Use of a Wuling preparation in the preparation of a medicine for preventing or treating non-alcoholic fatty liver

Abstract

The application provides application of a Wuling preparation in preparation of a medicine for preventing or treating non-alcoholic fatty liver. The medicine effective components of the Wuling preparation include Bupleurum chinense, Ganoderma lucidum, Salvia miltiorrhiza and Schisandra chinensis, the components are simple, but the four medicines have synergistic effect, and play an effective role in preventing or treating non-alcoholic fatty liver.

Description

Technical Field

[0001] This application relates to the field of traditional Chinese medicine, specifically to the application of a Wuling preparation in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Fatty liver is characterized by excessive lipid deposition in hepatocytes. It is defined as fatty liver when lipid accumulation in hepatocytes exceeds 5% of the liver's wet weight, or when more than one-third of hepatocytes are histologically degenerated. Non-alcoholic fatty liver disease (NAFLD) is a clinicopathological syndrome characterized by diffuse macrovesicular steatosis of hepatocytes, caused by factors other than alcohol and other known liver damage. Intrahepatic lipid accumulation is the main cause of NAFLD, and its pathological manifestations include vacuolar steatosis of hepatocytes, inflammation and necrosis of hepatocytes, and ballooning degeneration. With the significant increase in the global incidence of obesity and metabolic syndrome, NAFLD has become the most common chronic metabolic liver disease worldwide and is currently the leading chronic liver disease in my country.

[0003] Nonalcoholic 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, it progresses to nonalcoholic steatohepatitis (NASH), hepatocellular fibrosis, and cirrhosis. Furthermore, as a significant component of metabolic syndrome, it is closely associated with cardiovascular and cerebrovascular diseases, severely impacting people's health and quality of life, and placing a heavy burden on society.

[0004] Currently, the treatment of fatty liver mainly focuses on removing the cause, actively treating the primary disease, and adhering to a reasonable diet, with medication playing only an auxiliary role. Clinically, drugs used to treat fatty liver are mainly divided into lipid-lowering drugs, liver-protecting lipid-lowering drugs, and traditional Chinese medicine; however, these drugs have complex ingredients and complex manufacturing processes. Summary of the Invention

[0005] This application provides the use of a Wuling preparation in the preparation of a drug for the prevention or treatment of non-alcoholic fatty liver disease.

[0006] The first aspect of this application provides the use of a Wuling preparation in the preparation of a medicament for the prevention or treatment of non-alcoholic fatty liver disease.

[0007] In some embodiments, the Five Spirits Preparation comprises, by weight: 200-400 parts Bupleurum, 100-200 parts Ganoderma, 200-350 parts Salvia miltiorrhiza, and 250-350 parts Schisandra chinensis.

[0008] In some embodiments, the Five Spirits Preparation comprises, by weight: 300-350 parts Bupleurum, 150-180 parts Ganoderma, 300-350 parts Salvia miltiorrhiza, and 300-350 parts Schisandra chinensis.

[0009] In some embodiments, the Five Spirits Preparation comprises, by weight: 342 parts Bupleurum, 173 parts Ganoderma, 342 parts Salvia miltiorrhiza, and 342 parts Schisandra chinensis.

[0010] In some implementations, the dosage form of Wuling preparations is decoction, pills, oral liquid, tablets, capsules, granules, ointment or powder.

[0011] In some embodiments, the Wuling preparation is selected from any one of Wuling capsules, Wuling pills, and Wuling powder.

[0012] In some implementations, the Wuling preparation is administered at an adult dose of 3g-6g per dose, three times a day.

[0013] The active ingredients in Wuling preparations 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 the prevention or treatment of non-alcoholic fatty liver disease.

[0014] Instruction manual illustrations

[0015] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0016] Figure 1 A typical image of the intensity of fat staining in zebrafish liver after treatment with Wuling capsules in Example 1 of this application is shown, in which the yellow dashed box represents the liver.

[0017] Figure 2 A typical diagram of zebrafish liver tissue structure after treatment with Wuling capsules in Example 1 of this application is shown. The yellow dashed box represents the zebrafish liver of the analyzed site, the red arrows indicate hepatocyte enlargement, and the black arrows indicate fat vacuolar degeneration. Detailed Implementation

[0018] The embodiments of this application will be described in further detail below with reference to the examples. The detailed description of the following embodiments is used to illustrate the principles of this application, but should not be used to limit the scope of this application, that is, this application is not limited to the described embodiments.

[0019] As analyzed in the background section of this application, although there are some traditional Chinese medicine compositions in the prior art for treating non-alcoholic fatty liver disease, these compositions are complex. Furthermore, the applicant has researched drugs currently used clinically to treat chronic hepatitis, hepatitis B, cirrhosis, hepatitis B-related liver fibrosis, and compensated cirrhosis, and found that drugs effective for these conditions do not necessarily guide treatment of non-alcoholic fatty liver disease. For example, Compound Turtle Shell Softening Liver Tablets have significant efficacy for these conditions, but no efficacy for non-alcoholic fatty liver disease.

[0020] In one embodiment of this application, a Wuling preparation is provided for use in the preparation of a drug for the prevention or treatment of non-alcoholic fatty liver disease.

[0021] The active ingredients of the Wuling preparation include Bupleurum, Ganoderma lucidum, Salvia miltiorrhiza, and Schisandra chinensis. While the composition is simple, the four herbs work synergistically to effectively prevent or treat non-alcoholic fatty liver disease. Bupleurum, being pungent and bitter, effectively regulates liver qi, soothes the liver, and relieves stagnation, serving as the principal herb. Ganoderma lucidum, being sweet and neutral, invigorates qi and strengthens the spleen, supporting the body's vital energy. Salvia miltiorrhiza invigorates blood, removes blood stasis, and relieves pain, serving as the assistant herb. Schisandra chinensis, being sour, sweet, and warm, invigorates qi and nourishes yin, assisting Ganoderma lucidum in invigorating qi and strengthening the spleen, calming the mind, relieving depression, and preventing Bupleurum's dispersing and purging effects from depleting qi and damaging yin, thus protecting the liver and lowering enzymes. When used together, these herbs treat both the liver and spleen, regulate qi and blood, and combine astringent and dispersing effects. When used together, these herbs work synergistically to soothe the liver and relieve depression, invigorate qi and strengthen the spleen, and promote blood circulation and remove blood stasis. They can significantly improve symptoms such as liver area pain, abdominal distension, fatigue, and loss of appetite in patients with non-alcoholic fatty liver disease, and reduce blood biochemical indicators.

[0022] To further improve the synergistic effect of the four herbs, in some embodiments of this application, the preferred five-herb preparation, by weight, comprises: 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. More preferably, by weight, the five-herb preparation comprises: 300-350 parts of Bupleurum chinense, 150-180 parts of Ganoderma lucidum, 300-350 parts of Salvia miltiorrhiza, and 300-350 parts of Schisandra chinensis. Even more preferably, by weight, the five-herb preparation comprises: 342 parts of Bupleurum chinense, 173 parts of Ganoderma lucidum, 342 parts of Salvia miltiorrhiza, and 342 parts of Schisandra chinensis.

[0023] The Wuling preparation of this application can be used in any conventional dosage form. In some embodiments, the dosage form of the above-mentioned Wuling preparation is a decoction, pills, oral liquid, tablets, capsules, granules, ointment, or powder. The preparation processes of the above-mentioned dosage forms are relatively mature, which facilitates the application of Wuling preparation in the preparation of drugs for the prevention or treatment of non-alcoholic fatty liver disease.

[0024] In some embodiments, the Wuling preparation is selected from any one of Wuling capsules, Wuling pills, and Wuling powder. All of the above preparations are existing pharmaceutical preparations, which are more conducive to the implementation of this application.

[0025] The inventors of this application, through repeated experimental verification, obtained the following formula, and using the effective safe concentration of zebrafish in this application, the equivalent theoretical human dosage was calculated using the following formula:

[0026] Human (g / time) = [Zebrafish concentration (μg / mL) × 6 / 100] × 2.

[0027] Based on the above conversion, in some implementations, the Wuling preparation is administered at a dosage of 3g-6g per dose for adults, three times a day. This dosage has a significant therapeutic effect on non-alcoholic fatty liver disease with minimal side effects.

[0028] The beneficial effects of this application will be further illustrated below with reference to embodiments and comparative examples, but the scope of the present invention is not limited to these embodiments.

[0029] Example 1

[0030] The Wuling capsules used were provided by Tsinghua Deren Xi'an Xingfu Pharmaceutical Co., Ltd.

[0031] 1. Experimental Principles and Methods

[0032] Experimental Principle: Zebrafish and humans share high similarity in liver structure, function, and genetics. Except for immune Kupffer cells, zebrafish contain other cell types found in the human liver and possess the same functions, including bile secretion, glycogen and lipid storage, insulin response, xenobiotic and ammonia metabolism, and the secretion of serum proteins such as complement and clotting factors, transferrin, and albumin-like proteins. Zebrafish livers are fully developed within 5 days of fertilization, allowing for comprehensive liver function assessments. Zebrafish are an important model for studying liver diseases. Feeding zebrafish a high-sugar, high-fat diet often provides more energy than the fish needs. This excess energy is stored as fat in the liver, leading to a continuous increase in liver fat content, inducing hepatocyte steatosis, and ultimately resulting in fatty liver disease.

[0033] Sample preparation information:

[0034] Wuling Capsules use standard dilution water as the solvent.

[0035] Positive control: Atorvastatin calcium tablets (hereinafter referred to as atorvastatin calcium), white tablets, batch number FR7909, Pfizer Pharmaceuticals Ltd., solvent is DMSO.

[0036] Laboratory animals:

[0037] Zebrafish were all raised in fish culture water at 28 °C (water quality: 200 mg of instant sea salt was added to every 1 L of reverse osmosis water, with a conductivity of 450 - 550 μS / cm; pH of 6.5 - 8.5; hardness of 50 - 100 mg / L CaCO3). They were provided by the fish breeding center of our company, and the experimental animal use license number was: SYXK(Zhe)2022 - 0004. The feeding management complied with the requirements of international AAALAC accreditation (accreditation number: 001458), and the IACUC ethical review number was: IACUC - 2023 - 7689 - 01.

[0038] Instruments, consumables and reagents:

[0039] 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 Berambo Biotechnology Co., Ltd., China); Double - person single - sided purification workbench (SW - CJ - 215KS, Shanghai Sujing Industrial Co., Ltd., China); Microtome (KD2258, Jinhua Cody Medical Devices Co., Ltd., China); Biological microscope (CX31, OLYMPUS, Japan).

[0040] Dimethyl sulfoxide (DMSO, batch number BCCD8942, Sigma, Switzerland); Methyl cellulose (batch number C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Absolute ethanol (batch number 20230329, Sinopharm Chemical Reagent Co., Ltd., China); Oil Red O (batch number SHBN4926, Sigma, USA); 1,2 - propanediol (batch number 20211117, Sinopharm Chemical Reagent Co., Ltd., China); 4% tissue cell fixative (batch number 20221014, Beijing Solarbio Science & Technology Co., Ltd., China); Xylene (batch number C14165111, Shanghai Macklin Biochemical Co., Ltd., China); PBS buffer (batch number 70115000, Biosharp, China); Mayer hematoxylin staining solution (batch number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); Eosin staining solution (batch number 20220120, Shanghai Yihe Biotechnology Co., Ltd., China); Neutral gum (batch number 330A021, Solarbio, China); High - efficiency sectioning paraffin (melting point 54 - 56 °C, batch number 20201020, Shanghai Huayong Paraffin Co., Ltd., China); High - efficiency sectioning paraffin (melting point 62 - 64 °C, batch number 20210828, Shanghai Huayong Paraffin Co., Ltd., China).

[0041] The experimental method is as follows:

[0042] (1) Determine the MTC of Wuling Capsules

[0043] Zebrafish of strain AB with a melanin allele mutation 5 days post-fertilization were randomly selected and placed in beakers, with 30 zebrafish treated in each beaker. Five concentration groups were set up for sample testing. Samples were added to microplates / beakers in solution form. A normal control group and a model control group were also set up, for a total of seven experimental groups, with each beaker containing 25 mL. The concentration groups were given Wuling capsules (concentrations shown in Table 1) in water. Except for the normal control group, all other experimental groups were given a high-sugar, high-fat diet in water to establish a zebrafish dietary (high-sugar, high-fat) induced fatty liver model (specifically, a 1.5 mg / mL egg yolk powder solution was given during the day, and a 30 mg / mL glucose solution was given at night to establish a zebrafish dietary fatty liver model). After treatment at 28℃ for 2 days, the number of zebrafish deaths and toxicity were recorded in each experimental group to determine the MTC of the samples. The results are recorded in Table 1.

[0044] Table 1. Results of the concentration exploratory experiment on the adjuvant protective efficacy of Wuling Capsules against food-induced fatty liver (n=30)

[0045]

[0046]

[0047] It is evident that the Zebrafish MTC of Wuling Capsules provides auxiliary protection against food-induced fatty liver disease.

[0048] (2) Degree of hepatic steatosis

[0049] Zebrafish of the Albino strain with a 5dpf melanin allele mutation were randomly selected and treated in beakers, with 30 zebrafish per beaker. Wuling capsules (concentration shown in Table 2) were administered in water-soluble form, and the samples were added to the beakers in solution form. Six groups were set up: a normal control group, a model control group, a positive control (positive control: atorvastatin calcium 11.6 μg / mL concentration), and three sample detection concentration groups (MTC 50 μg / mL, 1 / 2 MTC 25.0 μg / mL, and 1 / 4 MTC 12.5 μg / mL, respectively). Each beaker had a volume of 25 mL. Except for the normal control group, all other experimental groups were given a high-sugar, high-fat diet to establish a zebrafish dietary (high-sugar, high-fat) induced fatty liver model (specifically, a 1.5 mg / mL egg yolk powder solution was administered during the day, and a 30 mg / mL glucose solution was administered at night to establish a zebrafish dietary fatty liver model). After treatment at 28℃ for 2 days, whole-body fat was stained with Oil Red O. Following staining, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were collected using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of the zebrafish liver was analyzed. The statistical analysis results of this index were used to evaluate the adjuvant protective efficacy of Wuling capsules against dietary fatty liver. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software; p < 0.05 indicated statistical significance. Images of liver fat staining intensity were recorded in [image file name missing]. Figure 1 The data is recorded in Table 2.

[0050] Table 2

[0051]

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

[0053] It is evident that Wuling Capsules have an auxiliary protective effect against food-induced fatty liver.

[0054] (3) Liver pathological sections

[0055] Wild-type Albino strain zebrafish (5 dpf) were randomly selected, with 30 zebrafish treated in each beaker. Samples were added to the beakers in solution form, with each beaker containing 25 mL. Six groups were established: a normal control group, a model control group, a positive control (atorvastatin calcium at a concentration of 11.6 μg / mL), and three sample concentration groups (MTC 50 μg / mL, 1 / 2 MTC 25.0 μg / mL, and 1 / 4 MTC 12.5 μg / mL, respectively). Except for the normal control group, all experimental groups were given a high-sugar, high-fat diet to establish a zebrafish dietary fatty liver model (specifically, a 1.5 mg / mL egg yolk powder solution was given during the day, and a 30 mg / mL glucose solution was given at night to establish a dietary fatty liver model). After treatment at 28℃ for 2 days, the zebrafish in each group underwent fixation, dehydration, embedding, sectioning, and H&E staining for histopathological observation and analysis.

[0056] See pathological slide photos Figure 2 Under the above experimental conditions, histopathological examination of zebrafish livers in the normal control group showed that the hepatocytes were regular and clear, with normal structure and clear edges. Histopathological examination of zebrafish livers in the model control group showed that the hepatocyte structure was blurred, the hepatocyte nuclei were enlarged (indicated by the red arrow), and fatty vacuolar degeneration appeared in the liver tissue (indicated by the black arrow), indicating successful model establishment. Histopathological examination of zebrafish livers in the positive control group (atorvastatin calcium group) showed that the hepatocyte structure was clearer than that in the model control group, hepatocyte enlargement was reduced, and fatty vacuolar degeneration in the liver tissue was reduced, indicating that atorvastatin calcium has an ameliorative effect on dietary fatty liver induced by high sugar and high fat in zebrafish. Histopathological examination of zebrafish livers in the Wuling capsule concentration groups (12.5, 25.0, and 50.0 μg / mL) showed that the hepatocyte structure was clearer than that in the model control group, hepatocyte enlargement was reduced, and fatty vacuolar degeneration in the liver tissue was reduced, indicating that Wuling capsule has an auxiliary protective effect on dietary fatty liver induced by high sugar and high fat in zebrafish, specifically manifested in the recovery of liver tissue.

[0057] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. The application of a Wuling preparation in the preparation of a drug for the prevention or treatment of food-induced fatty liver, wherein, by weight, the Wuling preparation is made from 342 parts of Bupleurum chinense, 173 parts of Ganoderma lucidum, 342 parts of Salvia miltiorrhiza, and 342 parts of Schisandra chinensis, wherein the food-induced fatty liver has the pathological characteristics of fatty vacuolar degeneration of liver tissue.

2. The application according to claim 1, characterized in that, The dosage forms of the Wuling preparations are decoction, pills, oral liquid, tablets, capsules, granules, ointments, or powders.

3. The application according to claim 1 or 2, characterized in that, The Wuling preparation is selected from any one of Wuling capsules, Wuling pills, and Wuling powder.

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