A flavored composition of Digeda-4 for treating non-alcoholic fatty liver disease, and its preparation method and application

Abstract

The present invention provides a kind of Digeda-4 flavored composition for treating non-alcoholic fatty liver disease and its preparation method and application. The composition is made of raw materials: rib column flower, gardenia, superbus, picrorhizon, raw liquorice, turmeric powder, mulberry, rhubarb. The composition of the present invention can effectively reduce the weight of rats with non-alcoholic fatty liver disease model, reduce the content of TC, TG, AST, ALT in rat serum, reduce the content of MDA in rat liver, increase the content of SOD, and also increase the protein expression level of PPARα, PPARβ, LXRα in rat liver, and reduce the protein expression level of PPARγ, p-AKT, AKT; it has multi-component, multi-target, multi-channel effects, and can effectively prevent and treat the relevant symptoms of rats with non-alcoholic fatty liver disease model. The composition of the present invention is composed of a variety of raw materials, which can cooperate with each other, and this synergistic effect significantly enhances the therapeutic effect of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to a Digeda-4 flavored composition for treating non-alcoholic fatty liver disease, and a preparation method and application thereof. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress liver disease characterized by fatty degeneration and lipid deposition of hepatocytes in patients without a history of excessive drinking. The disease has a high incidence and is highly harmful. It is an important link in the development of liver fibrosis, cirrhosis, and even hepatocellular carcinoma. As an increasingly serious chronic liver disease in contemporary society, NAFLD has become a major challenge in the field of global public health. In recent years, the incidence of NAFLD has been increasing year by year, and the age of onset has gradually become younger, seriously affecting the health and quality of life of patients.

[0003] Clinically, the pathogenesis of NAFLD is still unclear, but its pathophysiology is believed to be unrelated to alcohol and related to obesity, insulin resistance, oxidative stress and mitochondrial dysfunction, the interaction of cytokines and adipokines, apoptosis, and overgrowth of small intestinal bacteria (see Tian Huiping et al., "Therapeutic Effects of Mongolian Medicine Digeda-4 on Non-alcoholic Fatty Liver Model Rats", Chinese Pharmacology and Clinical Medicine, 2010, pp. 115-116). The "two-hit" theory is now generally accepted. This hypothesis believes that "one-hit" insulin resistance leads to liver fatty degeneration, which in turn leads to fat metabolism disorders, accumulation of TG (triglycerides), TC (total cholesterol), FFA (free fatty acids), etc., and liver damage. The "two-hit" is centered on oxidative stress and lipid peroxidation. The massive increase in reactive oxygen species (ROS) triggered by oxidative stress leads to lipid peroxidation, inflammation, hepatocyte apoptosis and fibrosis.

[0004] At present, the western medicine for the treatment of NAFLD mainly adopts simple lipid-lowering and blood sugar-lowering drugs, such as statins, fibrates, niacin, metformin, pioglitazone, etc., while the pathology of NAFLD is not just about blood sugar and lipid-lowering. Although the treatment with western medicine alone has a certain therapeutic effect, it has a single effect and often produces side effects, which damages liver and kidney function. In recent years, traditional Chinese medicine has done a lot of research work in the prevention and treatment of NAFLD, and has also made certain progress. Traditional Chinese medicine treatment of NAFLD is mainly based on invigorating qi and strengthening spleen, removing dampness and activating blood circulation, etc. However, they all have more or less shortcomings, such as difficulty in obtaining materials, high price, too many medicinal flavors, rough preparation process, slow onset, etc. Therefore, there is an urgent need for a drug for the treatment of non-alcoholic fatty liver disease with good therapeutic effect, no side effects, and fewer medicinal flavors. Summary of the invention

[0005] The purpose of the present invention is to provide a digeda-4 flavored composition for treating non-alcoholic fatty liver disease, and its preparation method and application. The composition, with digeda-4 as the basic formula, is screened through a large number of experiments for flavored drug raw materials and compatibility dosages and preparation methods. Animal experiments have shown that the composition can effectively prevent and treat the relevant symptoms of NAFLD model rats and has a good therapeutic effect on non-alcoholic fatty liver disease.

[0006] Specifically, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides a modified Digeda-4 composition for treating non-alcoholic fatty liver disease, wherein the composition is prepared from the following raw materials in parts by weight: 5-15 parts of scutellaria baicalensis, 5-15 parts of gardenia, 5-15 parts of superbus, 5-15 parts of picrorhizome, 1-10 parts of raw licorice, 5-15 parts of turmeric powder, 3-13 parts of mulberry, and 8-20 parts of rhubarb.

[0008] As an optional manner, in the above composition, the composition is made of the following raw materials in parts by weight: 8-14 parts of flos lycopersicum, 8-14 parts of gardenia, 8-14 parts of superbus, 8-14 parts of picrorhizome, 5-9 parts of raw licorice, 8-14 parts of turmeric powder, 7-12 parts of mulberry, and 10-18 parts of rhubarb.

[0009] As an optional manner, in the above composition, the composition is made of the following raw materials in parts by weight: 13 parts of flos lycopersicum, 13 parts of gardenia, 13 parts of superbus, 13 parts of picrorhizome, 8 parts of raw licorice, 13 parts of turmeric powder, 11 parts of mulberry, and 16 parts of rhubarb.

[0010] In a second aspect, the present invention provides a method for preparing the Digeda-4 flavored composition described in the first aspect, the preparation method comprising the following steps:

[0011] (1) Weigh 5-15 parts of scutellaria baicalensis, 5-15 parts of gardenia, 5-15 parts of superbus, 5-15 parts of picrorhizome, 1-10 parts of raw liquorice, 5-15 parts of turmeric powder, 3-13 parts of mulberry, and 8-20 parts of rhubarb;

[0012] (2) removing impurities from the stigmata, gardenia, superbus, picrorhizome, and rhubarb, and then mixing and ultrafine grinding to prepare a mixed ultrafine powder;

[0013] (3) mixing raw licorice, turmeric powder and mulberries, adding water to perform a first decoction, decoct for 1-2.5 hours, filter to obtain a primary filtrate and filter residue, add water to the filter residue to perform a second decoction, decoct for 1-2.5 hours, filter to obtain a secondary filtrate, combine the primary filtrate and the secondary filtrate, and concentrate to obtain a concentrated solution;

[0014] (4) The mixed ultrafine powder obtained in step (2) is used as a base material, and the concentrated solution obtained in step (3) is sprayed into the mixed ultrafine powder, and the mixed powder is granulated and mixed to obtain the composite particles.

[0015] As an optional manner, in the above preparation method, in step (2), the fineness of the ultrafine powder is 300 mesh.

[0016] As an optional manner, in the above preparation method, in step (3), the time for the first decoction and the second decoction are both 2 hours.

[0017] In a third aspect, the present invention provides use of the composition described in the first aspect or a composition prepared by the preparation method described in the second aspect in preparing a drug for treating non-alcoholic fatty liver disease.

[0018] As an optional mode, in the above application, the drug has at least one of the effects (a) to (e):

[0019] (a) Improve liver pathological symptoms and reduce liver lipid accumulation;

[0020] (b) Improve dyslipidemia and reduce the levels of TC (total cholesterol) and TG (triglycerides) in serum;

[0021] (c) Reduce the lipotoxicity of liver fat accumulation to liver cells and reduce the levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase);

[0022] (d) Reduce oxidative stress damage in the liver;

[0023] (e) Enhanced PPARα (peroxisome proliferator-activated receptor α), PPARβ (peroxisome proliferator-activated receptor β), and LXRα (liver X receptor α) protein expression levels.

[0024] As an optional mode, in the above application, the drug dosage form is granules, tablets, capsules or oral liquid.

[0025] As an optional mode, in the above application, preferably, the pharmaceutical dosage form is a granule.

[0026] The formula analysis of the flavored composition of the present invention is as follows:

[0027] In the prescription, rib column flower and gardenia are the main medicines. Rib column flower tastes bitter and is cold in nature. It enters the liver and gallbladder meridians, and has the effects of clearing away heat and dampness, and detoxifying. Gardenia tastes bitter and is cold in nature. It enters the heart, liver and stomach meridians, and has the effects of purging fire and relieving restlessness, clearing heat and dampness, cooling blood and stopping bleeding; supernumerary biennis, picrorhizon and turmeric are the assistant medicines. Supernumerary biennis tastes bitter and is cold in nature. It enters the heart, small intestine and bladder meridians, and has the effects of clearing heat, promoting diuresis and relieving stranguria. Picrorhizon tastes bitter and is cold in nature. It enters the liver, stomach and large intestine meridians, and has the effects of clearing heat and dampness, purging fire and detoxifying. Turmeric tastes pungent and bitter, is warm in nature, and enters the liver and spleen meridians, and has the effects of promoting blood circulation and qi, and relieving menstruation and pain; rhubarb and mulberry are adjuvants that can purge accumulation, clear heat and purge fire, cool blood and detoxify, promote blood circulation and remove blood stasis, nourish yin and blood, and promote body fluid and moisten dryness; raw licorice nourishes the spleen and replenishes qi, clears heat and detoxifies, and harmonizes the various medicines. The combination of these medicines can clear away heat, remove dampness, promote qi and activate blood circulation.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention uses Digeda-4 as the basic formula, and through a large number of experiments to screen the raw materials of flavored drugs, the dosage of the compatibility, and the preparation method, the Digeda-4 flavored composition of the present invention is obtained. The composition is a multi-component, multi-target, and multi-channel drug, which plays an important role in the treatment of non-alcoholic fatty liver disease. The flavored composition of the present invention is composed of a variety of raw materials. These raw materials have certain pharmacological effects when they are independent, but when they are combined together, these raw materials can synergize with each other to produce a more powerful drug effect. This synergistic effect not only enhances the therapeutic effect of non-alcoholic fatty liver disease, but also reduces the risk of side effects. Secondly, the flavored composition of the present invention not only reduces the content of TC, TG, AST, and ALT in serum, reduces the content of MDA (malondialdehyde) in the liver, and increases the content of SOD (superoxide dismutase), but also increases the expression levels of PPARα, PPARβ, and LXRα proteins in liver tissue, and reduces the expression levels of PPARγ (peroxisome proliferator-activated receptor γ), p-AKT (phosphorylated protein kinase B), and AKT (protein kinase B) proteins; improves liver pathological symptoms and dyslipidemia, and reduces the lipotoxicity of liver fat accumulation to liver cells and the oxidative stress damage of the liver. This multi-target, multi-channel treatment strategy can effectively prevent and treat the related symptoms of NAFLD model rats and has a good therapeutic effect on non-alcoholic fatty liver disease. In addition, no obvious adverse reactions were observed in animal experiments, and the composition has good safety.

[0030] (2) The preparation method of the flavored Digeda-4 composition of the present invention utilizes and optimizes modern preparation technology, adopts ultrafine grinding technology, dynamic decoction technology, and one-step granulation technology, so that the preparation method of the present invention has the advantages of simple preparation process, less destruction of effective ingredients, high efficiency and energy saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0032] Figure 1 The effect of the composition of the present invention on the body weight of rats;

[0033] Figure 2 This is a HE staining (hematoxylin-eosin staining) image of a rat liver section treated with the composition of the present invention;

[0034] Figure 3 This is an Oil Red O staining image of a rat liver section treated with the composition of the present invention;

[0035] Figure 4 This is an analysis diagram of the positive area of ​​Oil Red O staining of rat liver sections after being treated with the composition of the present invention;

[0036] Figure 5 The standard curve regression equations for liver TC, TG, LDL-C (low-density lipoprotein cholesterol), HDL-C (high-density lipoprotein cholesterol), FFA, ALT, AST, and FAS (fatty acids);

[0037] Figure 6 This is a schematic diagram of the contents of TC, TG, LDL-C, HDL-C, FFA, ALT, AST and FAS in the serum of rats after being treated with the composition of the present invention;

[0038] Figure 7 This is a schematic diagram of the content of MDA and SOD in the liver of rats treated with the composition of the present invention;

[0039] Figure 8 This is a graph showing the expression levels of PPARα, PPARβ, PPARγ, AKT, p-AKT, iNOS (inducible nitric oxide synthase), and LXRα proteins in the liver of rats treated with the composition of the present invention;

[0040] Fig. 9 The figure is a comparative bar graph of the expression levels of PPARα, PPARβ, PPARγ, LXRα, iNOS, AKT and p-AKT proteins in the liver of rats treated with the composition of the present invention. DETAILED DESCRIPTION

[0041] The embodiments of the present invention are described in detail below. The embodiments are given to better illustrate the contents of the present invention and are only used to explain the present invention, but should not be understood as limiting the present invention.

[0042] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels. Embodiment 1:

[0043] A modified Digeda-4 composition for treating non-alcoholic fatty liver disease is prepared from the following raw materials in parts by weight: 50g of flos strychnis, 50g of gardenia, 50g of superbus, 50g of picrorhizome, 30g of raw liquorice, 50g of turmeric powder, 45g of mulberry and 60g of rhubarb.

[0044] The preparation method of the composition comprises the following steps:

[0045] (1) Weigh the following ingredients according to the above formula: Flos Corydalis, Gardenia, Dianthus Superbus, Rhizoma Picrorhizoma, Licorice, Turmeric Powder, Mulberry, and Rhubarb;

[0046] (2) removing impurities from the spatholobi, gardenia, superbus, picrorhizome, and rhubarb, and then mixing and ultrafine grinding them to obtain a mixed ultrafine powder, wherein the fineness of the ultrafine powder is 300 meshes;

[0047] (3) mixing raw licorice, turmeric powder and mulberry, adding water to perform a first decoction, decoct for 2 hours, filter to obtain a primary filtrate and filter residue, add water to the filter residue to perform a second decoction, decoct for 2 hours, filter to obtain a secondary filtrate, combine the primary filtrate and the secondary filtrate, and concentrate to obtain a concentrated solution;

[0048] (4) The mixed ultrafine powder obtained in step (2) is used as a base material, and the concentrated solution obtained in step (3) is sprayed into the mixed ultrafine powder, and the mixed powder is granulated and mixed to obtain the composite particles.

[0049] Comparative Example 1:

[0050] A Digeda-4 composition is prepared from the following raw materials in parts by weight: 50g of Corydalis erythrorhizon, 50g of Gardenia jasminoides, 50g of Dianthus superbus, and 50g of Picrorhizon.

[0051] The preparation method of the composition comprises the following steps:

[0052] (1) Weigh the following ingredients according to the above formula:

[0053] (2) removing impurities from the spatholobi, gardenia, superbus and picrorhizome, drying them, mixing them and then ultrafine grinding them to obtain mixed ultrafine powder, wherein the fineness of the ultrafine powder is 300 meshes;

[0054] (3) The mixed ultrafine powder obtained in step (2) is granulated by a wet method and dried to obtain digeda-4 granules. Embodiment 2:

[0055] Pharmacodynamics experiments

[0056] 1. Experimental Materials

[0057] 1.1 Experimental animals

[0058] Wistar male rats, weighing 150-160 g, license number: SCXK (Beijing) 2019-0010, were purchased from Beijing Sibeifu and kept in the animal room on the sixth floor of Jinhe Building, Inner Mongolia Medical University.

[0059] 1.2 Experimental drugs

[0060] Compound methionine choline tablets (batch number: H22024764) were purchased from Tonghua Dongbao Pharmaceutical Co., Ltd.; rib flower (batch number: C23021001), gardenia (batch number: 2209003), superbus (batch number: 29122080), picrorhizome (batch number: 20230510), turmeric powder (batch number: c061230201), rhubarb (batch number: c169220601), mulberry (batch number: 8220104601), and raw licorice (batch number: 8221209403) were all provided by Inner Mongolia Daoran Traditional Chinese Medicine Clinic.

[0061] 1.3 Experimental reagents

[0062] Hematoxylin-eosin stain was purchased from Shanghai Ruchuang Biotechnology Co., Ltd.; Oil Red O stain was purchased from Shanghai Ruchuang Biotechnology Co., Ltd.; ALT kit (JYM0722Ra), TC kit (JYM0689Ra), TG kit (JYM0365Ra), HDL-C kit (JYM0797Ra), LDL-c kit (JYM0795Ra), FFA kit (JYM0208Ra), AST kit (JYM0994Ra), FAS kit (JYM0722Ra), SOD kit (A001-1), MDA kit (A003-1) were all purchased from Wuhan Pure Biological Co., Ltd.; GAPDH (antibody ), batch number: 60004-1-Ig, purchased from Wuhan Sanying Company; PPARα (antibody), batch number: AF5301, PPARβ (antibody), batch number: DF7442, PPARγ (antibody), batch number: AF6284, iNOS (antibody), batch number: AF0119, AKT (antibody), batch number: AF0386, all purchased from Affinity Company; LXRα (antibody), batch number: A3974, p-AKT (antibody), batch number: AP1259, all purchased from Abclonal Company; high-fat feed, model: D12451 (batch number: SCXK (Beijing) 2018-0006), purchased from Xiaoshu Youtai (Beijing) Biotechnology Co., Ltd.

[0063] 2. Experimental Methods

[0064] 2.1 Adaptive feeding of experimental animals

[0065] In order to prevent rat stress, the purchased healthy Wistar rats were adaptively fed so that they could adapt to the new environment. First, 90 male Wistar rats were divided into cages, 5 in each cage, a total of 18 cages, and the cages were placed in a ventilated environment with sufficient light every day. The general light period was from 6 am to 6 pm. During the adaptive feeding process, the rats should be kept quiet and given clean feed and drinking water every day. The time lasted for a week, and the rats were divided into groups after one week.

[0066] 2.2 Establishment of high-fat diet (HFD)-induced nonalcoholic fatty liver disease model in rats

[0067] 90 Wistar rats were randomly divided into a blank group (K) (10 rats) and a modeling group (80 rats). The rats in the blank group were fed with ordinary feed, and the rats in the modeling group were fed with high-fat feed. After 10 weeks of feeding, the rats in the modeling group were significantly overweight compared with the rats in the blank group. Ten rats in the modeling group were randomly selected. After sampling, the liver tissue sections of the rats in the modeling group showed obvious pathological changes, indicating that the modeling was successful. Then 60 rats were randomly selected from the rats with successful modeling and randomly divided into a model group (M), a positive control group (Y), a high-dose group of Digda-4 with added flavor (G), a medium-dose group of Digda-4 with added flavor (Z), a low-dose group of Digda-4 with added flavor (D), and a Digda-4 group (D-4), with 10 rats in each group.

[0068] 2.3 Grouped medication

[0069] During the administration period, the rats in the blank group continued to be fed with ordinary feed, and the rats in the modeling group continued to be fed with high-fat feed.

[0070] Blank group (K) and model group (M): The mice were gavaged with an equal volume of distilled water (1 mL / 100 g) once a day in the morning for 10 weeks.

[0071] Positive control group (Y): Compound methionine choline tablets were administered orally at a dose of 0.0387 mg / kg once a day in the morning for 10 weeks.

[0072] Digeda-4 group (D-4): Digeda-4 granules prepared in Comparative Example 1 were intragastrically administered at a dose of 0.81 g / kg once a day in the morning for 10 weeks.

[0073] Digeda-4 flavored high-dose group (G): Digeda-4 flavored granules prepared in Example 1 were intragastrically administered at a dose of 1.62 g / kg once a day in the morning for 10 weeks.

[0074] Digeda-4 flavored medium dose group (Z): Digeda-4 flavored granules prepared in Example 1 were intragastrically administered at a dose of 0.81 g / kg, once a day in the morning, for 10 weeks.

[0075] Digeda-4 flavored low-dose group (D): Digeda-4 flavored granules prepared in Example 1 were intragastrically administered at a dose of 0.405 g / kg, once a day in the morning, for 10 weeks.

[0076] 2.4 Experimental Materials

[0077] The rats were weighed every 7 days during the modeling and treatment period. After the modeling and treatment, the rats were fasted and anesthetized with 10% chloral hydrate (0.35 mL / 100 g) by intraperitoneal injection. The abdominal cavity was cut open to collect blood from the abdominal aorta, centrifuged (4000 rpm, 15 min), and the serum was stored at -80°C for subsequent testing. The rats were dissected, and the liver tissue was removed. Half of it was fixed in 10% formalin solution, and the other half was frozen and stored in a -80°C refrigerator for subsequent testing.

[0078] 2.5 HE staining

[0079] 1) Sampling: Take fresh liver tissue and immerse it in 10% neutral formalin for more than 24 hours. Take the liver tissue out of the fixative and trim the target liver tissue with a scalpel in a fume hood. Put the trimmed liver tissue and the corresponding label in the dehydration box and mark the specimen code on the specimen bottle. Place it in a container filled with water to slightly clean the fixative on the tissue.

[0080] 2) Dehydration: Place the embedding frame into the basket of the dehydrator and dehydrate the liver tissue with graded alcohols in sequence: 75% alcohol for 2 hours → 85% alcohol for 1 hour → 90% alcohol for 1 hour → 95% alcohol for 40 minutes → anhydrous ethanol I for 30 minutes → anhydrous ethanol II for 30 minutes → alcohol benzene for 10 minutes → xylene I for 5 minutes → xylene II for 5 minutes → wax I for 1 hour → wax II for 1 hour → wax III for 1 hour.

[0081] 3) Embedding: Embed the wax-soaked liver tissue in an embedding machine. First, place the melted wax into the embedding frame. Before the wax solidifies, take the liver tissue out of the dehydration box and place it into the embedding bottom mold and embedding frame according to the requirements of the embedding surface. Cool it in a -20° freezer. After the wax solidifies, take the wax block out of the embedding frame and trim the wax block.

[0082] 4) Slice: Place the trimmed wax block on a paraffin slicer and slice it to a thickness of 3µm. Float the slices on the 42℃ warm water of the slicer to flatten the liver tissue, pick up the tissue with a slide, and bake it in a 60℃ oven. After the water is dried and the wax is melted, take it out and store it at room temperature for later use.

[0083] 5) Dewax the paraffin sections to water: place the sections in xylene I for 20 min → xylene II for 20 min → anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% alcohol for 5 min → 90% alcohol for 5 min → 80% alcohol for 5 min → 70% alcohol for 5 min → wash with distilled water.

[0084] 6) Hematoxylin staining of cell nuclei: Stain sections with hematoxylin for 5 minutes, wash with water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with water, blue with 1% ammonia water, and rinse with running water.

[0085] 7) Eosin staining of cytoplasm: Slice and stain in eosin solution for 5 minutes.

[0086] 8) Dehydration and sealing: Place the slices in 95% alcohol II for 15 min → anhydrous ethanol I for 10 min → anhydrous ethanol II for 10 min → xylene I for 10 min → xylene II for 10 min to dehydrate and make them transparent. Take the slices out of xylene, dry them slightly, and seal them with neutral gum.

[0087] 9) Microscopy and image acquisition and analysis were performed using a scanner (3D Histech, Pannoramic MIDI).

[0088] 2.6 Oil Red O staining

[0089] 1) Rewarming: Take the slices out of the refrigerator and return to room temperature.

[0090] 2) Staining: Fix with 50% ethanol for 30 seconds, change to the special rack for oil red, put in the oil red dye solution (freshly prepared and used) and dye for 8-10 minutes. Be careful not to let the slides dry (the ratio of oil red dye solution to pure water is 3:2).

[0091] 3) Differentiation: Differentiate in 50% ethanol for 1 min, transfer to a small iron rack, and transfer to tap water, taking care not to dry the slices.

[0092] 4) Re-staining: Stain with hematoxylin for 30 seconds, no differentiation, turns blue in running water.

[0093] 5) Sealing: Seal the slides with glycerol gelatin, avoid bubbles and store at 4°C.

[0094] 6) Microscopic examination: The sections were observed and images were collected under a Nikon inverted microscope. Lipid droplets were stained red and cell nuclei were stained blue.

[0095] 2.7 Elisa test

[0096] According to the requirements of the Elisa kit, the contents of TC, TG, LDL-C, HDL-C, FFA, ALT, AST, and FAS in rat serum were detected.

[0097] 1) Place the test kit at room temperature and allow it to equilibrate for 1 hour.

[0098] 2) Dilution and addition of standard samples: Set up 10 standard sample wells on the enzyme-labeled coated plate.

[0099] 3) Sample addition: Set up blank wells (blank control wells do not add samples and enzyme-labeled reagents, and the rest of the steps are the same) and test sample wells. First add 40μL of sample diluent to the test sample wells on the enzyme-labeled plate, and then add 10μL of the test sample (the final sample dilution is 5 times). Add the sample to the bottom of the well of the enzyme-labeled plate, try not to touch the well wall, and gently shake to mix.

[0100] 4) Add enzyme: Add 50 µL of enzyme-labeled reagent to each well, except for the blank well.

[0101] 5) Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes.

[0102] 6) Liquid preparation: Dilute 30 (20 times of 48T) times of concentrated washing solution with 30 (20 times of 48T) times of distilled water and set aside.

[0103] 7) Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times and pat dry.

[0104] 8) Color development: First add 50µL of color developer A to each well, then add 50µL of color developer B, gently shake to mix, and incubate at 37℃ in the dark for 10 minutes.

[0105] 9) Termination: Add 50 µL of stop solution to each well to terminate the reaction (the blue color immediately turns to yellow).

[0106] 10) Determination: Use the blank well as zero setting, and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. The determination should be performed within 15 minutes after adding the stop solution.

[0107] 2.8 Determination of liver tissue oxidation indexes

[0108] According to the requirements of the kit, the SOD and MDA contents in rat liver tissue were detected.

[0109] 1) Sample preparation: Wash rat liver tissue with physiological saline to remove blood and other impurities.

[0110] 2) Preparation of tissue homogenate: The processed liver tissue was weighed and cut into small pieces, physiological saline was added, and the tissue was homogenized using a homogenizer.

[0111] 3) Extraction of MDA and SOD: Centrifuge the homogenized liver tissue and take the supernatant as the test sample. Add the extract to the supernatant to extract MDA and SOD. Mix the extract with the supernatant, shake well, and centrifuge to obtain the MDA and SOD extracts.

[0112] 4) Reagent mixing: Mix all reagents thoroughly to ensure the uniformity and accuracy of the reagents.

[0113] 5) Set up the well plate and mark the control and sample wells on the 96-well plate.

[0114] 6) Sample addition: add rat liver sample to each sample well and add control substance to the control well. The sample addition amount should be accurate.

[0115] 7) Incubate the reaction, cover the plate, and place the plate in a 37°C incubator for incubation. During the incubation process, avoid light and vibration on the plate.

[0116] 8) Washing operation: After the incubation, pour out the liquid in the well plate, fill the well plate with washing solution, let it stand and then pour it out. Repeat the washing step 5 times to ensure that there is no residue in the well.

[0117] 9) Enzyme labeling reagent: According to the instructions of the kit, add an appropriate amount of enzyme labeling reagent to each well. Gently shake the well plate to fully mix the enzyme labeling reagent with the substances in the well.

[0118] 10) Incubate again and place the well plate in a 37°C incubator for incubation.

[0119] 11) Wash again. After the incubation is completed, repeat the washing operation to ensure that there is no residue in the well.

[0120] 12) Color development reaction: add an appropriate amount of color developer to each well, gently shake the plate to fully mix the color developer and the substance in the well, and let it stand at room temperature to allow the reaction to proceed fully.

[0121] 13) Stop the reaction by adding an appropriate amount of stop solution to each well to stop the color development reaction. Gently shake the plate to allow the stop solution to fully mix with the substances in the well.

[0122] 14) Determine the absorbance. Use a microplate reader to measure the absorbance value of each well and calculate the content of MDA and SOD in the sample according to the formula.

[0123] 2.9 Determination of related protein expression levels in liver tissue

[0124] The expression levels of PPARs, AKT, p-AKT, iNOS, and LXRα proteins in liver tissue were determined by Western Blot.

[0125] 1) Protein sample preparation

[0126] Cut the tissue block with scissors, wash the liver tissue block with pre-cooled PBS (phosphate buffer) 2-3 times, remove blood stains, cut into small pieces and place in a homogenizer tube. Add 3-4 small 2mm magnetic beads, add 10 times the volume of tissue RIPA (cell lysis buffer) and place in a homogenizer. Select the program to thoroughly homogenize for 60s, take out the homogenized sample tube, ice bath for 30min, shake every 5min to ensure complete tissue lysis. Centrifuge (12000rpm, 10min), and collect the supernatant.

[0127] 2) Concentration determination

[0128] Take the undenatured protein solution and measure the protein concentration using a BCA protein concentration determination kit. For the specific method, refer to the instructions of the BCA protein concentration determination kit.

[0129] 3) SDS-PAGE electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis)

[0130] (1) Clean the glass plate.

[0131] (2) Place the glass plate in the glue maker, insert wedges to fix it, and check the bottom to prevent glue leakage.

[0132] (3) After adding TEMED (tetramethylethylenediamine), shake the gel immediately. Fill the separation gel to an appropriate height, and then slowly and evenly add pure water into the gap until it is full. When the separation gel solidifies, pour off the water on the top of the gel and absorb the remaining water with absorbent paper.

[0133] (4) Prepare 5% concentrated gel. After adding TEMED, shake the gel immediately and pay attention to bubbles.

[0134] (5) Place the gel casting apparatus in the tank and start spotting. After spotting is complete, cover the electrophoresis tank and start electrophoresis. When the protein is in the stacking gel, use 75V. When you see the bromophenol blue band enter the interface between the stacking gel and the separation gel, increase the voltage to 120V and continue electrophoresis. Stop electrophoresis when the bromophenol blue is 0.5cm from the bottom of the gel casting apparatus.

[0135] 4) Transfer

[0136] (1) Prepare 6 sheets of 7 cm × 9 cm filter paper and a PVDF membrane (polyvinylidene fluoride membrane) cut to 8.5 cm × 5.5 cm using a paper cutter. The PVDF membrane must be activated with methanol before use.

[0137] (2) Place the transfer clamp, two sponge pads, and a small glass test tube in a gel cutting basin filled with transfer solution.

[0138] (3) Open the transfer clamp and lay it flat. Place transfer sponges on both sides and place three layers of filter paper neatly to remove the bubbles.

[0139] (4) Use a gel spatula to cut the separation gel from the edge of the concentrated gel to 0.5 cm below the bromophenol blue, and place it on the filter paper on the negative side of the transfer clip. Slowly and evenly cover the PVDF membrane on the gel from one side. Gently cover the filter paper and sponge on the positive side of the transfer clip on the PVDF membrane. Clamp the transfer clip, put it into the transfer core of the electrophoresis tank with transfer solution, cover the electrophoresis tank lid, place it in an ice basin to cool, adjust the power program to 300mA constant current mode, and start transfer for 1 hour.

[0140] 5) Immune response

[0141] (1) Add 5% BSA (bovine serum albumin) prepared with 0.5% TBST to the antibody incubation tank of the PVDF membrane and shake slowly on a destaining shaker at room temperature for 1 hour.

[0142] (2) Dilute the primary antibody with 5% BSA dissolved in TBST according to the antibody instructions, add it to the corresponding incubation tank, shake slowly, and incubate at 4°C for 16 hours. Recover the primary antibody for later use.

[0143] (3) Dilute the secondary antibody with TBST according to the instructions, add it to the incubation tank correctly, and incubate at room temperature for 30 minutes. Rinse the PVDF membrane incubated with secondary antibody quickly with TBST three times.

[0144] 6) Chemiluminescence

[0145] Mix the ECLIA (electrochemiluminescence immunoassay) reagent and ECL (enhanced chemiluminescence reagent) in equal volumes in a centrifuge tube, place it on the workbench of the luminescence imaging system, gently place the workbench in the darkroom of the luminescence imaging system, adjust the lens, and select the appropriate exposure time.

[0146] 7) Gel image analysis

[0147] Save the exposed images, organize them with PhotoShop, and use the software processing system to analyze the optical density value of the target band.

[0148] 8) WB results and analysis

[0149] The grayscale values ​​of the AlphaEaseFC analysis were imported into Graphpadprism 10 software to make a bar graph and perform grayscale analysis.

[0150] 2.10 Data Statistical Processing

[0151] SPSS 26.0 statistical software was used for data analysis. The data were expressed as X±S. The normal distribution was judged by one-way analysis of variance (ANOVA). If the data were normal, the rank sum test was used. If the data were not normal, the LSD test was used. If the data were equal, the Dunnetts test was used. When P < 0.05, there was a significant difference, which was statistically significant.

[0152] 3. Experimental Results

[0153] 3.1 Effect of the composition on rat body weight

[0154] The body weight of rats was measured every 7 days during the modeling and drug administration phases, and the changes were recorded (the results are shown in Tables 1 and Figure 1 The body weight index can effectively reflect the weight gain changes of rats after feeding high-fat diet. From Table 1 and Figure 1 It can be seen that in the first week of modeling, the body weight of rats in the model group showed a significant upward trend compared with the blank group ( * p <0.05 or ** p <0.01); In the first week of administration, the body weight of rats in the medium and low dose groups of Digda-4 plus flavor was significantly reduced compared with the model group ( ∆ p <0.05 or ∆∆ p <0.01); after administration, the body weight of rats in the Digda-4 supplemented high, medium and low dose groups was significantly reduced compared with the model group ( ∆ p <0.05 or ∆∆ p <0.01); and the body weights of rats in the Digedada-4 supplementary high, medium and low dose groups were significantly lower than those of rats in the positive control group and the Digedada-4 group.

[0155] Table 1: Effect of the composition of the present invention on body weight of rats (n=10)

[0156] Grouping K M Y G Z D D-4 The first week of modeling 182.1±10.31 <![CDATA[197.9±14.65 ** ]]> <![CDATA[196.5±8.34 ** ]]> <![CDATA[199.2±8.77 ** ]]> <![CDATA[201.84±12.68 ** ]]> <![CDATA[200.87±8.80 ** ]]> <![CDATA[198.4±11.07 ** ]]> First week of medication 358.8±45.21 <![CDATA[570.7±68.70 ** ]]> 560.8±38.56 548.9±60.51 <![CDATA[521.96±25.81 ∆ ]]> <![CDATA[519.48±51.49 ∆ ]]> 547.4±48.26 Last dose 445.1±41.20 <![CDATA[572.33±62.81 ** ]]> 559.62±59.41 <![CDATA[507.4±50.16 ∆∆ ]]> <![CDATA[518.97±46.66 ∆ ]]> <![CDATA[515.45±35.31 ∆ ]]> 535.6±46.47

[0157] 3.2 HE staining results of liver sections

[0158] HE staining results Figure 2 As shown. Figure 2It can be seen that compared with the overall structure of liver tissue in the blank group, the overall structure of liver tissue in the model group, positive control group, high-dose digda-4 plus flavor group, medium-dose digda-4 plus flavor group, low-dose digda-4 plus flavor group, and digda-4 group was abnormal to varying degrees, with loose liver cell structure, and a large number of liver cells with obvious fatty degeneration (round vacuoles) in the tissue. Compared with the overall structure of liver tissue in the model group, the overall abnormality of liver tissue in the positive control group, high-dose digda-4 plus flavor group, medium-dose digda-4 plus flavor group, and low-dose digda-4 plus flavor group was milder, with loose liver cell structure, and obvious fatty degeneration (round vacuoles) in some liver cells in the tissue, but the overall situation was improved.

[0159] 3.3 Oil Red O staining results of liver sections

[0160] Oil red O staining results Figure 3 The results of Oil Red O staining positive area analysis are shown in Figure 4 As shown, from Figure 4 It can be seen that the blank group was 0%, the model group was 28%, the positive control group was 15%, the high-dose group of Digda-4 plus flavor was 6%, the medium-dose group of Digda-4 plus flavor was 11%, the low-dose group of Digda-4 plus flavor was 18%, and the Digda-4 group was 22%. This shows that compared with the blank group, the positive area of ​​the model group increased greatly. Compared with the model group, the positive area of ​​the positive control group, the high-dose group of Digda-4 plus flavor, the medium-dose group of Digda-4 plus flavor, and the low-dose group of Digda-4 plus flavor were all significantly decreased. Among the drug-treated groups, the high-dose group of Digda-4 plus flavor had the best effect. The results showed that after drug treatment, the number of liver fat droplets in rats in the high, medium and low-dose groups of Digda-4 plus flavor was significantly reduced, and the liver fat droplets were improved.

[0161] 3.4 Elisa test results

[0162] The results of the detection of TC, TG, LDL-C, HDL-C, FFA, ALT, AST, and FAS in rat serum are shown in Table 2. Figure 6 As shown, the regression equation of the standard curve is drawn as Figure 5 As shown in Table 2, Figure 6 It can be seen that compared with the blank group, the HDL-C content in the serum of the rats in the model group was significantly reduced, and the TC, TG, LDL-C, AST, ALT, FFA, and FAS contents were significantly increased, and the differences were statistically significant ( * p <0.05 or ** p <0.01). Compared with the model group, the serum ALT and AST levels of rats in the positive control group were significantly reduced, with significant differences ( ∆ p<0.05), the levels of serum TC, TG, ALT, AST, and FAS in the high-dose group of Digeda-4 were significantly reduced, and the level of HDL-C was significantly increased, with significant differences ( ∆ p <0.05 or ∆∆ p <0.01); the levels of serum TC, TG, LDL-C, ALT, AST, FFA, and FAS in rats in the medium-dose group of Digeda-4 were significantly reduced, with significant differences ( ∆ p <0.05or( ∆∆ p <0.01); the levels of serum TC, TG, ALT, AST and FFA in rats in the low-dose group of Digeda-4 were significantly reduced, and the difference was statistically significant ( ∆ p <0.05 or ∆∆ p <0.01).

[0163] Table 2: Effects of the composition of the present invention on the levels of TC, TG, LDL-C, HDL-C, FFA, ALT, AST and FAS in rat serum (n=6)

[0164] Grouping K M Y G Z D D-4 TC 7.29±3.12 <![CDATA[17.04±2.19 ** ]]> 11.26±3.73 <![CDATA[10.53±4.27 ∆ ]]> <![CDATA[7.75±4.01 ∆∆ ]]> <![CDATA[10.52±3.23 ∆ ]]> 14.49±3.49 TG 36.67±11.25 <![CDATA[75.60±11.85 ** ]]> 64.46±9.71 <![CDATA[47.49±13.63 ∆ ]]> <![CDATA[38.01±8.86 ∆∆ ]]> <![CDATA[49.93±11.37 ∆ ]]> 62.64±7.53 LDL-C 18.49±3.11 <![CDATA[32.94±6.97 * ]]> 31.57±5.43 25.99±4.20 <![CDATA[20.18±3.03 ∆ ]]> 34.24±6.26 26.74±5.19 HDL-C 31.60±2.82 <![CDATA[16.08±3.91 ** ]]> 24.56±4.37 <![CDATA[28.88±3.33 ∆ ]]> 24.14±3.30 24.12±3.87 17.50±5.43 ALT 486.31±98.25 <![CDATA[806.95±43.54 * ]]> 650.24±62.47 <![CDATA[546.60±125.64 ∆ ]]> <![CDATA[550.30±118.81 ∆ ]]> <![CDATA[522.20±84.45 ∆∆ ]]> 683.06±96.46 AST 25.55±3.49 <![CDATA[46.45±4.35 ** ]]> 31.05±5.66 <![CDATA[28.84±4.93 ∆∆ ]]> <![CDATA[32.70±3.00 ∆ ]]> <![CDATA[25.54±4.44 ∆∆ ]]> 38.87±6.62 FFA 26.18±6.14 <![CDATA[47.61±5.80 ** ]]> 44.00±5.28 35.43±7.85 <![CDATA[33.51±6.53 ∆ ]]> <![CDATA[28.21±7.75 ∆∆ ]]> 41.18±9.12 FAS 40.50±2.70 <![CDATA[70.66±6.29 ** ]]> 55.70±15.74 <![CDATA[44.60±3.35 ∆∆ ]]> <![CDATA[42.40±6.15 ∆∆ ]]> 58.29±9.95 57.66±10.03

[0165] 3.5 Results of liver tissue oxidation index measurements

[0166] The results of SOD and MDA content detection in rat liver tissue are shown in Table 3. Figure 7 As shown in Table 3, Figure 7 It can be seen that compared with the blank group, the SOD content in the liver of the rats in the model group was significantly reduced, and the MDA content was significantly increased, and the difference was statistically significant ( * p <0.05 or ** p <0.01). Compared with the model group, the MDA content in the rat liver of the high-dose and medium-dose Digda-4 groups was significantly reduced, and the difference was statistically significant ( ∆ p <0.05 or ∆∆ p <0.01); the SOD content in the liver of rats in the high-dose group of Digeda-4 was significantly increased, and the difference was statistically significant ( ∆ p <0.05).

[0167] Table 3: Effect of the composition of the present invention on the content of SOD and MDA in rat liver (n=6)

[0168] Grouping K M Y G Z D D-4 SOD 238.24±40.85 <![CDATA[94.11±44.68 ** ]]> 170.66±54.32 <![CDATA[184.53±53.75 ∆ ]]> 161.69±41.37 145.08±58.74 114.96±49.93 MDA 11.51±5.43 <![CDATA[30.92±6.26 ** ]]> 19.23±4.35 <![CDATA[13.16±4.29 ∆∆ ]]> <![CDATA[15.95±5.74 ∆ ]]> 22.35±6.38 20.10±3.12

[0169] 3.6 Results of determination of related protein expression levels in liver tissue

[0170] The results of Western Blot assay are shown in Table 4. Figure 8 , Fig. 9 As shown in Table 4, Figure 8 , Fig. 9 It can be seen that compared with the blank group, the expression levels of PPARα, PPARβ, and LXRα proteins in the liver tissue of the rats in the model group were significantly decreased; the expression levels of PPARγ, AKT, P-AKT, and iNOS proteins were significantly increased, and the differences were statistically significant ( * p <0.05 or ** p <0.01). Compared with the model group, the expression levels of PPARα, PPARβ, and LXRα proteins in the liver tissue of rats in the positive control group were significantly increased, and the expression levels of PPARγ, AKT, p-AKT, and iNOS proteins were significantly decreased, and the differences were statistically significant ( ∆ p <0.05 or ∆∆ p <0.01); in the rat liver tissue of the high-dose group of Digeda-4 plus flavor, the expression levels of PPARα, PPARβ, and LXRα proteins were significantly increased, and the expression levels of PPARγ, iNOS, p-AKT, and AKT proteins were significantly decreased, and the differences were statistically significant ( ∆ p <0.05 or ∆∆ p <0.01); the expression levels of PPARα and PPARβ proteins in the liver tissue of rats in the medium-dose group of Digeda-4 were significantly increased, and the expression levels of PPARγ, p-AKT, and AKT proteins were significantly decreased, and the differences were statistically significant ( ∆ p <0.05 or ∆∆ p <0.01); the expression level of PPARα protein in the liver tissue of rats in the low-dose group of Digeda-4 plus flavor was significantly increased, and the expression levels of PPARγ, p-AKT, AKT and iNOS proteins were significantly decreased, and the differences were statistically significant ( ∆ p <0.05 or ∆∆ p <0.01).

[0171] Table 4: Effects of the composition of the present invention on the expression levels of PPARα, PPARβ, PPARγ, LXRα, AKT, iNOS, and p-AKT proteins in rat liver (n=3)

[0172] Grouping K M Y G Z D D-4 PPARα 2.29±0.62 <![CDATA[0.42±0.06 * ]]> <![CDATA[1.48±0.27 ∆ ]]> <![CDATA[1.31±0.25 ∆∆ ]]> <![CDATA[1.75±0.36 ∆∆ ]]> <![CDATA[1.11±0.37 ∆ ]]> 0.76±0.20 PPARβ 0.48±0.09 <![CDATA[0.13±0.00 ** ]]> 0.19±0.03 <![CDATA[0.25±0.050 ∆ ]]> <![CDATA[0.40±0.06 ∆∆ ]]> 0.22±0.04 0.13±0.01 PPARγ 0.32±0.07 <![CDATA[1.99±0.41 ** ]]> 0.65±0.12 <![CDATA[1.02±0.19 ∆ ]]> <![CDATA[0.81±0.13 ∆ ]]> <![CDATA[1.03±0.22 ∆ ]]> 1.46±0.28 LXRα 0.92±0.32 <![CDATA[0.16±0.04 * ]]> 0.91±0.38 <![CDATA[0.98±0.38 ∆ ]]> 0.74±0.32 0.63±0.29 0.56±0.25 AKT 0.54±0.09 <![CDATA[1.77±0.45 ** ]]> <![CDATA[0.74±0.15 ∆∆ ]]> <![CDATA[0.91±0.21 ∆∆ ]]> <![CDATA[1.11±0.24 ∆ ]]> <![CDATA[0.78±0.17 ∆∆ ]]> 1.62±0.35 p-AKT 0.19±0.12 <![CDATA[1.36±0.70 * ]]> <![CDATA[0.36±0.26 ∆ ]]> <![CDATA[0.43±0.31 ∆ ]]> <![CDATA[0.48±0.40 ∆ ]]> <![CDATA[0.53±0.39 ∆ ]]> 1.02±0.63 iNOS 0.38±0.04 <![CDATA[2.33±0.15 ** ]]> 0.76±0.17 <![CDATA[1.62±0.31 ∆∆ ]]> 1.27±0.32 <![CDATA[1.04±0.20 ∆∆ ]]> 1.80±0.30

[0173] In summary, the Digeda-4 flavored composition of the present invention can effectively reduce the body weight of rats with non-alcoholic fatty liver disease model, reduce the content of TC, TG, AST, and ALT in rat serum, reduce the content of MDA in rat liver, increase the content of SOD, and also increase the expression levels of PPARα, PPARβ, and LXRα proteins in rat liver tissue, and reduce the expression levels of PPARγ, p-AKT, and AKT proteins; it has the effects of multiple components, multiple targets, and multiple channels, and can effectively prevent and treat the relevant symptoms of rats with non-alcoholic fatty liver disease model. The Digeda-4 flavored composition of the present invention is composed of a variety of raw materials, which can cooperate with each other, and this synergistic effect significantly enhances the therapeutic effect of non-alcoholic fatty liver disease. In addition, in animal experiments, no obvious adverse reactions were observed, and the composition has good safety.

[0174] Obviously, the above embodiments are only examples for clearly explaining the present invention, and are not intended to limit the implementation methods of the present invention. Those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A Digeda-4 flavored composition for treating non-alcoholic fatty liver disease, characterized in that: The composition is prepared from the following raw materials in parts by weight: 5-15 parts of flos lycopersicum, 5-15 parts of gardenia, 5-15 parts of superbus, 5-15 parts of picrorhizome, 1-10 parts of raw liquorice, 5-15 parts of turmeric powder, 3-13 parts of mulberry and 8-20 parts of rhubarb.

2. The composition according to claim 1, characterized in that The composition is prepared from the following raw materials in parts by weight: 8-14 parts of flos lycopersicum, 8-14 parts of gardenia, 8-14 parts of superbus, 8-14 parts of picrophora, 5-9 parts of raw liquorice, 8-14 parts of turmeric powder, 7-12 parts of mulberry and 10-18 parts of rhubarb.

3. The composition according to claim 2, characterized in that The composition is prepared from the following raw materials in parts by weight: 13 parts of flos scabra, 13 parts of gardenia, 13 parts of superbus, 13 parts of picrophora, 8 parts of raw liquorice, 13 parts of turmeric powder, 11 parts of mulberry and 16 parts of rhubarb.

4. The method for preparing the Digeda-4 flavored composition according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) Weigh 5-15 parts of scutellaria baicalensis, 5-15 parts of gardenia, 5-15 parts of superbus, 5-15 parts of picrorhizome, 1-10 parts of raw liquorice, 5-15 parts of turmeric powder, 3-13 parts of mulberry, and 8-20 parts of rhubarb; (2) removing impurities from the stigmata, gardenia, superbus, picrorhizome, and rhubarb, and then mixing and ultrafine grinding to prepare a mixed ultrafine powder; (3) mixing raw licorice, turmeric powder and mulberries, adding water to perform a first decoction, decoct for 1-2.5 hours, filter to obtain a primary filtrate and filter residue, add water to the filter residue to perform a second decoction, decoct for 1-2.5 hours, filter to obtain a secondary filtrate, combine the primary filtrate and the secondary filtrate, and concentrate to obtain a concentrated solution; (4) The mixed ultrafine powder obtained in step (2) is used as a base material, and the concentrated solution obtained in step (3) is sprayed into the mixed ultrafine powder, and the mixed powder is granulated and mixed to obtain the composite particles.

5. The preparation method according to claim 4, characterized in that: In step (2), the fineness of the ultrafine powder is 300 mesh.

6. The preparation method according to claim 4 or claim 5, characterized in that: In step (3), the first decoction and the second decoction are both performed for 2 hours.

7. Use of the composition according to any one of claims 1 to 3 or the composition prepared by the preparation method according to any one of claims 4 to 6 in the preparation of a medicament for treating non-alcoholic fatty liver disease.

8. The use according to claim 7, characterized in that: The drug has at least one of the following effects: (a) Improve liver pathological symptoms and reduce liver lipid accumulation; (b) Improve dyslipidemia and reduce the levels of total cholesterol (TC) and triglycerides (TG) in serum; (c) Reduce the lipotoxicity of liver fat accumulation on liver cells and reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST); (d) Reduce oxidative stress damage in the liver; (e) Enhanced the expression levels of peroxisome proliferator-activated receptor α (PPARα), peroxisome proliferator-activated receptor β (PPARβ), and liver X receptor α (LXRα) proteins.

9. The use according to claim 7 or claim 8, characterized in that: The drug dosage form is granules, tablets, capsules or oral liquid.

10. The use according to claim 9, characterized in that: The pharmaceutical dosage form is granules.

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