A pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease

Abstract

The present invention discloses a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease. The pharmaceutical composition comprises the following components: magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside, wherein the mass ratio between the magnolol, the gentisic acid, the baicalin, the syringin, the wogonoside, the honokiol, the prunasin, and the pinoresinol 4-O-β-glucoside is (5.00 to 33.00):(12.00 to 110.00):(12.00 to 110.00):(4.50 to 42.00):(2.00 to 9.00):(0.10 to 1.10):(0.80 to 5.50):(6.00 to 40.00). The pharmaceutical composition of the present invention can improve macrovesicular steatosis, inflammatory cell infiltration and ballooning degeneration of hepatocytes, and can be used for preventing and treating non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility, characterized by abnormal lipid deposition in the liver. Excluding other secondary factors such as excessive alcohol consumption, drugs, and genetic diseases that cause fatty liver, its disease spectrum includes non-alcoholic simple fatty liver (NAFL), non-alcoholic steatohepatitis (NASH), and cirrhosis. NAFLD has become the most common chronic liver disease globally, with a prevalence of approximately 25-30% in the general adult population. NASH is the key pathological stage for NAFL to progress to fibrosis and cirrhosis, and is one of the main causes of hepatocellular carcinoma. NASH-related cirrhosis has become the main cause of liver transplantation in women and people over 65 years old. Lifestyle changes (weight loss, exercise, healthy diet) remain the basic measures for treating NAFLD currently. Multinational guidelines recommend glucagon-like peptide-1 analogue semaglutide and pioglitazone for patients with type 2 diabetes complicated with NASH confirmed by liver biopsy, and vitamin E for patients with NASH confirmed by liver biopsy. However, the all-cause mortality increases with long-term high-dose use of vitamin E, and at the same time, it is associated with an increased incidence of prostate cancer in men, so its safety needs to be evaluated. In March 2024, the US Food and Drug Administration approved the liver-targeted thyroid hormone receptor β-selective agonist resmetrom for the treatment of NASH with significant fibrosis, but the safety of its long-term use still needs to be observed. In addition, the preliminary results of clinical trials of new drugs such as pan-PPAR agonists, fibroblast growth factor 21 analogues, and GLP-1 receptor / glucose-dependent insulinotropic polypeptide and GLP-1 receptor dual agonists (tirzepatide) are satisfactory, but still require a large amount of clinical data support. The farnesoid X receptor agonist obeticholic acid, which was previously highly concerned and expected, was not approved for NASH treatment in the United States and was withdrawn from the market in Europe, further indicating that the research and development of effective drugs for NAFLD still need to be efforts.

[0003] The pathological mechanism of NAFLD is complex. Currently, the drug design targeting a single target is likely to be one of the reasons for the failure of most drugs that showed potential in preclinical studies after entering clinical trials. Traditional Chinese medicine (TCM) has a clinical practice basis for the treatment of NAFLD, and successive clinical studies have also shown the potential efficacy of certain TCM formulas or patent TCM drugs for NAFLD, which may be related to the characteristics of multi-component and multi-target actions of TCM. In addition, some TCM extracts or monomers such as total glucosides of paeony in Paeonia lactiflora Pall., geniposide in Gardenia jasminoides Ellis, salvianolic acid B in Salvia miltiorrhiza Bunge, and berberine in Coptis chinensis Franch. also showed good intervention effects on NAFLD, but await confirmation in clinical trials.

[0004] There is no relevant report in the prior art on a composition comprising magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside in a certain proportion combination for preventing and / or treating liver diseases such as non-alcoholic fatty liver disease (non-alcoholic steatohepatitis). Summary of the Invention

[0005] Based on this, the present invention provides a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, which comprises the following components: magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside, wherein the mass ratio between the magnolol, the gentisic acid, the baicalin, the syringin, the wogonoside, the honokiol, the prunasin, and the pinoresinol 4-O-β-glucoside is (5.00 - 33.00):(12.00 - 110.00):(12.00 - 110.00):(4.50 - 42.00):(2.00 - 9.00):(0.10 - 1.10):(0.80 - 5.50):(6.00 - 40.00).

[0006] Furthermore, the mass ratio between the magnolol, the gentisic acid, the baicalin, the syringin, the wogonoside, the honokiol, the prunasin, and the pinoresinol 4-O-β-glucoside is not 30.00:31.64:23.73:5.34:6.33:0.18:2.81:8.90.

[0007] Furthermore, the mass ratio between the magnolol, the gentisic acid, the baicalin, the syringin, the wogonoside, the honokiol, the prunasin, and the pinoresinol 4-O-β-glucoside is not 22.50:42.19:31.64:40.00:3.56:0.18:2.11:8.90.

[0008] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is not 12.66:17.80:42.19:30.00:2.67:0.32:2.11:37.50.

[0009] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is approximately 16.88: approximately 13.35: approximately 100.00: approximately 30.00: approximately 3.56: approximately 1.00: approximately 1.19: approximately 6.67.

[0010] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is approximately 5.34: approximately 75.00: approximately 75.00: approximately 16.88: approximately 4.75: approximately 0.13: approximately 5.00: approximately 37.5.

[0011] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is approximately 7.12: approximately 23.73: approximately 17.80: approximately 16.88: approximately 2.67: approximately 1.00: approximately 1.58: approximately 11.87.

[0012] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is approximately 7.12: approximately 13.35: approximately 13.35: approximately 40.00: approximately 8.44: approximately 0.32: approximately 0.89: approximately 15.82.

[0013] Furthermore, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (16.00 - 32.00):(53.00 - 106.00):(16.00 - 60.00):(5.00 - 24.00):(6.00 - 16.00):(0.50 - 0.80):(3.50 - 4.00):(11.00 - 17.00).

[0014] Further, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is about 30.00: about 100.00: about 56.25: about 5.34: about 6.33: about 0.56: about 3.75: about 11.87.

[0015] Further, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is about 16.88: about 56.25: about 17.80: about 22.50: about 15.00: about 0.75: about 3.75: about 15.82.

[0016] Further, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0017] Further, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained-release agent, retention aid, lubricant, dispersant, plasticizer, light-blocking agent, and antioxidant.

[0018] Further, the dosage form of the pharmaceutical composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0019] Further, the pharmaceutical composition further comprises one or more drugs for preventing and / or treating non-alcoholic fatty liver disease.

[0020] Further, the drug is selected from one or more of the following: ursodeoxycholic acid, farnesoid X receptor agonist, liver-selective thyroid hormone receptor β agonist, pan-PPAR agonist, fibroblast growth factor 21 analog, GLP-1 and GLP-1 receptor dual agonist, silymarin, polyene phosphatidylcholine, bicyclol, glycyrrhizin preparation, and reduced glutathione.

[0021] Further, the farnesoid X receptor agonist is obeticholic acid.

[0022] Further, the liver-selective thyroid hormone receptor β agonist is Resmetirom.

[0023] Further, the pan-PPAR agonist is Lanifibranor.

[0024] Further, the fibroblast growth factor 21 analogs are Efruxifermin and Pegozafermin.

[0025] Furthermore, the GLP-1 and GLP-1 receptor dual agonist is tirzepatide.

[0026] Furthermore, the glycyrrhizin preparation is magnesium isoglycyrrhizinate, compound glycyrrhizin, and diammonium glycyrrhizinate.

[0027] According to another aspect of the present invention, there is provided the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease.

[0028] Furthermore, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic hepatic fibrosis, and non-alcoholic liver cirrhosis.

[0029] Furthermore, the pharmaceutical composition prevents and / or treats non-alcoholic fatty liver disease in one or two of the following ways: reducing the serum alanine aminotransferase level and reducing the liver tissue triglyceride level.

[0030] Advantages of the present invention:

[0031] The pharmaceutical composition of the present invention can improve macrovesicular steatosis, inflammatory cell infiltration, and ballooning degeneration of hepatocytes, and can be used for preventing and treating non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0033] Figure 1 It is a schematic diagram of the change results of serum ALT and liver TG content in mice. Among them, A is serum ALT; B is liver TG; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0035] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention pertains or in the field in which the term is applied. Although any methods, conditions, materials, or substances similar or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, materials, or substances are described herein.

[0036] The present invention is expected to cover all alternatives, variations, and equivalents that may be included in the existing field of the invention as defined by the claims. Those skilled in the art will recognize many methods and substances similar or equivalent to those described herein, which can be applied to the practice of the present invention. The present invention is in no way limited to the description of methods and substances.

[0037] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0038] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "including", "comprising", "having", "containing", or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus containing the listed elements need not be limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0039] As described in the background art section, there are no reports in the prior art on a composition containing magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside in a certain proportion combination that can be used for preventing and / or treating liver diseases such as non-alcoholic fatty liver disease (non-alcoholic steatohepatitis). To solve the above problems, the present invention provides a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, which pharmaceutical composition contains the following components: magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside, wherein the mass ratio between the magnolol, the gentisic acid, the baicalin, the syringin, the wogonoside, the honokiol, the prunasin, and the pinoresinol 4-O-β-glucoside is (5.00 - 33.00):(12.00 - 110.00):(12.00 - 110.00):(4.50 - 42.00):(2.00 - 9.00):(0.10 - 1.10):(0.80 - 5.50):(6.00 - 40.00).

[0040] In the present invention, when a quality or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, it should be understood that all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value are specifically disclosed, regardless of whether the range is separately disclosed or not. For example, when the range "5.00 to 33.00" is disclosed, the described range should be interpreted as including ranges "5.00 to 33.00", "5.00 to 30.00", "5.00 to 25.00", "5.00 to 20.00", "5.00 to 15.00", "5.00 to 10.00", "10.00 to 33.00", "10.00 to 30.00", "10.00 to 25.00", "10.00 to 20.00", "10.00 to 15.00", "15.00 to 33.00", "15.00 to 30.00", "15.00 to 25.00", "15.00 to 20.00", "20.00 to 33.00", "20.00 to 30.00", "20.00 to 25.00", "25.00 to 33.00", "25.00 to 30.00", "30.00 to 33.00", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0041] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is not 30.00:31.64:23.73:5.34:6.33:0.18:2.81:8.90.

[0042] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is not 22.50:42.19:31.64:40.00:3.56:0.18:2.11:8.90.

[0043] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is not 12.66:17.80:42.19:30.00:2.67:0.32:2.11:37.50.

[0044] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-glucoside is about 16.88: about 13.35: about 100.00: about 30.00: about 3.56: about 1.00: about 1.19: about 6.67.

[0045] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 16.88" includes ±5% of 16.88, or from 16.036 to 17.724; "about 13.35" includes ±5% of 13.35, or from 12.6825 to 14.0175; "about 100.00" includes ±5% of 100.00, or from 95.00 to 105.00; "about 30.00" includes ±5% of 30.00, or from 28.50 to 31.50; "about 3.56" includes ±5% of 3.56, or from 3.382 to 3.738; "about 1.00" includes ±5% of 1.00, or from 0.95 to 1.05; "about 1.19" includes ±5% of 1.19, or from 1.1305 to 1.2495; "about 6.67" includes ±5% of 6.67, or from 6.3365 to 7.0035.

[0046] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-glucoside is about 5.34: about 75.00: about 75.00: about 16.88: about 4.75: about 0.13: about 5.00: about 37.5.

[0047] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 5.34" includes ±5% of 5.34, or from 5.073 to 5.607; "about 75.00" includes ±5% of 75.00, or from 71.25 to 78.75; "about 16.88" includes ±5% of 16.88, or from 16.036 to 17.724; "about 4.75" includes ±5% of 4.75, or from 4.5125 to 4.9875; "about 0.13" includes ±5% of 0.13, or from 0.1235 to 0.1365; "about 5.00" includes ±5% of 5.00, or from 4.75 to 5.25; "about 37.5" includes ±5% of 37.5, or from 35.625 to 39.375.

[0048] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-glucoside is about 7.12: about 23.73: about 17.80: about 16.88: about 2.67: about 1.00: about 1.58: about 11.87.

[0049] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 7.12" includes ±5% of 7.12, or from 6.764 to 7.476; "about 23.73" includes ±5% of 23.73, or from 22.5435 to 24.9165; "about 17.80" includes ±5% of 17.80, or from 16.91 to 18.69; "about 16.88" includes ±5% of 16.88, or from 16.036 to 17.724; "about 2.67" includes ±5% of 2.67, or from 2.5365 to 2.8035; "about 1.00" includes ±5% of 1.00, or from 0.95 to 1.05; "about 1.58" includes ±5% of 1.58, or from 1.501 to 1.659; "about 11.87" includes ±5% of 11.87, or from 11.2765 to 12.4635.

[0050] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-glucoside is about 7.12: about 13.35: about 13.35: about 40.00: about 8.44: about 0.32: about 0.89: about 15.82.

[0051] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 7.12" includes ±5% of 7.12, or from 6.764 to 7.476; "about 13.35" includes ±5% of 13.35, or from 12.6825 to 14.0175; "about 40.00" includes ±5% of 40.00, or from 38.00 to 42.00; "about 8.44" includes ±5% of 8.44, or from 8.018 to 8.862; "about 0.32" includes ±5% of 0.32, or from 0.304 to 0.336; "about 0.89" includes ±5% of 0.89, or from 0.8455 to 0.9345; "about 15.82" includes ±5% of 15.82, or from 15.029 to 16.611.

[0052] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-D-glucoside is (16.00 - 32.00):(53.00 - 106.00):(16.00 - 60.00):(5.00 - 24.00):(6.00 - 16.00):(0.50 - 0.80):(3.50 - 4.00):(11.00 - 17.00).

[0053] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-D-glucoside is about 30.00: about 100.00: about 56.25: about 5.34: about 6.33: about 0.56: about 3.75: about 11.87.

[0054] In the present invention, "about" means a value within the range of ±5% of a specific value. For example, "about 30.00" includes ±5% of 30.00, or from 28.50 to 31.50; "about 100.00" includes ±5% of 100.00, or from 95.00 to 105.00; "about 56.25" includes ±5% of 56.25, or from 53.4375 to 59.0625; "about 5.34" includes ±5% of 5.34, or from 5.073 to 5.607; "about 6.33" includes ±5% of 6.33, or from 6.0135 to 6.6465; "about 0.56" includes ±5% of 0.56, or from 0.532 to 0.588; "about 3.75" includes ±5% of 3.75, or from 3.5625 to 3.9375; "about 11.87" includes ±5% of 11.87, or from 11.2765 to 12.4635.

[0055] In a preferred embodiment, the mass ratio among magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-D-glucoside is about 16.88: about 56.25: about 17.80: about 22.50: about 15.00: about 0.75: about 3.75: about 15.82.

[0056] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 16.88" includes ±5% of 16.88, or from 16.036 to 17.724; "about 56.25" includes ±5% of 56.25, or from 53.4375 to 59.0625; "about 17.80" includes ±5% of 17.80, or from 16.91 to 18.69; "about 22.50" includes ±5% of 22.50, or from 21.375 to 23.625; "about 15.00" includes ±5% of 15.00, or from 14.25 to 15.75; "about 0.75" includes ±5% of 0.75, or from 0.7125 to 0.7875; "about 3.75" includes ±5% of 3.75, or from 3.5625 to 3.9375; "about 15.82" includes ±5% of 15.82, or from 15.029 to 16.611.

[0057] In a preferred embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0058] In a preferred embodiment, the excipient is selected from one or more of the following: diluent, wetting agent, binder, disintegrant, inclusion agent, flavoring agent, sustained release agent, retention aid, lubricant, dispersant, plasticizer, light shielding agent, and antioxidant.

[0059] In a preferred embodiment, the dosage form of the pharmaceutical composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

[0060] In a preferred embodiment, the pharmaceutical composition further comprises one or more drugs for preventing and / or treating non-alcoholic fatty liver disease.

[0061] In a preferred embodiment, the drug is selected from one or more of the following: ursodeoxycholic acid, farnesoid X receptor agonist, liver-selective thyroid hormone receptor β agonist, pan-PPAR agonist, fibroblast growth factor 21 analog, GLP-1 and GLP-1 receptor dual agonist, silymarin, polyene phosphatidylcholine, bicyclol, glycyrrhizin preparation, and reduced glutathione.

[0062] In a preferred embodiment, the farnesoid X receptor agonist is obeticholic acid.

[0063] In a preferred embodiment, the liver-selective thyroid hormone receptor β agonist is Resmetirom.

[0064] In a preferred embodiment, the pan-PPAR agonist is Lanifibranor.

[0065] In a preferred embodiment, the fibroblast growth factor 21 analogs are efruxifermin and pegozafermin.

[0066] In a preferred embodiment, the GLP-1 and GLP-1 receptor dual agonist is tirzepatide.

[0067] In a preferred embodiment, the glycyrrhizin preparations are magnesium isoglycyrrhizinate, compound glycyrrhizin, and diammonium glycyrrhizinate.

[0068] According to another aspect of the present invention, there is provided the use of the above-mentioned pharmaceutical composition in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease.

[0069] In a preferred embodiment, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic hepatic fibrosis, and non-alcoholic cirrhosis.

[0070] In a preferred embodiment, the pharmaceutical composition prevents and / or treats non-alcoholic fatty liver disease in one or both of the following ways: reducing the serum alanine aminotransferase level and reducing the liver tissue triglyceride level.

[0071] The present invention will be further illustrated below with reference to specific examples. It should be understood that these examples are only for illustrating the present invention and not for limiting the scope of the present invention. The experimental methods without specific conditions noted in the following examples are generally carried out under conventional conditions or conditions recommended by the manufacturer.

[0072] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred methods and materials described herein are only for illustrative purposes.

[0073] The above-mentioned features mentioned in the present invention, or the features mentioned in the examples, can be combined arbitrarily. All the features disclosed in this patent specification can be used in combination with any composition form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equivalent or similar features.

[0074] Examples

[0075] 1. Experimental Instruments

[0076] The information of the instrument and equipment is shown in Table 1.

[0077] Table 1 Instrument and Equipment Information Table

[0078]

[0079]

[0080] 2. Experimental Materials

[0081] 2.1 Experimental Animals

[0082] Adult male SPF-grade C57BL / 6 mice, 6 - 8 weeks old, raised in an environment of 22 - 25 °C, with a relative indoor humidity of 55 - 60%, natural light, and free access to water.

[0083] 2.2 Drugs

[0084] Eight components, magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin were purchased from Chengdu Efa Biotechnology Co., Ltd., and their purities were all > 98%.

[0085] 2.3 Reagents

[0086] Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD, product number: A06071302, Research Diets Inc), triglyceride kit (product number: A010017, Zhejiang Dong'ou Diagnostic Products Co., Ltd.).

[0087] 3. Experimental Methods

[0088] 3.1 Uniform Design Experimental Scheme

[0089] Magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside were set as 8 investigated factors, and an 8-factor 16-level uniform design table was selected, namely U 16 (16 8)Table (as shown in Table 2). Let X1, X2, X3, X4, X5, X6, X7, X8 represent magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside respectively. Each factor takes 8 dose levels, decreasing by a factor of 0.75. The upper limit of the dose level of X1 (magnolol) is 30 (mg / kg body weight) (see the literature: Zhang H, Ju B, Zhang X, Zhu Y, Nie Y, Xu Y, et al. Magnolol Attenuates Concanavalin A-induced Hepatic Fibrosis, Inhibits CD4(+) T Helper 17 (Th17) Cell Differentiation and Suppresses Hepatic Stellate Cell Activation: Blockade of Smad3 / Smad4 Signalling. Basic Clin Pharmacol Toxicol 2017;120:560-570), the upper limit of the dose level of X2 (gentisic acid) is 100 (mg / kg body weight) (see the literature: Han X, Guo J, Gao Y, Zhan J, You Y and Huang W. Gentisic acid prevents diet-induced obesity in mice by accelerating the thermogenesis of brown adipose tissue. Food Funct 2021;12:1262-1270), the upper limit of the dose level of X3 (baicalin) is 100 (mg / kg body weight) (see the literature: Miao L, Zhang X, Zhang H, Cheong M S, Chen X, Farag M A, et al. Baicalin ameliorates insulin resistance and regulates hepatic glucose metabolism via activating insulin signaling pathway in obese pre-diabetic mice. Phytomedicine 2024;124:155296), the upper limit of the dose level of X4 (syringin) is 40 (mg / kg body weight) (see the literature: Deng Z, Ren C, Tang C, Chen S, Li J, Wei J, et al.Syringin alleviates hepatic fibrosis by enhancing autophagic flux and attenuating ER stress-TRIB3 / SMAD3 in diabetic mice. Tissue Cell 2023;83:102159), X5 (wogonoside) the upper limit of the dose level is 20 (mg / kg body weight) (see the literature: Jiang G, Chen D, Li W, Liu C, Liu J and Guo Y. Effects of wogonoside on the inflammatory response and oxidative stress in mice with nonalcoholic fatty liver disease. Pharm Biol 2020;58:1177-1183), X6 (honokiol) the upper limit of the dose level is 1 (mg / kg body weight) (see the literature: Choi D Y, Lee JW, Lin G, Lee YK, Lee Y H, Choi I S, et al. Obovatol attenuates LPS-induced memory impairments in mice via inhibition of NF-κB signaling pathway. Neurochem Int 2012;60:68-77), X7 (prunasin) the upper limit of the dose level is 5 (mg / kg body weight) (see the literature: Zhang C, Lin J, Zhen C, Wang F, Sun X, Kong X, et al. Amygdalin protects against acetaminophen-induced acute liver failure by reducing inflammatory response and inhibiting hepatocyte death. Biochem Biophys Res Commun 2022;602:105-112), X8 (pinoresinol 4-O-β-glucoside) the upper limit of the dose level is 50 (mg / kg body weight) (see the literature: Sousa G M, Cazarin C B B, Maróstica Junior M R, Lamas C A, Quitete V, Pastore G M, et al.The effect of α-terpineol enantiomers on biomarkers of rats fed a high-fat diet. Heliyon 2020;6:e03752). In the uniform design table (as shown in Table 2), dose levels 9-16 are repeats of dose levels 1-8. According to U16(16... 8 ) It was divided into 16 groups in total. The medication was administered for 4 weeks in total.

[0090] Table 2 U 16 (16 8 ) Uniform design table

[0091] 1 2 3 4 5 6 7 8 1 1 5 11 4 13 3 9 6 2 13 16 6 2 11 7 14 8 3 7 7 4 11 15 1 12 13 4 9 14 9 12 16 13 2 5 5 10 3 8 3 6 5 1 14 6 6 1 16 15 10 8 3 9 7 2 15 7 13 3 9 8 15 8 3 11 2 5 9 16 4 11 9 16 4 3 9 12 10 6 2 10 15 13 12 8 2 2 5 10 11 11 9 1 16 5 4 10 4 12 8 8 14 1 4 14 7 3 13 5 2 5 7 1 12 13 7 14 12 10 15 6 14 11 11 16 15 14 6 10 14 7 15 15 12 16 4 12 13 10 8 6 16 1

[0092] The uniform design plan is shown in Table 3.

[0093] Table 3 Uniform design plan

[0094]

[0095]

[0096]

[0097] 3.2. Preparation of animal models and administration of drugs

[0098] After 1 week of adaptive feeding of C57BL / 6J mice, they were randomly divided into a normal control group, a model group, and 16 groups of uniform design 1, 2, 3... 16 (groups UA-UP), with 7 mice in each group. The normal control group was given normal feed for 6 weeks, and the model group and the uniform design groups were all given CDAHFD feed for 6 weeks. From the 3rd week, groups 1, 2, 3... 16 of the uniform design (groups UA-UP) were given the corresponding drugs respectively, and the normal control group and the model group were both given an equal amount of sterilized drinking water by gavage. Samples were taken at the end of the 6th week.

[0099] 3.3. Sample collection

[0100] 3.3.1. Serum

[0101] Samples were taken at the end of the 6th week. After fasting for 12 h, the mice were sacrificed by decapitation after taking blood from the eye socket. The blood was allowed to stand at 4 °C for 4 h, centrifuged at 3000 r / min for 15 min, and 200 μL of serum was taken and stored in a 1.5 ml ep tube at -80 °C.

[0102] 3.3.2. Liver

[0103] The mouse liver was dissected from the abdominal cavity, rinsed with normal saline, blotted dry on filter paper, photographed and weighed. Subsequently, two pieces of liver tissue approximately 1 cm × 1 cm × 1 cm in size were taken from the central part of the large liver lobe. One piece was placed in a pathology frame and preserved in formalin. The remaining liver tissue was snap-frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0104] 3.4. Index Detection

[0105] 3.4.1. Detection of Liver Triglyceride (TG)

[0106] Before measuring liver TG, liver tissue homogenate was prepared in advance: Weigh 50 mg of liver tissue and place it in a 2 mL centrifuge tube containing 3 steel beads. Add 375 μL each of absolute ethanol and acetone, and grind with a fully automatic sample grinder at 65 Hz for 60 s × 3 times to obtain liver tissue homogenate. Let it stand overnight in a 4°C refrigerator. The next day, centrifuge for 15 minutes (4°C, 3000 rpm / min), and take the supernatant for measuring TG content, operating according to the instructions.

[0107] 3.4.2. Detection of Serum ALT

[0108] Serum ALT was detected using a kit, operating according to the instructions.

[0109] 3.4.3. HE Staining

[0110] After fixing fresh liver tissue in rapid fixative for 48 h, dehydration and paraffin embedding were carried out. Paraffin tissue sections (4 μm) were placed at 70°C for 30 min, and then dewaxed: successively placed in xylene ① for 10 min, xylene ② for 10 min, absolute ethanol ① for 5 min, absolute ethanol ② for 2 min, 95% ethanol for 2 min, 85% ethanol for 2 min, 70% ethanol for 2 min, and washed in double-distilled water 6 times. Finally, staining was started: The dewaxed slides were successively placed in hematoxylin for 15 min, washed in double-distilled water 6 times, differentiated in hydrochloric acid alcohol for 2 - 3 s, washed in double-distilled water 6 times, stained with eosin for 2 - 3 min, washed in intensifying solution 6 times, dried the excess water around the tissue, and sealed with neutral resin. Let it stand at room temperature for 1 - 2 days and then scanned.

[0111] 4. Experimental Results

[0112] 4.1. Successful Establishment of the Mouse NAFLD Model

[0113] During the experiment, the mice were in good condition and none died. The liver weight of the mice in the model group was significantly higher than that of the normal group (P < 0.01), and the liver weight of the mice in the UJ group was significantly lower than that of the model group (P < 0.01). The liver weight / body weight ratio of the mice in the model group was significantly higher than that of the normal group (P < 0.01), and there were no significant changes in each drug treatment group compared with the model group.

[0114] 4.2. Changes in Serum ALT and Liver TG of Mice

[0115] The serum ALT of the mice in the model group was significantly higher than that in the normal group (P<0.01), and the serum ALT of the mice in the UF, UG, UH, UK, and UL groups was significantly lower than that in the model group (P<0.05, P<0.01)( Figure 1 A). The liver TG of the mice in the model group was significantly higher than that in the normal group (P<0.01), and the liver TG of the mice in the UL and UO groups was significantly lower than that in the model group (P<0.05, P<0.01)( Figure 1 B).

[0116] 4.3 Histopathological changes of the liver

[0117] In the liver tissue sections of the mice in the normal group, the hepatic lobule structure was clear, the hepatic plates were arranged neatly in cords, and there was no infiltration of inflammatory cells. In the liver tissue sections of the rats in the model group, macrovesicular steatosis of hepatocytes, infiltration of inflammatory cells, and ballooning degeneration were seen. The UF, UG, UH, UK, UL, and UO groups were significantly alleviated to varying degrees compared with the model group, and the changes in other groups were not significant.

[0118] The embodiments of the present invention have been introduced in detail above. Specific examples are used in this article to elaborate on the principle and implementation manner of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. At the same time, based on the idea of the present invention, changes or deformations made by those skilled in the art in the specific implementation manner and application scope of the present invention all fall within the protection scope of the present invention. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, characterized in that, The pharmaceutical composition comprises the following components: magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside, wherein the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (16.036-17.724):(12.6825-14.0175):(95.00-105.00):(28.50-31.50):(3.382-3.738):(0.95-1.05):(1.1305-1.2495):(6.3365-7.0035); or the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (5.073-5.607):(71.25-78.75):(71.25-78.75):(16.036-17.724):(4.5125-4.9875):(0.1235-0.1365):(4.75-5.25):(35.625-39.375); or the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (6.764-7.476):(22.5435-24.9165):(16.91-18.69):(16.036-17.724):(2.5365-2.8035):(0.95-1.05):(1.501-1.659):(11.2765-12.4635); or the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (6.764-7.476):(12.6825-14.0175):(12.6825-14.0175):(38.00-42.00):(8.018-8.862):(0.304-0.336):(0.8455-0.9345):(15.029-16.611); or the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin, and pinoresinol 4-O-β-glucoside is (28.50-31.50):(95.00-105.00):(53.4375-59.0625):(5.073-5.607):(6.0135-6.6465):(0.532-0.588):(3.5625-3.(11.2765 to 12.4635); or the mass ratio between magnolol, gentisic acid, baicalin, syringin, wogonoside, honokiol, prunasin and pinoresinol 4-O-β-glucoside is (16.036 to 17.724):(53.4375 to 59.0625):(16.91 to 18.69):(21.375 to 23.625):(14.25 to 15.75):(0.7125 to 0.7875):(3.5625 to 3.9375):(15.029 to 16.611).

2. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

3. The pharmaceutical composition according to claim 2, wherein, The excipients are selected from one or more of the following: diluents, wetting agents, binders, disintegrants, inclusion agents, flavoring agents, sustained-release agents, glidants, lubricants, dispersants, plasticizers, light-blocking agents, and antioxidants.

4. The pharmaceutical composition according to claim 1, wherein The dosage form of the pharmaceutical composition is powder, tablet, dripping pill, capsule, film, lozenge, granule, injection, or oral liquid.

5. The pharmaceutical composition according to claim 1, characterized in that, The pharmaceutical composition further comprises one or more drugs for preventing and / or treating non-alcoholic fatty liver disease.

6. The pharmaceutical composition according to claim 5, characterized in that, The drugs are selected from one or more of the following: ursodeoxycholic acid, farnesoid X receptor agonist, liver-selective thyroid hormone receptor beta agonist, fibroblast growth factor 21 analog, silymarin, polyene phosphatidylcholine, bicyclol, glycyrrhizin preparation, and reduced glutathione.

7. The pharmaceutical composition according to claim 6, wherein The farnesoid X receptor agonist is obeticholic acid.

8. The pharmaceutical composition according to claim 6, wherein The liver-selective thyroid hormone receptor beta agonist is resmetirom.

9. The pharmaceutical composition according to claim 6, wherein The fibroblast growth factor 21 analogs are elafibranor and pelafibranor.

10. The pharmaceutical composition according to claim 6, characterized in that, The glycyrrhizin preparations are magnesium isoglycyrrhizinate, compound glycyrrhizin, and diammonium glycyrrhizinate.

11. Use of the pharmaceutical composition according to any one of claims 1 to 10 in the preparation of a medicament for preventing and / or treating non-alcoholic fatty liver disease.

12. The use according to claim 11, characterized in that, The non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic hepatic fibrosis, and non-alcoholic liver cirrhosis.

13. The use according to claim 11, characterized in that, The pharmaceutical composition prevents and / or treats non-alcoholic fatty liver disease by one or both of the following ways: reducing the level of serum alanine aminotransferase and reducing the level of triglyceride in liver tissue.

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