Use of wuling formulation in the preparation of drug for preventing or treating non-alcoholic fatty liver disease

Abstract

The use of a Wuling formulation in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease. Effective medicine components of the Wuling formulation comprise radix bupleuri, glossy ganoderma, radix salviae miltiorrhizae and fructus schisandrae chinensis. Said formulation has simple components, but the four medicines are synergistic and play an effective role in preventing or treating non-alcoholic fatty liver disease.

Description

Application of a Wuling preparation in preparing a medicine for preventing or treating non-alcoholic fatty liver disease

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310919939.7 filed on July 25, 2023, entitled “Application of a Wuling preparation in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease” and Chinese patent application No. 202410263883.9 filed on March 7, 2024, entitled “Application of a Wuling preparation in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease”. The entire contents of the above two applications are incorporated herein by reference. Technical Field

[0003] The present application relates to the field of traditional Chinese medicine, and specifically to the use of a Wuling preparation in the preparation of a medicine for preventing or treating non-alcoholic fatty liver disease. Background Art

[0004] Fatty liver is characterized by excessive lipid deposition in hepatocytes. When intracellular lipid accumulation exceeds 5% of the wet liver weight, or when histologically, more than one-third of the hepatocytes per unit area are fatty, it is considered fatty liver. Non-alcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome characterized by diffuse macrovesicular fatty changes in the hepatocytes, caused by factors other than alcohol and other clear liver-damaging factors. Intrahepatic lipid accumulation is the primary cause of NAFLD, and its pathological manifestations include vacuolar steatosis of hepatocytes, intralobular inflammation and hepatocyte necrosis, and ballooning. With the global prevalence of obesity and metabolic syndrome increasing dramatically, NAFLD has become the most common chronic metabolic liver disease worldwide and the leading chronic liver disease in my country.

[0005] Non-alcoholic fatty liver disease (NAFLD) is one of the most common precursors to chronic liver diseases (such as liver fibrosis and liver cancer) and metabolic disorders (such as obesity, type 2 diabetes, and atherosclerosis). Patients with NAFLD cannot recover from this condition; instead, the disease progresses to non-alcoholic steatohepatitis (NASH), hepatocellular fibrosis, and cirrhosis. As a key component of metabolic syndrome, NAFLD is closely associated with cardiovascular and cerebrovascular diseases, severely impacting people's health and quality of life, and placing a heavy burden on society.

[0006] Currently, the treatment of fatty liver disease still focuses on eliminating the cause, actively treating the underlying disease, and maintaining a balanced diet, with medications serving only as an adjunct. Currently, the drugs used clinically to treat fatty liver disease are primarily categorized as lipid-lowering drugs, liver-protecting and lipid-lowering drugs, and traditional Chinese medicines. However, these drugs have complex ingredients and complex preparation processes.

[0007] Summary of the Invention

[0008] The present application provides a use of a Wuling preparation in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

[0009] A first aspect of the present application provides a use of a Wuling preparation in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

[0010] In some embodiments, the Wuling preparation includes, by weight: 200-400 parts of Bupleurum chinense, 100-200 parts of Ganoderma lucidum, 200-350 parts of Salvia miltiorrhiza, and 250-350 parts of Schisandra chinensis.

[0011] In some embodiments, the Wuling preparation includes, by weight: 300-350 parts of Bupleurum, 150-180 parts of Ganoderma Lucidum, 300-350 parts of Salvia miltiorrhiza, and 300-350 parts of Schisandra chinensis.

[0012] In some embodiments, the Wuling preparation includes, by weight: 342 parts of Bupleurum chinense, 173 parts of Ganoderma lucidum, 342 parts of Salvia miltiorrhiza, and 342 parts of Schisandra chinensis.

[0013] In some embodiments, the dosage form of the Wuling preparation is a decoction, pill, oral solution, tablet, capsule, granule, paste or powder.

[0014] In some embodiments, the Wuling preparation is selected from any one of Wuling capsules, Wuling pills, and Wuling powder.

[0015] In some embodiments, the Wuling preparation is administered at a dosage of 3 g to 6 g per time for adults, and is administered three times a day.

[0016] The active ingredients of Wuling preparation include Bupleurum, Ganoderma lucidum, Salvia miltiorrhiza and Schisandra chinensis. The ingredients are simple, but the four herbs work synergistically to play an effective role in preventing or treating non-alcoholic fatty liver disease.

[0017] Figures in the specification

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on the drawings without creative work.

[0019] Figure 1 shows a typical graph of fat staining intensity in zebrafish liver after treatment with Wuling Capsules in Example 1 of the present application, where the yellow dotted box represents the liver.

[0020] Figure 2 shows a typical diagram of the zebrafish liver tissue structure after treatment with Wuling Capsules in Example 1 of the present application, wherein the yellow dotted box is the analysis site zebrafish liver, the red arrow points to hepatocyte enlargement, and the black arrow points to fatty vacuolar degeneration. DETAILED DESCRIPTION

[0021] The following embodiments of the present invention are further described in detail with reference to the following examples. The detailed description of the following examples is used to illustrate the principles of the present invention, but is not intended to limit the scope of the present invention, that is, the present invention is not limited to the described examples.

[0022] As analyzed in the background of this application, while some traditional Chinese medicine compositions for treating non-alcoholic fatty liver disease exist in the prior art, these compositions are complex. Furthermore, the applicant has studied currently available drugs that are effective in treating chronic hepatitis, hepatitis B, cirrhosis, hepatitis B liver fibrosis, and compensated cirrhosis. The applicant has found that drugs effective for these conditions are not effective for treating non-alcoholic fatty liver disease. For example, Compound Biejiaruangan Tablets, while effective for these conditions, are ineffective for non-alcoholic fatty liver disease.

[0023] In one embodiment of the present application, a Wuling preparation is provided for use in preparing a medicament for preventing or treating non-alcoholic fatty liver disease.

[0024] The active ingredients of the Wuling preparation include Bupleurum chinense, Ganoderma lucidum, Salvia miltiorrhiza, and Schisandra chinensis. Despite its simple composition, these four herbs work synergistically to effectively prevent or treat non-alcoholic fatty liver disease. Bupleurum chinense, with its pungent and bitter properties, regulates liver qi and relieves liver depression, making it the main herb. Ganoderma lucidum, with its sweet and neutral properties, invigorates qi and strengthens the spleen, tonifies the middle and strengthens the body's health. Salvia miltiorrhiza activates blood circulation, removes stasis, promotes menstruation, and relieves pain, making it the auxiliary herb. Schisandra chinensis, with its sweet and sour properties, nourishes qi and yin, and complements Ganoderma lucidum's qi-tonifying and middle-tonifying effects, calming the mind and alleviating depression. It also prevents Bupleurum chinense's dispersing and ascending properties from depleting qi and damaging yin, protecting the liver and lowering enzymes, serving as an adjuvant. These herbs, when used together, treat both the liver and spleen, regulating qi and blood, and exerting both astringent and dispersing effects. The combination of these medicines can relieve liver depression, invigorate qi and strengthen spleen, and promote blood circulation and remove blood stasis. It can significantly improve the symptoms of non-alcoholic fatty liver disease such as liver pain, abdominal distension, fatigue, loss of appetite, etc., and reduce blood biochemical indicators.

[0025] To further improve the synergistic effect of the four herbs, in some embodiments of the present application, the Wuling preparation preferably includes, by weight, 200-400 parts of Bupleurum, 100-200 parts of Ganoderma, 200-350 parts of Salvia miltiorrhiza, and 250-350 parts of Schisandra chinensis. More preferably, by weight, the Wuling preparation includes, by weight, 300-350 parts of Bupleurum, 150-180 parts of Ganoderma, 300-350 parts of Salvia miltiorrhiza, and 300-350 parts of Schisandra chinensis. Even more preferably, by weight, the Wuling preparation includes, by weight, 342 parts of Bupleurum, 173 parts of Ganoderma, 342 parts of Salvia miltiorrhiza, and 342 parts of Schisandra chinensis.

[0026] The Wuling preparation of the present application can be used in any conventional dosage form. In some embodiments, the dosage form of the Wuling preparation is a decoction, pill, oral liquid, tablet, capsule, granule, paste, or powder. The preparation processes of the above dosage forms are relatively mature, which facilitates the use of the Wuling preparation in the preparation of a medicament for preventing or treating non-alcoholic fatty liver disease.

[0027] In some embodiments, the Wuling preparation is selected from any one of Wuling Capsules, Wuling Pills, and Wuling Powder. Each of the above preparations is an existing pharmaceutical preparation, which is more conducive to the implementation of the application of this application.

[0028] The inventors of this application have repeatedly demonstrated through experiments that the following formula was obtained. The equivalent theoretical human dose was calculated using the effective safety concentration of zebrafish in this application using the following formula:

[0029] Human (g / time) = [zebrafish concentration (μg / mL) × 6 / 100] × 2.

[0030] Based on the above conversion, in some embodiments, the Wuling preparation is administered at a dosage of 3g-6g per dose for adults, and is administered three times per day. The above dosage has a significant therapeutic effect on non-alcoholic fatty liver disease and has minimal side effects on the human body.

[0031] The following examples and comparative examples will further illustrate the beneficial effects of the present application, but the scope of the present invention is not limited to these examples.

[0032] Example 1

[0033] The Wuling capsules used were provided by Tsinghua Deren Xi'an Xingfu Pharmaceutical Co., Ltd.

[0034] 1. Experimental Principles and Methods

[0035] Principle: Zebrafish and humans share a high degree of similarity in liver structure, function, and genetics. Besides immune Kupffer cells, zebrafish possess other cell types found in the human liver and share common functions, including bile secretion, glycogen and lipid storage, insulin response, xenobiotic and ammonia metabolism, and secretion of serum proteins such as complement and coagulation factors, transferrin, and albumin-like proteins. Zebrafish liver function is fully developed within five days of fertilization, allowing for full assessment of liver function. Zebrafish are an important model for studying liver diseases. High-sugar, high-fat diets often provide zebrafish with more energy than they need. This excess energy is stored as fat in the liver, leading to a continuous increase in liver fat content, inducing hepatic steatosis, and ultimately, fatty liver disease.

[0036] Sample preparation information:

[0037] Wuling Capsules, the solvent is standard dilution water.

[0038] Positive control: Atorvastatin calcium tablets (hereinafter referred to as atorvastatin calcium), white tablets, batch number FR7909, Pfizer Pharmaceuticals Co., Ltd., solvent is DMSO.

[0039] Experimental animals:

[0040] Zebrafish were reared in aquaculture water at 28°C (water quality: 200 mg of instant sea salt was added to each liter of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / L CaCO3). They were bred and provided by our company's fish farming center. The experimental animal use license number is: SYXK(Zhejiang)2022-0004. The breeding and management of zebrafish complies with the requirements of the international AAALAC accreditation (certification number: 001458) and the IACUC ethics review number is: IACUC-2023-7689-01.

[0041] Instruments, consumables and reagents:

[0042] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, America); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China); double-person single-sided clean bench (SW-CJ-215KS, Shanghai Sujing Industrial Co., Ltd., China); slicer (KD2258, Jinhua Kedi Medical Equipment Co., Ltd., China); biological microscope (CX31, OLYMPUS, Japan).

[0043] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); methylcellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); anhydrous ethanol (batch number 20230329, Sinopharm Chemical Reagent Co., Ltd., China); Oil Red O (batch number SHBN4926, Sigma, USA); 1,2-propylene glycol (batch number 20211117, Sinopharm Chemical Reagent Co., Ltd., China); 4% tissue cell fixative (batch number 20221014, Beijing Solebold Technology Co., Ltd., China); xylene (batch number C14165111, Shanghai MacLean Biochemical Technology Co., Ltd., China); Co., Ltd., China); PBS buffer (Batch No. 70115000, Biosharp, China); Mayer's hematoxylin staining solution (Batch No. 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); eosin staining solution (Batch No. 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); neutral gum (Batch No. 330A021, Solarbio, China); high-efficiency section paraffin (melting point 54-56°C, Batch No. 20201020, Shanghai Huayong Paraffin Co., Ltd., China); high-efficiency section paraffin (melting point 62-64°C, Batch No. 20210828, Shanghai Huayong Paraffin Co., Ltd., China).

[0044] The experimental method is as follows:

[0045] (1) Determine the MTC of Wuling Capsule

[0046] AB strain zebrafish with melanin allele mutations at 5 days post-fertilization (dpf) were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker. Five test concentration groups were set up, and samples were added to microtiter plates / beakers in solution. A normal control group and a model control group were also set up, for a total of seven experimental groups, with a capacity of 25 mL per beaker. The test concentration group was given Wuling Capsules (concentrations shown in Table 1) in water. Except for the normal control group, the remaining experimental groups were given a high-sugar, high-fat diet in water to establish a zebrafish diet-induced fatty liver model (specifically, a 1.5 mg / mL egg yolk powder solution was given in water during the day and a 30 mg / mL glucose solution was given in water at night to establish a zebrafish diet-induced fatty liver model. After two days of treatment at 28°C, the number of zebrafish deaths and toxicity in each experimental group were counted to determine the MTC of the samples. The results are recorded in Table 1.

[0047] Table 1 Results of experimental study on the concentration of Wuling Capsule for auxiliary protection of food-induced fatty liver (n=30)

[0048] It can be seen that the auxiliary protective effect of Wuling Capsule on food-induced fatty liver is 50.0μg / mL in zebrafish.

[0049] (2) Degree of liver fatty degeneration

[0050] 5-day-old zebrafish of the Albino strain with a melanin allele mutation were randomly selected and placed in beakers, with 30 zebrafish per beaker. Wuling capsules (concentrations shown in Table 2) were administered in water, and samples were added to the beakers as solutions. A normal control group, a model control group, a positive control (positive control atorvastatin calcium concentration of 11.6 μg / mL), and three test concentration groups (MTC 50 μg / mL, 1 / 2MTC 25.0 μg / mL, and 1 / 4MTC 12.5 μg / mL) were set up for a total of six groups. Each beaker had a capacity of 25 mL. Except for the normal control group, all other experimental groups were administered a high-sugar, high-fat diet to establish a zebrafish diet-induced fatty liver model (specifically, a 1.5 mg / mL egg yolk powder solution was administered in water during the day and a 30 mg / mL glucose solution was administered in water at night to establish a zebrafish diet-induced fatty liver model). After two days of treatment at 28°C, whole-body fat staining was performed using Oil Red O. Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of the zebrafish livers was analyzed. Statistical analysis of this indicator was used to evaluate the protective efficacy of Wuling Capsules against food-induced fatty liver disease. Statistical analysis results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software. A p < 0.05 indicated statistical significance. Images of liver fat staining intensity are shown in Figure 1, and the data are recorded in Table 2.

[0051] Table 2

[0052] *** indicates p < 0.001 compared with the model control group.

[0053] It can be seen that Wuling Capsule has auxiliary protective effect on food-induced fatty liver.

[0054] (3) Liver pathological sections

[0055] 5dpf wild-type Albino zebrafish were randomly selected, and 30 zebrafish were treated in each beaker. Samples were added to the beakers in the form of solutions. The capacity of each beaker was 25mL. At the same time, a normal control group, a model control group, a positive control (positive control atorvastatin calcium concentration of 11.6μg / mL) and three test concentration groups of samples (MTC 50μg / mL, 1 / 2MTC 25.0μg / mL and 1 / 4MTC 12.5μg / mL) were set up, for a total of six groups. Except for the normal control group, the remaining experimental groups were given a high-sugar and high-fat diet to establish a zebrafish food (high-sugar and high-fat diet)-induced fatty liver model (specifically, 1.5mg / mL egg yolk powder solution was given in the water during the day and 30mg / mL glucose solution was given in the water at night to establish a zebrafish diet-induced fatty liver model). After treatment at 28°C for 2 days, the zebrafish in each group were fixed, dehydrated, embedded, sectioned, and stained with H&E for histopathological observation and analysis.

[0056] Pathological section photos are shown in Figure 2. Under the above experimental conditions, pathological histological observation of the zebrafish liver in the normal control group showed that the hepatocytes were regular and clear, with normal hepatocyte structure and clear edges. Pathological histological examination of the zebrafish liver in the model control group showed that the hepatocyte structure was fuzzy, the hepatocyte nuclei were enlarged (indicated by the red arrow), and the liver tissue showed fatty vacuolar degeneration (indicated by the black arrow), indicating that the model was successfully established. Pathological histological examination of the zebrafish liver in the positive control atorvastatin calcium group showed that the hepatocyte structure was clearer than that in the model control group, the hepatocyte enlargement was reduced, and the fatty vacuolar degeneration of the liver tissue was reduced, indicating that atorvastatin calcium has an improving effect on high-sugar and high-fat-induced diet-induced fatty liver in zebrafish. Histopathological examination of zebrafish livers in the Wuling Capsule concentration groups of 12.5, 25.0 and 50.0 μg / mL showed that the liver cell structure was clearer than that in the model control group, the liver cell enlargement was reduced, and the fatty vacuolar degeneration of the liver tissue was reduced, indicating that Wuling Capsule has an auxiliary protective effect on zebrafish diet-induced fatty liver induced by high sugar and high fat, which is specifically manifested in the recovery of liver tissue.

[0057] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and any embodiments having substantially the same structure and effect as the technical concept within the scope of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the present application, any other embodiments that can be conceived by those skilled in the art and that combine some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. Use of a Wuling preparation in the preparation of a drug for preventing or treating non-alcoholic fatty liver disease.

2. The use according to claim 1, characterized in that: In parts by weight, the Wuling preparation comprises: 200-400 parts of Bupleurum, 100-200 parts of Ganoderma Lucidum, 200-350 parts of Salvia Miltiorrhiza, and 250-350 parts of Schisandrae Chinensis.

3. The use according to claim 2, characterized in that: In parts by weight, the Wuling preparation comprises: 300-350 parts of Bupleurum, 150-180 parts of Ganoderma Lucidum, 300-350 parts of Salvia miltiorrhiza, and 300-350 parts of Schisandra chinensis.

4. The use according to claim 3, characterized in that: In parts by weight, the Wuling preparation includes: 342 parts of Bupleurum, 173 parts of Ganoderma Lucidum, 342 parts of Salvia miltiorrhiza, and 342 parts of Schisandra chinensis.

5. The use according to any one of claims 1 to 4, characterized in that The dosage form of the Wuling preparation is a decoction, pill, oral liquid, tablet, capsule, granule, paste or powder.

6. The use according to claim 5, characterized in that: The Wuling preparation is selected from any one of Wuling capsules, Wuling pills and Wuling powder.

7. The use according to any one of claims 1 to 6, characterized in that The Wuling preparation is administered at a dosage of 3g-6g / time for adults, and is administered three times a day.

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