Use of kuding tea extract in preparation of preparations for preventing and treating non-alcoholic fatty liver

Abstract

The application discloses application of an Ilicium fortunei extract in preparation of a product for preventing and treating non-alcoholic fatty liver disease. The Ilicium fortunei extract is prepared through a water extraction-alcohol precipitation-dialysis impurity removal and macroporous resin decolorization process, has a sugar content of 36.23+1.56%, a sugar acid content of 46.00+1.77%, a protein content of 1.03+0.02% and a polyphenol content of 0.07+0.001%. Experimental results show that the extract can significantly reduce the body weight, liver weight and serum transaminase level of a mouse with metabolic disorder induced by a high-fat diet, improve glucose tolerance and insulin resistance, reduce liver steatosis and inflammatory damage, and effectively regulate intestinal flora structure and restore microbial community balance, thereby playing an intervention role on metabolic related fatty liver disease. The application provides a scientific basis for a new use of the Ilicium fortunei extract in prevention and treatment of metabolic related fatty liver disease, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to new applications of bitter tea extract, specifically the application of bitter tea extract in the preparation of products for the prevention and treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Metabolic-associated fatty liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is currently the most common chronic liver disease worldwide, affecting approximately 38% of adults globally. Its prevalence continues to rise rapidly, placing a heavy burden on society, the economy, and healthcare systems. While lifestyle interventions are effective in the early stages of MASLD, their effectiveness significantly decreases in advanced-stage patients. Currently, only one drug, Resmetirom, is approved for the treatment of metabolic-associated steatohepatitis (MASH), which must be used in combination with exercise and dietary interventions, and its fibrosis remission rate remains below 30%.

[0003] Kuding tea extract (LRP) has the effects of lowering blood lipids, increasing coronary blood flow, increasing myocardial blood supply, and resisting atherosclerosis. It has a good preventive and therapeutic effect on symptoms such as dizziness, headache, chest tightness, fatigue, and insomnia in patients with cardiovascular and cerebrovascular diseases. However, systematic research on the use of Kuding tea extract in the prevention and treatment of metabolic-related fatty liver disease is still lacking. Therefore, developing a safe and effective Kuding tea extract for the preparation of products for the treatment of metabolic-related fatty liver disease is of great clinical significance and urgency. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of bitter tea extract in the preparation of products for the prevention and treatment of non-alcoholic fatty liver disease (NAFLD), offering a new approach to the research and development of drugs for the prevention and treatment of NAFLD. The extraction method for bitter tea extract is simple, promoting the full development and utilization of food resources, and providing a medicine for the prevention and treatment of NAFLD. To achieve the above objectives, this invention is implemented through the following technical solutions: Technical solution of the present invention: Application of bitter tea extract in the preparation of products for the prevention and treatment of non-alcoholic fatty liver disease.

[0005] The aforementioned bitter tea extract contained 36.23 ± 1.56% sugar, 46.00 ± 1.77% uronic acid, 1.03 ± 0.02% protein, and 0.07 ± 0.001% polyphenols.

[0006] The preparation method of the aforementioned bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 50-150 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:7-15, boil and extract for 1-5 h, repeat the extraction 1-4 times, combine the 1-4 extracts, filter with a 100-300 mesh sieve, concentrate to 80-150 kg at 35℃-75℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 30℃-60℃ to obtain bitter tea extract A, for use. (2) Dialysis to remove impurities: Take bitter tea extract A and prepare an aqueous solution of 35-65 mg / mL. Centrifuge at 6000-10000 rpm for 5-18 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 0℃-10℃ for 18-30 h. Change the pure water every 2-6 h. After dialysis, take the solution in the bag to obtain bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, add ethanol and soak for 7-16 h, then wash repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix. Decolorize with magnetic stirring at room temperature for 1-8 h. After stirring, centrifuge at 6000-10000 rpm for 1-10 min, take the supernatant, concentrate under reduced pressure at 30℃-80℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 1-5 h and the drying temperature to 40℃ for 8-16 h to freeze dry to obtain bitter tea extract. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 1-5:1.

[0007] Specifically, the preparation method of the aforementioned bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 80-120 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:8-12, boil and extract for 2-3 h, repeat the extraction 2-3 times, combine the 2-3 extracts, filter with a 150-250 mesh sieve, concentrate to 90-110 kg at 45℃-65℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 35℃-55℃ to obtain bitter tea extract A, for use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 40-55 mg / mL, centrifuge at 7000-9000 rpm for 7-13 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 2℃-5℃ for 20-26 h, changing the pure water every 3-5 h. After dialysis, take the solution in the bag to obtain bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, add ethanol and soak for 10-14 h, then wash repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix. Decolorize with magnetic stirring at room temperature for 2-6 h. After stirring, centrifuge at 7000-9000 rpm for 2-7 min. Take the supernatant and concentrate it under reduced pressure at 40℃-60℃. Place it in a freeze dryer, set the cold trap temperature to -20℃ for 1-3 h, and the drying temperature to 40℃ for 10-14 h for freeze drying to obtain bitter tea extract. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 2-4:1.

[0008] Specifically, the preparation method of the aforementioned bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 100 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:10, boil and extract for 2.5 h, repeat the extraction twice, combine the two extracts, filter with a 200 mesh sieve, concentrate to 100 kg at 55℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature of 40-55℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 50 mg / mL, centrifuge at 8000 rpm for 10 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 4℃ for 24 h. Change the pure water every 4 h. After the dialysis is completed, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 12 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 4 h. After stirring, centrifuge at 8000 rpm for 5 min, take the supernatant, concentrate it under reduced pressure at 55℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 2 h and the drying temperature to 40℃ for 12 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 3:1.

[0009] The aforementioned application is characterized in that: the product includes pharmaceuticals.

[0010] The aforementioned preparation method involves combining bitter tea extract with acceptable excipients from the drug, processing them according to conventional methods, and then producing the corresponding pharmaceutical formulation.

[0011] The dosage forms of the aforementioned products include tablets, capsules, granules, decoctions, or pills.

[0012] The raw materials of the aforementioned products include the bitter tea extract as described in any one of claims 3-5.

[0013] The aforementioned product is mainly prepared from the bitter tea extract according to any one of claims 3-5.

[0014] Beneficial effects of this invention: This invention optimizes the extraction process of bitter tea, successfully obtaining a bitter tea extract, and experimentally verifies its role in the prevention and treatment of non-alcoholic fatty liver disease (NAFLD). The results show that the bitter tea extract can effectively intervene in related pathological processes, providing reliable experimental evidence and reference for the further development and application of bitter tea in the prevention and treatment of NAFLD and related diseases. Attached Figure Description

[0015] Figure 1 This is a molecular weight distribution diagram of LRP; Figure 2 The diagram shows the monosaccharide composition of LRP; Figure 3 The infrared spectrum analysis diagram of LRP; Figure 4 The image shows the UV spectrum of LRP. Figure 5 A schematic diagram of the experimental design and workflow for LRP-induced abnormal glucose metabolism in mice induced by a high-fat diet (HFD); Figure 6 A graph showing the trend of weight change in each group of mice; Figure 7 Liver weight of mice in each group; Figure 8 The food intake of mice in each group; Figure 9 For glucose tolerance test (GTT); Figure 10 Insulin tolerance test (ITT); Figure 11 For the area under the GTT curve (AUC) analysis (*P < 0.05, **P < 0.01, ***P < 0.001); Figure 12 For the area under the ITT curve (AUC) analysis (*P < 0.05, **P < 0.01, ***P < 0.001); Figure 13 The total cholesterol (TC) content in the serum of mice in each group; Figure 14 Serum triglyceride (TG) levels in each group of mice; Figure 15Serum alanine aminotransferase (ALT) activity in mice of each group was measured. Figure 16 The serum aspartate aminotransferase (AST) activity of mice in each group was measured. Figure 17 The total TC content in the liver tissue of mice in each group; Figure 18 The TG content in the liver tissue of mice in each group; Figure 19 HE and Oil Red staining were performed on liver tissues of mice in each group. Figure 20 Oil Red staining MAS score of liver tissue from each group of mice; Figure 21 The Chao1 index represents the gut microbiota. Figure 22 The Simpson index for gut microbiota; Figure 23 The Shannon index represents the gut microbiota. Figure 24 PCA analysis of gut microbiota β diversity; Figure 25 The relative abundance of gut microbiota at the phylum level; Figure 26 A heatmap of the gut microbiota at the phylum level; Figure 27 The relative abundance of gut microbiota at the genus level; Figure 28 A heatmap of gut microbiota at the genus level; Figure 29 LEfSe analysis for differential gut microbiota; Figure 30 The relative abundance of Muribaculaceae; Figure 31 The relative abundance of Parabacteroides; Figure 32 The relative abundance of Bacteroides; Figure 33 The relative abundance of Blautia; Figure 34 Relative abundance of Lachnospiraceae NK4A136 group; Figure 35 The relative abundance of Faecalibaculum; Figure 36 The relative abundance of Odoribacter; Figure 37 The relative abundance of Coriobacteriaceae UCG-002; Figure 38The relative abundance of the Eubacterium coprostanoligenes group; Figure 39 Relative abundance of the Prevotellaceae NK3B31 group; Figure 40 The relative abundance of Bacteroidota; Figure 41 Relative abundance of Firmicutes; Figure 42 The relative abundance of Actinobacteriota; Figure 43 The relative abundance of Verrucomicrobiota; Detailed Implementation

[0016] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0017] Example 1: Preparation method of bitter tea extract (1) Water extraction and alcohol precipitation: Take 100 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:10, boil and extract for 2.5 h, repeat the extraction twice, combine the two extracts, filter with a 200 mesh sieve, concentrate to 100 kg at 55℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature of 40-55℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 50 mg / mL, centrifuge at 8000 rpm for 10 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 4℃ for 24 h. Change the pure water every 4 h. After the dialysis is completed, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 12 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 4 h. After stirring, centrifuge at 8000 rpm for 5 min, take the supernatant, concentrate it under reduced pressure at 55℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 2 h and the drying temperature to 40℃ for 12 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 3:1.

[0018] Example 2: Preparation method of bitter tea extract (1) Water extraction and alcohol precipitation: Take 50 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:7, boil and extract for 1 h, repeat the extraction once, combine the extracts, filter with a 100 mesh sieve, concentrate to 80 kg at 35℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 30℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 35 mg / mL, centrifuge at 6000 rpm for 5 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 0℃ for 18 h. Change the pure water every 2 h. After dialysis, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 7 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 1 h. After stirring, centrifuge at 6000 rpm for 1 min, take the supernatant, concentrate it under reduced pressure at 30℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 1 h and the drying temperature to 40℃ for 8 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 1:1.

[0019] Example 3: Preparation method of bitter tea extract (1) Water extraction and alcohol precipitation: Take 80 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:8, boil and extract for 2 h, repeat the extraction twice, combine the two extracts, filter with a 150 mesh sieve, concentrate to 90 kg at 45℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 35℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 40 mg / mL, centrifuge at 7000 rpm for 7 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, dialyze at 2℃ for 20 h, change the pure water every 3 h, take the solution in the bag after dialysis, and you will get bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 10 h, and wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 2 h. After stirring, centrifuge at 7000 rpm for 2 min, take the supernatant, concentrate it under reduced pressure at 40℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 3 h and the drying temperature to 40℃ for 10 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 2:1.

[0020] Example 4: Preparation method of bitter tea extract (1) Water extraction and alcohol precipitation: Take 120 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:12, boil and extract for 3 h, repeat the extraction 3 times, combine the 3 extracts, filter with a 250 mesh sieve, concentrate to 110 kg at 65℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 55℃ to obtain bitter tea extract A, for use. (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 55 mg / mL, centrifuge at 9000 rpm for 13 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 5℃ for 26 h. Change the pure water every 5 h. After the dialysis is completed, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 14 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 6 h. After stirring, centrifuge at 9000 rpm for 7 min, take the supernatant, concentrate it under reduced pressure at 60℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 4 h and the drying temperature to 40℃ for 14 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 4:1.

[0021] Example 5: Preparation method of bitter tea extract (1) Water extraction and alcohol precipitation: Take 150 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:15, boil and extract for 5 h, repeat the extraction 4 times, combine the 4 extracts, filter with a 300 mesh sieve, concentrate to 150 kg at 75℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 60℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 65 mg / mL, centrifuge at 10000 rpm for 18 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, dialyze at 10℃ for 30 h, change the pure water every 6 h, take the solution in the bag after dialysis, and you will get bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 16 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 8 h. After stirring, centrifuge at 10000 rpm for 10 min, take the supernatant, concentrate it under reduced pressure at 80℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 5 h and the drying temperature to 40℃ for 16 h to freeze dry to obtain bitter tea extract. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 5:1.

[0022] Example 6: Preparation of tablets Take 100 g of the bitter tea extract obtained in Example 1, pulverize it through a 100-mesh sieve, and mix it evenly with 50 g of microcrystalline cellulose, 30 g of lactose, and 10 g of crospovidone. Add an appropriate amount of 10% povidone K30 ethanol solution to form a soft mass, granulate it through a 20-mesh sieve, dry it at 50°C, and after granulation, add 1 g of magnesium stearate, mix evenly, and compress it into tablets to obtain tablets. Each tablet contains 100 mg of bitter tea extract.

[0023] Treatment of conditions: Prevention and treatment of non-alcoholic fatty liver disease.

[0024] Instructions for use: Oral administration.

[0025] Dosage and administration: 1-2 tablets each time, twice a day.

[0026] Example 7: Preparation of Capsules Take 100 g of the bitter tea extract obtained in Example 1, pulverize it through a 100-mesh sieve, mix it evenly with 50 g of starch, 20 g of pregelatinized starch, and 20 g of microcrystalline cellulose, add an appropriate amount of 5% povidone K30 aqueous solution to granulate, pass it through a 24-mesh sieve, dry it at 50℃, and after granulation, fill it into gelatin capsules to obtain capsules. Each capsule contains 100 mg of bitter tea extract.

[0027] Treatment of conditions: Prevention and treatment of non-alcoholic fatty liver disease.

[0028] Instructions for use: Oral administration.

[0029] Dosage and administration: 1-2 capsules each time, twice a day.

[0030] Example 8: Preparation of Granules Take 100 g of the bitter tea extract obtained in Example 1, pulverize it through a 100-mesh sieve, mix it evenly with 80 g of sucrose powder and 40 g of dextrin, add an appropriate amount of 70% ethanol solution to make a soft mass, granulate it through a 14-mesh sieve, dry it at 50℃, granulate it, and package it to obtain granules. Each bag contains 100 mg of bitter tea extract.

[0031] Treatment of conditions: Prevention and treatment of non-alcoholic fatty liver disease.

[0032] Instructions for use: Oral administration.

[0033] Dosage and administration: 1-2 sachets each time, twice a day.

[0034] Example 9: Preparation of decoction paste Take 100 g of the bitter tea extract obtained in Example 1, add 500 mL of purified water, heat and stir to dissolve, filter, and concentrate the filtrate to a clear extract with a relative density of approximately 1.30 (60°C). Separately, take 200 g of sucrose, add 200 mL of water, heat to dissolve, filter, mix with the above clear extract, and continue to concentrate to a relative density of approximately 1.35 (60°C) to obtain the decoction. Each 20 g of decoction contains approximately 1 g of bitter tea extract.

[0035] Treatment of conditions: Prevention and treatment of non-alcoholic fatty liver disease.

[0036] Instructions for use: Oral administration.

[0037] Dosage and administration: 4-8 g each time, twice a day.

[0038] Example 10: Preparation of pills Take 100 g of the bitter tea extract obtained in Example 1, pulverize it through a 120-mesh sieve, mix it evenly with 50 g of starch and 30 g of microcrystalline cellulose, and make microcapsules with an appropriate amount of 50% ethanol. Dry the microcapsules, select the microcapsules, and coat them with a thin film to obtain the pills. Each 100 pills contain 5 g of bitter tea extract, and each pill contains 50 mg of bitter tea extract.

[0039] Treatment of conditions: Prevention and treatment of non-alcoholic fatty liver disease.

[0040] Instructions for use: Oral administration.

[0041] Dosage and administration: 2-4 pills each time, twice a day.

[0042] The inventors conducted numerous experiments, and the following is a study of specific implementation methods of the bitter tea extract described in this invention in the prevention and treatment of non-alcoholic fatty liver disease: Preparation method of bitter tea extract 1.1 Main experimental reagents Kuding tea was sourced from Yuqing County, Guizhou Province; 3500 Da dialysis bags were purchased from Viskase / United Carbon; D101 macroporous resin was sourced from; 8-week-old male mice were purchased from Beijing Vitonlius Laboratory Animal Technology Co., Ltd.; all animal experimental procedures were approved by the Animal Ethics Committee of Zhejiang University, approval number: ZJU20250925. 1.2 Preparation process of bitter tea extract (1) Water extraction and alcohol precipitation: Take 100 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:10, boil and extract for 2.5 h, repeat the extraction twice, combine the two extracts, filter with a 200 mesh sieve, concentrate to 100 kg at 55℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature of 40-55℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 50 mg / mL, centrifuge at 8000 rpm for 10 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 4℃ for 24 h. Change the pure water every 4 h. After the dialysis is completed, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 12 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 4 h. After stirring, centrifuge at 8000 rpm for 5 min, take the supernatant, concentrate it under reduced pressure at 55℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 2 h and the drying temperature to 40℃ for 12 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 3:1.

[0043] 2. Methods and results for detecting active ingredients 2.1 Determination of sugar content The sugar content in LRP was determined using the phenol-sulfuric acid method. A 0.1 mg / mL glucose standard solution and a 1 mg / mL LRP solution were prepared. 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mL of glucose standard solution and 0.2 mL of LRP solution were respectively pipetted into 10 mL EP tubes. Water was added to bring the volume to 2 mL. Then, 1 mL of 6% phenol solution and 5 mL of concentrated sulfuric acid were added sequentially. After thorough mixing, the mixture was allowed to stand for 30 min, and the absorbance was measured at 490 nm.

[0044] 2.2 Results of sugar content determination The sugar content in LRP was measured to be 36.23 ± 1.56%.

[0045] 2.3 Determination of uronic acid content The uronic acid content in LRP was determined using the m-hydroxybiphenyl method. A 0.1 mg / mL galacturonic acid standard solution and a 1 mg / mL LRP solution were prepared. 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 mL of the galacturonic acid standard solution and 0.2 mL of the LRP solution were respectively pipetted into 10 mL EP tubes. Water was added to bring the volume to 2 mL, and 6 mL of 0.0125 mol / L sodium tetraborate-sulfuric acid solution was added sequentially. The mixture was boiled in a water bath for 5 min, cooled to room temperature, and then 0.1 mL of 0.15 mg / mL m-hydroxybiphenyl solution was added. After reacting for 5 min, the absorbance was measured at 525 nm.

[0046] 2.4 Results of uronic acid content determination The uronic acid content in LRP was measured to be 46.00 ± 1.77%.

[0047] 2.5 Protein content determination The protein content in LRP was determined using the Coomassie Brilliant Blue method. A 0.1 mg / mL bovine serum albumin standard solution and a 1 mg / mL LRP solution were prepared. 100 mg of Coomassie Brilliant Blue G-250 was weighed and dissolved in 50 mL of ethanol, followed by the addition of 100 mL of phosphoric acid. The solution was then diluted to 1 L with distilled water to obtain the Coomassie Brilliant Blue reagent. 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of bovine serum albumin standard solution and 1.0 mL of LRP solution were transferred to the solution, respectively, and diluted to 1 mL with water. 5 mL of Coomassie Brilliant Blue solution was added to each solution sequentially. After reacting for 5 min, the absorbance was measured at 595 nm.

[0048] 2.6 Protein content determination results The protein content in LRP was measured to be 1.03 ± 0.02%.

[0049] 2.7 Polyphenol Content Determination The polyphenol content of LRP was determined using the Folin-Ciocalteu method. A 0.1 mg / mL gallic acid standard solution and a 1 mg / mL LRP solution were prepared. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 mL of gallic acid standard solution and 1 mL of LRP solution were respectively pipetted into 10 mL EP tubes, and water was added to bring the volume to 1 mL. 1 mL of Folin-Ciocalteu reagent and 2 mL of 10% sodium carbonate solution were added sequentially, and the mixture was diluted with water to 10 mL. The reaction was carried out in the dark for 1 h, and the absorbance was measured at 765 nm.

[0050] 2.8 Results of Polyphenol Content Determination The polyphenol content in LRP was measured to be 0.07 ± 0.001%.

[0051] 2.9 Determination of Molecular Weight Distribution The molecular weight distribution of LRP was determined by high-performance gel permeation chromatography (HPGPC). LRP was dissolved in pure water to prepare a 1 mg / mL solution, filtered through a 0.22 μm membrane, and then subjected to HPGPC analysis. The analysis was performed using an Agilent 1260 instrument coupled with an evaporative light scattering detector, employing OHpak SB-805 HQ (8.0 × 300 mm) and SB-804 HQ (8.0 × 300 mm) columns in series. Ultrapure water was used as the mobile phase at a flow rate of 0.8 mL / min. Calibration was performed using pullulan standards of known molecular weights (P5, P10, P20, P50, P100, P200, P400, P800).

[0052] 2.10 Molecular weight distribution results The molecular weight distribution range of LRP shows four peaks as follows: Figure 1 As shown, the retention times were 14.31, 16.82, 17.74, and 27.98 min, indicating that LRP is a mixed polysaccharide. Peak 1 was the major component of LRP, with Mw, Mn, and Mp values ​​of 1.362 × 10⁻⁶. 3 1.180×10 3 and 1.369×10 3 kDa.

[0053] 2.11 Determination of Monosaccharide Composition The monosaccharide composition of LRP was determined by pre-column PMP derivatization using HPLC. 5 mg of LRP was weighed and added to 3 mL of 2 M trifluoroacetic acid. Hydrolysis was carried out at 110 °C for 4 h. The hydrolysate was then evaporated to dryness under reduced pressure with methanol, and this process was repeated three times. The hydrolysate was dissolved in 0.2 mL of water, followed by the addition of 0.2 mL of 0.5 M PMP and 0.2 mL of 0.3 M sodium hydroxide. The reaction was carried out at 70 °C for 1.5 h, and then terminated with 0.2 mL of 0.3 M hydrochloric acid. The mixture was extracted three times with 2 mL of chloroform, and the aqueous phase was collected and analyzed using Nucifera C... 18H HPLC analysis was performed on a column (250 × 4.6 mm, 5 μm) using a 2% acetic acid-acetonitrile gradient elution (acetonitrile concentration increased from 17% to 21% from 0 to 10 min, and remained at 21% from 10 to 45 min), with a flow rate of 0.8 mL / min, a detection wavelength of 245 nm, and an injection volume of 10 μL.

[0054] 2.12 Results of Monosaccharide Composition Determination The monosaccharide composition and molar ratio of LRP Figure 2As shown, LRP is mainly composed of galactose (Gal), glucose (Glu), arabinose (Ara), and galacturonic acid (Gal-A), with contents of 14.10 ± 0.21%, 17.79 ± 0.26%, 25.66 ± 0.38%, and 37.65 ± 0.56%, respectively; it also contains small amounts of mannose (Man), rhamnose (Rha), glucuronic acid (Glu-A), and fucose (Fuc), with contents of 1.37 ± 0.03%, 2.58 ± 0.05%, 0.43 ± 0.01%, and 0.42 ± 0.01%, respectively.

[0055] 2.13 Infrared Spectroscopy Analysis Place the dried LRP sample in the sample cell at 4000-4000 cm⁻¹. -1 Record the spectrum within the wavenumber range.

[0056] 2.14 Infrared Spectroscopic Analysis Results The infrared spectrum of LRP is as follows Figure 3 As shown. 3287 cm -1 The broad asymmetric absorption peak at 2927 cm⁻¹ is attributed to the OH stretching vibration. -1 The absorption peak at 1740 cm⁻¹ corresponds to the CH stretching vibration; -1 and 1600 cm -1 The absorption peak at 1416 cm⁻¹ indicates the presence of a C=O group; -1 The absorption peak at 1076 cm⁻¹ corresponds to the bending vibration of the CH bond; -1 and 1032 cm -1 The absorption peaks at 834 cm⁻¹ are attributed to the stretching vibrations of COC and COH, respectively; -1 The absorption peak at that location indicates the presence of an α-glycosidic bond.

[0057] 2.15 Ultraviolet Spectroscopy Analysis Prepare a 1 mg / mL LRP aqueous solution and scan the spectrum in the wavelength range of 200-600 nm.

[0058] 2.16 Results of Ultraviolet Spectroscopy Analysis The ultraviolet spectrum of LRP is as follows: Figure 4 As shown, no obvious absorption peaks were observed at 260 nm and 280 nm, indicating that LRP contains only trace amounts of nucleic acids and proteins.

[0059] 3. Study on efficacy against non-alcoholic fatty liver disease 3.1 LRP alleviates HFD-induced metabolic disorders by regulating body weight, hepatic lipid deposition, and glucose metabolism. 3.1.1 Animal grouping Eight-week-old male mice were selected and housed in a specific pathogen-free (SPF) environment with a 12-hour / 12-hour light / dark cycle, and constant room temperature and relative humidity. After 7 days of acclimatization, the animals were randomly divided into 6 groups of 6 mice each: a normal control group, an HFD model group, a low-dose LRP group (200 mg / kg / d), a high-dose LRP group (400 mg / kg / d), a commercially available bitter tea polysaccharide control group (400 mg / kg / d), and a positive control group (3 mg / kg / d). The normal control group was given a standard basal diet, while the other groups were fed a high-fat diet (product number XT310). After establishing the model through 14 weeks of continuous high-fat feeding, the mice were subjected to gavage intervention for 8 weeks while maintaining the high-fat diet, for a total experimental period of 22 weeks. Changes in body weight and food intake were recorded weekly during the experiment (see [link to experimental data]). Figure 5 ).

[0060] 3.1.2 Experimental Methods In the 20th week, an insulin tolerance test (ITT) was conducted: after fasting for 12 hours, tail blood was collected from mice to measure basal blood glucose levels. Insulin was then injected intraperitoneally, and blood glucose levels were measured at 15, 30, 45, 60 and 90 minutes after administration.

[0061] In week 21, an oral glucose tolerance test (GTT) was performed: fasting blood glucose was measured after 6 hours of fasting. After oral administration of glucose solution, blood glucose changes were measured at 15, 30, 45, 60 and 90 minutes.

[0062] Mice were anesthetized with isoflurane at the end of week 22 before sacrifice, and body fat content was measured and the percentage of body weight to fat was calculated. The mice were then fasted overnight, anesthetized again, and blood samples were collected. Liver tissue was isolated; a portion was fixed in 4% paraformaldehyde for histological examination, while the remaining samples were rapidly frozen and stored at -80°C for subsequent gut microbiota analysis.

[0063] 3.1.3 Experimental Results After 14 weeks of continuous high-fat diet (HFD) feeding, the body weight of mice in the model group (HFD) and each LRP intervention group was significantly higher than that of the normal control group (CON). Furthermore, there were no significant differences among the LRP dosage groups, the commercially available bitter tea polysaccharide group (LRP-C), and the positive control Resmetirom (RES) group, indicating that the high-fat diet model was successfully established (see [link to relevant documentation]). Figure 6 During the subsequent 8-week intervention phase, compared with the HFD group, the rate of weight gain in mice in all intervention groups was significantly reduced. Among them, LRP-H showed the most significant inhibitory effect, which was comparable to RES and superior to LRP-C. LRP-C also significantly inhibited weight gain, but its effect was weaker than that of LRP-H, suggesting that LRP is superior to commercially available polysaccharides in controlling HFD-induced weight abnormalities.

[0064] Liver marker analysis showed (see) Figure 7 The liver weight of mice in the HFD group was significantly higher than that in the CON group, indicating significant hepatic hypertrophy and lipid deposition. All doses of LRP, LRP-C, and RES intervention significantly reduced liver weight, with LRP-H showing the greatest reduction, comparable to the RES group and superior to the LRP-C group, indicating that LRP has a significant effect in inhibiting hepatic lipid deposition. There was no significant difference in food intake among the groups (see...). Figure 8 This indicates that the improvement in weight and liver weight was not due to reduced food intake.

[0065] To assess the impact of LRP on glucose metabolism, glucose tolerance (GTT) and insulin tolerance (ITT) tests were conducted. GTT results showed (see...) Figures 9-10 The blood glucose levels in the HFD group mice were significantly higher than those in the CON group during the test, indicating a significant impairment in glucose tolerance. LRP intervention could reduce blood glucose levels, with both LRP-L and LRP-C significantly reducing the blood glucose curve, while LRP-H showed a decreasing trend, and its overall AUC reduction was comparable to that of RES. Overall comparison showed that the hypoglycemic effect of LRP-C was between that of LRP-L and LRP-H.

[0066] ITT results (see) Figures 11-12 The results showed that the glucose clearance rate was significantly reduced in the HFD group, indicating significant insulin resistance. LRP intervention improved insulin sensitivity in a dose-dependent manner. The AUC value of the LRP-H group decreased most significantly, followed by the RES group. LRP-C also showed significant improvement, but less than LRP-H, indicating that LRP is superior to commercially available polysaccharides in improving insulin resistance.

[0067] In summary, a high-fat diet successfully induced typical metabolic abnormalities in mice, including weight gain, hepatomegaly, and glucose metabolism disorders. LRP intervention effectively inhibited weight and liver weight gain, and improved impaired glucose tolerance and insulin resistance without affecting food intake. Among the interventions, LRP-H showed the best overall effect in weight control and insulin sensitivity improvement, comparable to the positive control RES and superior to commercially available bitter tea polysaccharide (LRP-C). These results suggest that LRP has stronger bioactivity and application potential in alleviating HFD-induced obesity and glucose metabolism disorders.

[0068] 3.2 LRP alleviates hepatic steatosis and improves histological morphological damage in HFD mice 3.2.1 Experimental Methods Using standardized biochemical assay kits provided by Nanjing Jiancheng Biotechnology Research Institute in China, we measured the serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels in mice, as well as the TG and TC contents in liver tissue, to evaluate the effect of LRP intervention on improving lipid dyslipidemia and liver function damage induced by a high-fat diet (HFD).

[0069] Simultaneously, liver histopathology and lipid deposition analysis were performed. Liver tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5-6 μm). Hematoxylin and eosin (H&E) staining was performed to observe hepatocyte morphology and inflammatory damage. Separately, liver tissue was embedded in OCT, frozen, sectioned, and stained with Oil Red O to assess neutral lipid deposition. The MASLD activity score (MAS) was calculated based on scores for steatosis (0-3 points), lobular inflammation (0-3 points), and hepatocyte ballooning degeneration (0-2 points). All stained sections were scanned and imaged using the SCAN I digital pathology scanning system (Servicebio, Wuhan) for subsequent quantitative analysis.

[0070] 3.2.2 Experimental Results Analysis of blood lipids and liver damage indicators showed (see...) Figures 13-16 Serum TC, TG, ALT, and AST levels in the HFD group were significantly higher than those in the CON group. All doses of LRP, LRP-C, and RES intervention could reduce blood lipids and transaminase levels. Among them, LRP-L and LRP-C had more significant effects on reducing TC and TG, while LRP-H had the most significant effect on improving ALT and AST, with effects comparable to and superior to LRP-C.

[0071] Results of liver tissue lipid content (see) Figures 17-18 The results showed that HFD significantly increased the levels of TC and TG in the liver, while LRP, LRP-C and RES treatments significantly reduced liver lipid deposition, with LRP-H showing the largest reduction, followed by RES and LRP-C.

[0072] Histopathological observation further confirmed the above results (see Figures 19-20HE staining showed that the liver tissue structure in the CON group was intact, while the HFD group showed obvious hepatocellular ballooning degeneration, inflammatory infiltration, and disordered lobular structure. LRP intervention significantly reduced the above pathological damage, with LRP-H showing the most significant improvement. LRP-C also provided some relief, but to a lesser extent than LRP-H. Oil Red O staining showed that the HFD group had a large amount of lipid droplet deposition in the liver tissue, while LRP, LRP-C, and RES treatments all significantly reduced lipid deposition, with LRP-H showing the most significant reduction. MAS scores showed that the HFD group increased to 6-7 points, LRP-C decreased to 4-5 points, LRP-L decreased to 4-5 points, and LRP-H further decreased to 3-4 points, comparable to the RES group.

[0073] In summary, LRP effectively inhibited weight gain and hepatic hypertrophy, improved glucose tolerance and insulin resistance, and reduced dyslipidemia and hepatic lipid deposition in HFD-induced metabolic disorder models. Overall comparisons showed that high-dose LRP was superior to commercially available bitter tea polysaccharide (LRP-C) in terms of weight control, insulin sensitivity, and liver protection, suggesting its greater potential for application in the prevention and treatment of high-fat diet-related metabolic disorders and MASLD.

[0074] 3.3 LRP improves HFD-induced gut microbiota balance 3.3.1 Experimental Methods Colonic contents were collected and rapidly frozen in liquid nitrogen, then stored at -80°C for later use. Following the manufacturer's instructions, microbial genomic DNA was extracted from the samples using the DNeasy Power Soil Kit (Qiagen, Hilden, Germany), and stored at -20°C. PCR amplification was performed using region-specific primers targeting the hypervariable V3-V4 region of the bacterial 16S rRNA gene. The reverse primer contained a specific barcode sequence to distinguish each sample; both forward and reverse primers were ligated with Illumina sequencing adapters. After purification, the PCR products were subjected to paired-end sequencing (PE250) on an Illumina NovaSeq 6000 platform (Illumina, San Diego, USA). Sequencing and preliminary data processing were performed by Shanghai Ouyi Biomedical Technology Co., Ltd.

[0075] Raw sequencing sequences were used for quality control, noise reduction, paired-end sequence assembly, and amplicon variant (ASV) identification via the DADA2 plugin in the QIIME2 platform. Species taxonomic annotation was performed using the q2-feature-classifier module based on the SILVA138 reference database. Alpha diversity indices (Chao1, Shannon, and Simpson) were calculated using Mothur software (version 1.30.1); beta diversity was assessed using principal coordinate analysis (PCoA) of the Bray-Curtis distance matrix. The LEfSe method was used to screen for dominant bacterial groups with significant differences at the phylum and genus levels.

[0076] 3.3.2 Experimental Results To investigate the effects of LRP on the gut microbiota, 16S rRNA sequencing analysis was performed on the colonic contents of mice in the CON, HFD, LRP-L, and LRP-H groups. Alpha diversity indices (Observed species, Chao1, Shannon, and Simpson) showed no significant differences among the groups (see [link to study]. Figures 21-23 In contrast, PCA-based beta diversity analysis showed a clear spatial separation between the HFD group and the LRP intervention group, suggesting that LRP treatment significantly altered the overall gut microbiota structure induced by HFD (see...). Figure 24 ).

[0077] At the phylum level, the dominant phyla in each group were mainly Bacteroidota, Firmicutes, and Desulfobacterota (see...). Figures 25-26 Compared with the CON group, HFD feeding significantly altered their relative abundance; LRP intervention could partially reverse this change, showing an increase in the relative abundance of Firmicutes and a decrease in Bacteroidota, while Actinobacteriota and Desulfobacterota also showed an increasing trend.

[0078] At the genus level, Muribauculaceae, Alistipes, Parabacteroides, and Bacteroides are the dominant bacterial groups (see [link to relevant documentation]). Figures 27-28 HFD feeding significantly reduced the abundance of Muribauculaceae, while LRP treatment restored it in a dose-dependent manner; Alistipes showed a similar trend. Conversely, Bacteroides were significantly elevated in the HFD group and decreased markedly after LRP intervention.

[0079] LEfSe analysis identified 30 bacterial communities with LDA scores greater than 4.0 and significant differences among groups (see [link to analysis]). Figure 29The study included 10 antibodies in the CON group, 7 in the HFD group, 10 in the LRP-L group, and 3 in the LRP-H group. Muribaculaceae, Lachnospiraceae NK4A136 group, and Blautia were enriched in the CON group and partially recovered after LRP intervention; while Tannerellaceae and Bacteroidaceae were mainly enriched in the HFD group. Quantitative analysis showed that HFD feeding significantly reduced the abundance of Muribaculaceae and Lachnospiraceae NK4A136 group, while increasing Bacteroides levels (see [link to study]. Figures 30-39 LRP treatment reversed the above changes, with Muribaculaceae and Blautia showing dose-dependent increases, while Bacteroides decreased significantly. Furthermore, Faecalibaculum, Odoribacter, and Coriobacteriaceae UCG002 were also regulated by LRP.

[0080] At the phylum level, HFD feeding increases the relative abundance of Bacteroidota and decreases Firmicutes, thereby causing an imbalance in the phylum ratio (see...). Figures 40-43 After LRP intervention, this imbalance was improved to some extent, as evidenced by an increase in the proportion of Firmicutes and a relative decrease in Bacteroidota. Simultaneously, the abundance of Actinobacteriota and Verrucomicrobiota also showed an upward trend, with more pronounced changes in the LRP-L group. These results indicate that LRP remodeled the gut microbiota structure in HFD mice by restoring beneficial bacteria and inhibiting potentially harmful bacteria.

[0081] In summary, LRP significantly corrected the gut microbiota imbalance induced by a high-fat diet in HFD mice by systematically reshaping the gut microbiota structure. Its regulatory effects were mainly manifested in restoring beneficial bacteria such as Muribaculaaceae, Lachnospiraceae NK4A136 group, and Blautia, increasing the proportion of dominant phyla such as Firmicutes, and inhibiting the excessive proliferation of potentially harmful bacteria such as Bacteroides, thereby rebuilding a healthier gut microecological balance. This process showed a certain dose-dependent effect, with higher doses of LRP showing more significant effects. The results of this invention indicate that LRP can improve HFD-related metabolic disorders and hepatic lipid deposition through gut microbiota regulation pathways, providing a microecological basis for its application in the prevention and treatment of metabolic liver diseases, and laying the foundation for gut microbiota-targeted intervention strategies and the screening of functional microbiota biomarkers. Future research should combine multi-omics and mechanistic studies to further elucidate the key pathways and targets of LRP-microbiota-host interactions.

Claims

1. Application of bitter tea extract in the preparation of products for the prevention and treatment of non-alcoholic fatty liver disease.

2. The application according to claim 1, characterized in that: The bitter tea extract contained 36.23 ± 1.56% sugar, 46.00 ± 1.77% uronic acid, 1.03 ± 0.02% protein, and 0.07 ± 0.001% polyphenols.

3. The application according to claim 1 or 2, characterized in that: The preparation method of the bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 50-150 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:7-15, boil and extract for 1-5 h, repeat the extraction 1-4 times, combine the 1-4 extracts, filter with a 100-300 mesh sieve, concentrate to 80-150 kg at 35℃-75℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 30℃-60℃ to obtain bitter tea extract A, for use. (2) Dialysis to remove impurities: Take bitter tea extract A and prepare an aqueous solution of 35-65 mg / mL. Centrifuge at 6000-10000 rpm for 5-18 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 0℃-10℃ for 18-30 h. Change the pure water every 2-6 h. After dialysis, take the solution in the bag to obtain bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, add ethanol and soak for 7-16 h, then wash repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix. Decolorize with magnetic stirring at room temperature for 1-8 h. After stirring, centrifuge at 6000-10000 rpm for 1-10 min, take the supernatant, concentrate under reduced pressure at 30℃-80℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 1-5 h and the drying temperature to 40℃ for 8-16 h to freeze dry to obtain bitter tea extract. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 1-5:

1.

4. The application according to claim 3, characterized in that: The preparation method of the bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 80-120 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:8-12, boil and extract for 2-3 h, repeat the extraction 2-3 times, combine the 2-3 extracts, filter with a 150-250 mesh sieve, concentrate to 90-110 kg at 45℃-65℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature pulse at 35℃-55℃ to obtain bitter tea extract A, for use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 40-55 mg / mL, centrifuge at 7000-9000 rpm for 7-13 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 2℃-5℃ for 20-26 h, changing the pure water every 3-5 h. After dialysis, take the solution in the bag to obtain bitter tea extract B, which can be used for later use. (3) Decolorization: Take D101 macroporous resin, add ethanol and soak for 10-14 h, then wash repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix. Decolorize with magnetic stirring at room temperature for 2-6 h. After stirring, centrifuge at 7000-9000 rpm for 2-7 min. Take the supernatant and concentrate it under reduced pressure at 40℃-60℃. Place it in a freeze dryer, set the cold trap temperature to -20℃ for 1-3 h, and the drying temperature to 40℃ for 10-14 h for freeze drying to obtain bitter tea extract. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 2-4:

1.

5. The application according to claim 4, characterized in that: The preparation method of the bitter tea extract is carried out according to the following steps: (1) Water extraction and alcohol precipitation: Take 100 kg of bitter tea leaves, add water, the mass ratio of bitter tea leaves to water is 1:10, boil and extract for 2.5 h, repeat the extraction twice, combine the two extracts, filter with a 200 mesh sieve, concentrate to 100 kg at 55℃, add 95% ethanol to make the final ethanol concentration in the solution 60%, let stand to precipitate, take the precipitate and dry it at a low temperature of 40-55℃ to obtain bitter tea extract A, for later use; (2) Dialysis to remove impurities: Take bitter tea extract A, prepare an aqueous solution of 50 mg / mL, centrifuge at 8000 rpm for 10 min, take the supernatant, transfer it to a dialysis bag with a molecular weight cutoff of 3500 Da, place it in pure water, and dialyze at 4℃ for 24 h. Change the pure water every 4 h. After the dialysis is completed, take the solution in the bag to obtain bitter tea extract B, which is ready for use. (3) Decolorization: Take D101 macroporous resin, soak it in ethanol for 12 h, and then wash it repeatedly with pure water until there is no ethanol smell. Take bitter tea extract B and D101 macroporous adsorption resin and mix them. Decolorize by magnetic stirring at room temperature for 4 h. After stirring, centrifuge at 8000 rpm for 5 min, take the supernatant, concentrate it under reduced pressure at 55℃, and place it in a freeze dryer. Set the cold trap temperature to -20℃ for 2 h and the drying temperature to 40℃ for 12 h to freeze dry, and the bitter tea extract is obtained. The volume ratio of bitter tea extract B to D101 macroporous adsorption resin is 3:

1.

6. The application according to any one of claims 1-5, characterized in that: The products include pharmaceuticals.

7. The application according to claim 6, characterized in that: The preparation method of the product is to combine bitter tea extract with acceptable excipients in the drug, process them according to conventional methods, and make the corresponding drug preparation.

8. The application according to claim 7, characterized in that: The dosage forms of the product include tablets, capsules, granules, decoctions, or pills.

9. A product for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that: The raw materials of the product include the bitter tea extract as described in any one of claims 3-5.

10. A product for the prevention and treatment of non-alcoholic fatty liver disease, characterized in that: The product is mainly prepared from the bitter tea extract according to any one of claims 3-5.

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