulating the expression of ACC protein in the model group. In contrast, the drug-treated group activated the expression of p-AMPKα / AMPKα and ACC proteins, thereby playing a role in energy regulation and inhibiting the expression of downstream lipid synthesis-related protein ACC.

[0091] In addition, the protein expression of SREBP-1c, which is involved in lipid metabolism, was observed in the liver tissue of the model group mice. Figure 5 C) and the expression of downstream lipid synthesis-related genes SCD1 and FASN ( Figure 4 C) Significantly upregulated, while all groups treated with Ganmutong significantly downregulated the expression of these three substances.

[0092] Figure 5 The experimental data corresponding to B and 5C are shown in Table 5.

[0093] Table 5. Summary of experimental data on p-AMPKα / AMPKα, Srepb1-c, and ACC protein expression levels.

[0094] control group Model group low-dose group High-dose group p-AMPKα / AMPKα 1.0718±0.0076 0.4821±0.0082 0.5189±0.0023 0.9130±0.0140 SREBP-1c 1.0000±0.0085 1.3059±0.0041 0.6030±0.0040 0.7446±0.0161 ACC 1.0000±0.0122 1.5471±0.0374 0.7961±0.0056 0.8567±0.0097

[0095] As shown in Table 5, regarding the relative expression level of p-AMPKα / AMPKα in mice, the low-dose group was 7.6% higher than the model group, and the high-dose group was 89.3% higher than the model group; regarding the relative expression level of SREBP-1c, the low-dose group was 53.8% lower than the model group, and the high-dose group was 43% lower than the model group; regarding the expression level of ACC in mice, the low-dose group was 48.5% lower than the model group, and the high-dose group was 44.6% lower than the model group.

[0096] Therefore, it can be seen that Ganmutong can activate AMPKα and inhibit the activity of SREBP-1c, an important regulator of lipid synthesis, thereby reducing the expression of lipid synthesis genes FASN and SCD1 and protein ACC, improving the antioxidant stress capacity of hepatocytes, and improving hepatic steatosis.

[0097] Examples 1-4 collectively demonstrate that: First, after intervention with Gynostemma pentaphyllum, the body weight and liver weight of mice were significantly reduced, the regularity of hepatocyte arrangement was effectively improved, and fat accumulation in liver tissue was significantly reduced; Second, the biochemical indicators of mice, including TC, TG, LDL-C, HDL-C, ALT, and AST, were effectively improved, proving that Gynostemma pentaphyllum has a protective effect on the liver; Third, Gynostemma pentaphyllum can not only improve hepatocyte damage by enhancing the liver's antioxidant stress capacity and inhibiting the expression of inflammatory factors, but also improve hepatic steatosis by reducing the expression of adipogenic genes. Fourth, Ganmutong can activate AMPKα and inhibit the activity of SREBP-1c, an important regulator of lipid synthesis, thereby reducing the expression of lipid synthesis genes FASN and SCD1 and protein ACC, improving the antioxidant stress capacity of hepatocytes, and improving hepatic steatosis. In other words, Ganmutong can alleviate and / or treat NAFLD model animals induced by a high-fat diet, and no discomfort, death, deformity or carcinogenesis occurred in the model mice during this process, which also confirms the safety of Ganmutong. This provides the possibility of Ganmutong in the preparation of health products and medicines with liver protection.

Claims

1. Application of Gynostemma pentaphyllum in non-alcoholic fatty liver disease (NAFLD).

2. The application of Aristolochia debilis in non-alcoholic fatty liver disease (NAFLD) according to claim 1, characterized in that: The application of the aforementioned Gynostemma pentaphyllum as the main component in the preparation of products for the treatment of non-alcoholic fatty liver disease (NAFLD).

3. The application of Aristolochia debilis in non-alcoholic fatty liver disease (NAFLD) according to claim 2, characterized in that: The products mentioned include pharmaceuticals or health supplements.

4. The application of Aristolochia debilis in non-alcoholic fatty liver disease (NAFLD) according to claim 2 or 3, characterized in that, The product is a preparation made with Aristolochia debilis as the main active ingredient, plus commonly used pharmaceutical excipients or auxiliary ingredients.

5. The application of Aristolochia debilis in non-alcoholic fatty liver disease (NAFLD) according to claim 4, characterized in that: The formulations include liquid formulations, solid formulations, or semi-solid formulations.

6. Application of Gynostemma pentaphyllum in obesity.

7. The application of Aristolochia debilis in obesity according to claim 6, characterized in that: The application of the aforementioned Gynostemma pentaphyllum as the main component in the preparation of products for treating obesity.

8. The application of Aristolochia debilis in obesity according to claim 7, characterized in that: The products mentioned include pharmaceuticals or health supplements.

9. The application of Aristolochia debilis in obesity according to claim 7 or 8, characterized in that, The product is a preparation made with Aristolochia debilis as the main active ingredient, plus commonly used pharmaceutical excipients or auxiliary ingredients.

10. The application of Aristolochia debilis in obesity according to claim 9, characterized in that: The formulations include liquid formulations, solid formulations, or semi-solid formulations.

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