Pharmaceutical composition containing honokiol and application of pharmaceutical composition in prevention and treatment of non-alcoholic fatty liver disease

Abstract

The invention discloses application of a compound with a formula I or a salt, a stereoisomer, a tautomer, a deuterated compound, a solvate, a metabolite, a prodrug or a mixture thereof in preparation of drugs for preventing and treating non-alcoholic fatty liver diseases. The compound disclosed by the invention can be used for remarkably inhibiting lipid deposition in AML12 cells induced by free fatty acid, improving fatty degeneration of liver cells, obviously relieving ballooning and reducing inflammatory cell infiltration. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a pharmaceutical composition comprising honokiol and use of the pharmaceutical composition in preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a metabolic stress-induced liver injury closely related to insulin resistance and genetic susceptibility. It is characterized by abnormal lipid deposition in the liver. Excluding other secondary factors such as excessive drinking, drugs, and fatty liver caused by genetic diseases, its disease spectrum includes nonalcoholic simple fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), and cirrhosis. NAFLD has become the most common chronic liver disease in the world, with a prevalence of about 25-30% in ordinary adults. NASH is the key pathological stage of NAFL progression to fibrosis and cirrhosis, and is one of the main causes of hepatocellular carcinoma. NASH-related cirrhosis has become the main reason for liver transplantation in women and people over 65 years old. Lifestyle changes (weight loss, exercise, healthy diet) are still the basic measures for the treatment of NAFLD. Guidelines from multiple countries recommend the use of glucagon-like peptide-1 analogs semaglutide and pioglitazone for patients with NASH confirmed by liver biopsy and type 2 diabetes, and vitamin E for patients with NASH confirmed by liver biopsy, but long-term high-dose use of vitamin E increases all-cause mortality and is associated with an increased incidence of prostate cancer in men, so its safety remains to be evaluated. In March 2024, the U.S. Food and Drug Administration approved the liver-targeted thyroid hormone receptor β-selective agonist Resmetrom for the treatment of NASH with significant fibrosis, but its long-term safety remains to be seen. In addition, the preliminary results of clinical trials of new drugs such as pan-PPAR agonists, fibroblast growth factor 21 analogs, and GLP-1 receptor / glucose-dependent insulinotropic polypeptide and GLP-1 receptor dual agonist (telportide) are satisfactory, but a large amount of clinical data is still needed to support them. The previously highly anticipated farnesoid X receptor agonist obeticholic acid has not been approved for the treatment of NASH in the United States and has been withdrawn from the market in Europe, which further suggests that effective drug development for NAFLD still requires efforts.

[0003] The pathological mechanism of NAFLD is complex. At present, drug design targeting a single target is likely one of the reasons why most drugs that showed potential in preclinical studies failed after entering clinical trials. Traditional Chinese medicine has a clinical practice basis for the treatment of NAFLD, and clinical studies have also shown the potential efficacy of certain Chinese herbal prescriptions or Chinese patent medicines on NAFLD, which may be related to the multi-component and multi-target characteristics of Chinese medicine. In addition, some Chinese herbal medicine extracts or monomers, such as total glycosides of white paeony in white peony, gardenia glycosides in gardenia, salvianolic acid B in salvia miltiorrhiza, and berberine in coptis chinensis, have also shown good intervention effects on NAFLD, but clinical trials are yet to be confirmed.

[0004] There is no report in the prior art that the compound of formula I claimed in the present invention or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof, or a composition comprising the above substances can be used to prevent and / or treat 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 use of a compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0006]

[0007] wherein R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C8 alkyl group, a C2 to C8 alkenyl group, a C2 to C8 alkynyl group, a C1 to C8 fluoroalkyl group, a C2 to C8 fluoroalkenyl group, a C2 to C8 fluoroalkynyl group, a C1 to C8 chloroalkyl group, a C2 to C8 chloroalkenyl group, a C2 to C8 chloroalkynyl group, a C1 to C8 bromoalkyl group, a C2 to C8 bromoalkenyl group, a C2 to C8 bromoalkynyl group, a C1 to C8 iodoalkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C 10 Aryl, substituted C6-C 10 Aryl, 3-8 membered heteroaryl, substituted 3-8 membered heteroaryl, monosaccharide, disaccharide or polysaccharide.

[0008] Further, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C3 alkyl group, a C2 to C3 alkenyl group, a C2 to C3 alkynyl group, a C1 to C3 fluoroalkyl group, a C2 to C3 fluoroalkenyl group or a C2 to C3 fluoroalkynyl group.

[0009] Further, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, hydroxyl, methyl, ethyl or propyl.

[0010] Furthermore, the compound of formula I is the honokiol shown in formula II,

[0011]

[0012] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, comprising a compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or a mixture thereof and gentisic acid or its salt, wherein the mass ratio of gentisic acid or its salt to the compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or a mixture thereof is (45 to 800): about 1.

[0013] Further, the mass ratio between the gentisic acid or its salt and the compound of formula I or its salt, stereoisomer, tautomer, deuterated product, solvate, metabolite, prodrug or mixture thereof is about 769.2: about 1, or about 200: about 1, or about 50: about 1.

[0014] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising a compound of formula I or a salt, stereoisomer, tautomer, deuterated product, solvate, metabolite, prodrug or a mixture thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0015] Furthermore, the compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels and inhibiting lipid deposition in AML12 cells.

[0016] Furthermore, the liver tissue pathological abnormality includes one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

[0017] Furthermore, the pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels.

[0018] Furthermore, the liver tissue pathological abnormality includes one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

[0019] Furthermore, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

[0020] Beneficial effects of the present invention:

[0021] The compound of the present invention can significantly inhibit lipid deposition in AML12 cells induced by free fatty acids, improve fatty degeneration of liver cells, significantly alleviate ballooning degeneration, and reduce inflammatory cell infiltration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without exceeding the scope of protection required by the present invention.

[0023] Figure 1 Schematic diagram of the structural formula of magnolol, honokiol and magnolol.

[0024] Figure 2 Schematic diagram of the effect of gentisic acid and honokiol on the relative viability of AML12 cells after 24h incubation. A. Relative cell viability; B. TG content in cells. G: gentisic acid, O: honokiol, *P<0.05, **P<0.01, ****P<0.0001.

[0025] Figure 3 Schematic diagram of the effects of gentisic acid and magnolia officinalis on CDAHFD-induced non-alcoholic fatty liver disease in mice. A, HE staining of liver tissue, B, TG of liver tissue, C, serum ALT. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Gl, low dose of gentisic acid, Gm, medium dose of gentisic acid, Gh, high dose of gentisic acid, Ol, low dose of magnolia officinalis, Om, medium dose of magnolia officinalis, Oh, high dose of magnolia officinalis.

[0026] Figure 4 Schematic diagram of the effects of GO prescription and different ratios of gentisic acid and magnolia officinalis phenol (UA-UF) on CDAHFD-induced non-alcoholic fatty liver disease in mice. A, HE staining of liver tissue, B, TG of liver tissue, C, serum ALT. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0027] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0028] Unless otherwise indicated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by those of ordinary skill in the field of the invention or the field in which the terms are used. Although any methods, conditions, substances or materials similar to or equivalent to those disclosed herein may be used in the practice of the present invention, preferred methods, conditions, substances or materials are described herein.

[0029] The present invention is intended to encompass all alternatives, variations and equivalents that may be included in the present invention as defined by the claims. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described.

[0030] Unless otherwise indicated, the following definitions used herein shall apply.For purposes of the present invention, the chemical elements are as per the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994, the entire contents of which are incorporated herein by reference.

[0031] 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.

[0032] In the present invention, the term "comprising" is synonymous with "including". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus comprising the listed elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0033] The term "salt" refers to salts formed between the compound of formula I and an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed between the compound of formula I and an inorganic base; or methylamine salts, ethylamine salts or ethanolamine salts formed between the compound of general formula I and an organic base.

[0034] The term "stereoisomers" refers to compounds that have identical chemical constitution, but differ in the way the atoms or groups are arranged in space. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotamers), geometric (cis / trans) isomers, atropisomers, and the like.

[0035] The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can be interconverted through a low energy barrier. If tautomerism is possible (such as in solution), a chemical equilibrium of tautomers can be reached. For example, proton tautomers (also known as prototropic tautomers) include interconversions through proton migration, such as keto-enol isomerization and imine-enamine isomerization.

[0036] When the tautomerism (e.g., keto-enol tautomerism) phenomenon of the compound of the present invention or its prodrug exists, it is claimed that both their respective single forms (e.g., keto or enol form) and mixtures thereof in any ratio are protected. The same applies to their stereoisomers, for example, enantiomers, diastereomers, conformers (rotamers), geometric (cis / trans) isomers, atropisomers, etc.

[0037] If necessary, tautomers can be separated according to methods known in the art (e.g., liquid chromatography). The same applies to their enantiomers, for example, by separation using a chiral stationary phase. In addition, enantiomers can be separated by conversion into diastereomers, i.e., coupling with an enantiomerically pure auxiliary compound, followed by separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of the compounds of the invention can be obtained by stereoselective synthesis using optically pure starting materials.

[0038] The term "deuterated compound" used in the present invention refers to a deuterated compound generated by replacing one or more hydrogen atoms in the compound of formula I with deuterium.

[0039] The term "solvate" as used herein refers to an association formed by one or more solvent molecules and a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, ethanolamine or mixtures thereof. The term "hydrate" refers to an association formed by a solvent molecule being water. When the solvent is water, the term "hydrate" may be used. In one embodiment, one molecule of a compound of the present invention may be combined with one water molecule, such as a monohydrate; in another embodiment, one molecule of a compound of the present invention may be combined with more than one water molecule, such as a dihydrate; in yet another embodiment, one molecule of a compound of the present invention may be combined with less than one water molecule, such as a hemihydrate. It should be noted that the hydrates of the present invention retain the biological effectiveness of the non-hydrated form of the compound.

[0040] The term "metabolite" as used herein refers to a product obtained by metabolism of a specific compound or its salt in vivo. The metabolite of a compound can be identified by techniques known in the art, and its activity can be characterized by experimental methods as described herein. Such products can be obtained by administering the compound through oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, etc. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting the compounds of the present invention with mammals for a period of time.

[0041] The term "prodrug" as used in the present invention refers to a compound that is converted in vivo into a compound of Formula I. Such conversion is affected by the hydrolysis of the prodrug in the blood or by the conversion of the prodrug into the parent structure by enzymes in the blood or tissues. The prodrug compounds of the present invention may be esters. In the prior invention, esters that can be used as prodrugs include phenyl esters, aliphatic (C 1-24 ) esters, acyloxymethyl esters, carbonates, carbamates and amino acid esters. For example, a compound of the present invention contains a hydroxyl group, which can be acylated to obtain a prodrug form of the compound. Other prodrug forms include phosphates, such as these phosphate compounds obtained by phosphorylation of the hydroxyl group on the parent.

[0042] Unless otherwise stated, all suitable salts, stereoisomers, tautomers, deuterated forms, solvates, metabolites and prodrugs of the compounds of the invention are included within the scope of the invention.

[0043] In the present invention, the term "C1 to C8" refers to having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms. "3-8 members" refers to having 3-8 ring atoms, and so on.

[0044] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety. For example, the term "C1 to C8 alkyl group" refers to a straight or branched alkyl group having 1 to 8 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl.

[0045] In the present invention, the term "alkenyl" refers to a straight chain or branched hydrocarbon moiety containing at least one double bond. For example, the term "C2 to C8 alkenyl" refers to a straight chain or branched alkenyl group containing one double bond having 2 to 8 carbon atoms, including but not limited to ethenyl, propenyl, n-butenyl, and isobutenyl.

[0046] In the present invention, the term "alkynyl" refers to a straight or branched alkynyl group containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl and the like.

[0047] In the present invention, the term "aryl" refers to a hydrocarbon moiety containing one or more aromatic rings. 10 The term "aryl" refers to an aromatic ring group having 6 to 10 carbon atoms and containing no heteroatoms in the ring, such as phenyl, naphthyl, etc.

[0048] Unless otherwise specified, the alkyl, alkenyl, alkynyl, heteroaryl and aryl groups described herein are substituted and unsubstituted groups, and possible substituents include, but are not limited to: hydroxy, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C6 alkoxy, aryl, heteroaryl, heteroaryloxy ... C10 alkylamino, C1-C20 dialkylamino, arylamino, diarylamino, C1-C10 alkylsulfamoyl, arylsulfamoyl, C1-C10 alkylimino, C1-C10 alkylsulfoimino, arylsulfoimino, thiol, C1-C10 alkylthio, C1-C10 alkylsulfonyl, arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidino, ureido, cyano, acyl, thioacyl, acyloxy, carboxyl and carboxylate groups.

[0049] In the present invention, the substitution is mono- or poly-substitution, and the poly-substitution is di-, tri-, tetra- or penta-substitution. The di-substitution means having two substituents, and so on.

[0050] As described in the background technology section, there is no report in the prior art that the compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof, or a composition comprising the above substances can be used to prevent and / or treat liver diseases such as non-alcoholic fatty liver disease (non-alcoholic fatty hepatitis). In order to solve the above problems, the present invention provides a use of a compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease,

[0051]

[0052] wherein R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C8 alkyl group, a C2 to C8 alkenyl group, a C2 to C8 alkynyl group, a C1 to C8 fluoroalkyl group, a C2 to C8 fluoroalkenyl group, a C2 to C8 fluoroalkynyl group, a C1 to C8 chloroalkyl group, a C2 to C8 chloroalkenyl group, a C2 to C8 chloroalkynyl group, a C1 to C8 bromoalkyl group, a C2 to C8 bromoalkenyl group, a C2 to C8 bromoalkynyl group, a C1 to C8 iodoalkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C 10 Aryl, substituted C6-C 10 Aryl, 3-8 membered heteroaryl, substituted 3-8 membered heteroaryl, monosaccharide, disaccharide or polysaccharide.

[0053] In a preferred embodiment, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C3 alkyl group, a C2 to C3 alkenyl group, a C2 to C3 alkynyl group, a C1 to C3 fluoroalkyl group, a C2 to C3 fluoroalkenyl group or a C2 to C3 fluoroalkynyl group.

[0054] In a preferred embodiment, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, hydroxyl, methyl, ethyl or propyl.

[0055] In a preferred embodiment, the compound of formula I is honokiol as shown in formula II,

[0056]

[0057] According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, comprising a compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or a mixture thereof and gentisic acid or its salt, wherein the mass ratio of gentisic acid or its salt to the compound of the above-mentioned formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or a mixture thereof is (45 to 800): about 1.

[0058] In the present invention, "about" refers to a value within the range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0059] In the present invention, when mass ratio or other value or parameter is expressed in a range, preferred range, or 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 and any range lower limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately. For example, when the range "45-800" is disclosed, the described range should be interpreted as including the range "45-800", "45-600", "45-400", "45-200", "45-100", "100-800", "100-600", "100-400", "100-200", "200-800", "200-600", "200-400", "400-800", "400-600", "600-800" and the like. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end value and all integers and fractions within the range.

[0060] In a preferred embodiment, the mass ratio between the gentisic acid or a salt thereof and the compound of formula I or its salt, stereoisomer, tautomer, deuterated form, solvate, metabolite, prodrug or a mixture thereof is about 769.2: about 1, or about 200: about 1, or about 50: about 1.

[0061] In the present invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 769.2" includes ±5% of 769.2, or from 730.74 to 807.66; "about 200" includes ±5% of 200, or from 190 to 210; "about 50" includes ±5% of 50, or from 47.5 to 52.5; "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0062] According to another aspect of the present invention, there is provided a use of a pharmaceutical composition comprising a compound of formula I or a salt, stereoisomer, tautomer, deuterated product, solvate, metabolite, prodrug or a mixture thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

[0063] In a preferred embodiment, the compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels and inhibiting lipid deposition in AML12 cells.

[0064] In a preferred embodiment, the liver tissue pathological abnormality includes one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

[0065] In a preferred embodiment, the pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels.

[0066] In a preferred embodiment, the liver tissue pathological abnormality includes one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

[0067] In a preferred embodiment, the non-alcoholic fatty liver disease includes one or more of the following: non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, non-alcoholic fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

[0068] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions or conditions recommended by the manufacturer.

[0069] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those 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 implementation methods and materials described herein are for demonstration purposes only.

[0070] The above features mentioned in the present invention or the features mentioned in the embodiments can be combined in any way. All the features disclosed in this patent specification can be used in combination with any combination form, and each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0071] Example 1 Effects of Honokiol, Gentisic Acid, Honokiol and Honokiol on Fatty Acid-Induced Fatty Degeneration of AML12 Mouse Hepatocytes

[0072] 1. Experimental Materials

[0073] 1.1 Culture medium, reagents, and drugs

[0074] DMEM:F12 (Gibco, catalog number: 11320033), the basic culture medium contains 10% fetal bovine serum, 1% ITS Liquid Media Supplement (Sigma, I3146), 40ng / ml dexamethasone (Sigma, catalog number D4902-100mg), and 1% double antibody.

[0075] Oleic acid (Sigma, catalog number O1383) and palmitic acid (Sigma, catalog number: A602448-0050) were prepared according to the reference method (Song Yuling, 2023) (Li Ran, 2022) and used as needed.

[0076] Honokiol (catalog number: AB3389, purity 98%), magnolol (catalog number: AB0682, purity 98%), honokiol (catalog number: AB1042, purity 98%), and gentisic acid (catalog number: AB1077, purity 98%) were purchased from Chengdu Aifa Biotechnology Co., Ltd.

[0077] The structural formulas of honokiol, magnolol and honokiol are as follows Figure 1 shown.

[0078] CCK8 kit (Biyuntian, catalog number: C0038), cell triglyceride detection kit (Beijing Pulilai, catalog number E1013), according to the instructions.

[0079] 1.2 Cell lines

[0080] AML12 mouse hepatocyte cell line was purchased from Shanghai Meiyan Biotechnology Co., Ltd.

[0081] 2. Modeling and grouping

[0082] 2.1 Cytotoxicity test:

[0083] The cells were seeded in a 96-well plate, and the culture medium was replaced when the cells grew to more than 80%.

[0084] 2.1.1. Cytotoxicity test of magnolol and gentisic acid

[0085] AML12 cells were divided into blank control group, honokiol group (200, 100, 50, 25 μM), and gentisic acid group (200, 100, 50, 25 μM), with 10 wells in each group. The corresponding drugs were added and incubated for 24 h, and the cell viability was detected by CCK8 kit (Biyuntian, Cat. No.: C0038).

[0086] 2.1.2. Cytotoxicity test of magnolol, magnolol and magnolol

[0087] AML12 cells were divided into blank control group, honokiol group (50 μM), magnolol group (50 μM), and magnolol group (50 μM), with 10 wells in each group. The corresponding drugs were added and incubated for 24 h, and the cell viability was detected by CCK8 kit.

[0088] 2.2. AML12 cell fat deposition experiment

[0089] AML-12 cells were cultured in 6-well plates and divided into normal group, model group, gentisic acid group (10 μM), honokiol group (10 μM), honokiol group (10 μM), and magnolol group (10 μM), with 6 wells in each group. Except for the cells in the normal group, the cells in the other groups were stimulated with palmitic acid (0.6 mM) and oleic acid (0.17 mM) for 24 h (Zhou, Pang, Tripathi, Ho, Widjaja, Shekeran, Cook, Suzuki, Diehl, Petretto, Singh and Yen, 2022). The drug group was also added with the corresponding drugs for 24 h. After 24 h, the culture medium was aspirated and discarded, washed twice with PBS, 1 ml of 0.25% trypsin was added to digest the cells for 1 min, and the cells in each well were collected into a 15 mL EP tube and fixed to 3 ml. Cell count was performed and the total number of cells was calculated. Centrifuge at 3000r / min for 5min, discard the supernatant, keep the precipitate, rinse with 1ml PBS, repeat twice. Resuspend with 1ml PBS for the last time and aspirate into a 1.5ml EP tube, centrifuge again to get the precipitate, and detect the intracellular TG content according to the instructions of the cell triglyceride detection kit.

[0090] 3. Experimental results

[0091] 3.1. Cytotoxicity of gentisic acid, honokiol, honokiol, and magnolol

[0092] Gentisic acid showed no cytotoxicity, honokiol showed cytotoxicity at a concentration of 100 μM, honokiol and magnolol showed cytotoxicity at a concentration of 50 μM, and honokiol, magnolol, and honokiol showed no cytotoxicity at a concentration of 10 μM (e.g. Figure 2 A).

[0093] 3.2 Effects of gentisic acid, magnolol, magnolol, and magnolol on free fatty acid-induced lipid deposition in AML12 cells

[0094] Gentisic acid and honokiol at a concentration of 10 μM significantly inhibited free fatty acid-induced lipid deposition in AML12 cells (eg Figure 2 B). Magnolol and honokiol showed cytotoxicity at a concentration of 50 μM (as shown in Figure 2 A), no inhibitory effect was observed on free fatty acid-induced lipid deposition in AML12 cells at a concentration of 10 μM (as shown in Figure 2 B).

[0095] Example 2 Effects of Hopoxetine and Gentisic Acid on Mice with Nonalcoholic Fatty Liver Disease

[0096] 1. Experimental Materials

[0097] 1.1 Experimental Animals

[0098] Male SPF grade C57BL / 6 mice, 6-8 weeks old, were housed in an environment of 22-25°C, 55-60% relative indoor humidity, natural light, and free access to drinking water.

[0099] 1.2 Drugs

[0100] Gentisic acid and magnolol.

[0101] 1.3 Reagents

[0102] Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD, catalog number: A06071302, Research Diets Inc), triglyceride kit (catalog number: A010017, Zhejiang Dongou Diagnostic Products Co., Ltd.).

[0103] 2. Experimental methods

[0104] 2.1 Animal model preparation and drug administration

[0105] After 1 week of adaptive feeding, the mice were randomly divided into a normal control group, a model group, a gentisic acid high (200 mg / kg body weight), medium (100 mg / kg body weight), and low-dose group (50 mg / kg), a honokiol high (2 mg / kg), medium (1 mg / kg body weight), and low-dose group (0.5 mg / kg body weight), and an obeticholic acid control group, with 8 mice in each group. The normal control group was fed with ordinary feed for 6 weeks, and the other groups were fed with CDAHFD feed for 6 weeks. From the third week onwards, the high, medium, and low-dose groups of gentisic acid, the high, medium, and low-dose groups of honokiol, and the obeticholic acid control group were given corresponding drugs, and the normal control group and the model group were gavaged with an equal amount of sterilized drinking water, and the samples were collected at the end of the sixth week.

[0106] 2.2 Sample Collection

[0107] 2.2.1 Serum

[0108] The samples were collected at the end of the 6th week. The mice were fasted for 12 hours, and their eyeballs were removed to collect blood. The mice were then killed by cervical dislocation. The blood was kept at 4°C for 4 hours and centrifuged at 3000 r / min for 15 minutes. 200 μL of serum was collected in a 1.5 ml epoxide tube and stored at -80°C.

[0109] 2.2.2 Liver

[0110] The mouse liver was removed from the abdominal cavity, rinsed with saline, dried on filter paper, photographed and weighed, and then two pieces of liver tissue with a size of about 1cm*1cm*1cm were taken from the central part of the liver lobe. One piece was placed in a pathology box and stored in formalin. The remaining liver tissue was quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator.

[0111] 2.3、Indicator detection

[0112] 2.3.1. Hematoxylin and eosin (H&E) staining of liver tissue

[0113] Staining was performed using a hematoxylin-eosin staining kit (Nanjing Jiancheng Bioengineering Institute, catalog number: C0105). Paraffin sections of liver tissue (4 μm) were baked on a 70°C slide baker (LEICA, Germany, model: HI1220) for 40 minutes and dewaxed in the following order: xylene, 10 minutes; xylene, 10 minutes; anhydrous ethanol, 5 minutes; anhydrous ethanol, 2 minutes; 95% ethanol, 2 minutes; 85% ethanol, 2 minutes; 70% ethanol, 2 minutes; tap water, wash 3 times; double distilled water, wash 3 times. Then, staining was performed in the following order: hematoxylin stain, 15 minutes; tap water, wash 3 times; double distilled water, wash 3 times; hydrochloric acid alcohol, 3 seconds; tap water, wash 3 times; double distilled water, wash 3 times; eosin, 10 seconds, 95% ethanol, 1 minute; 95% ethanol, 1 minute; anhydrous ethanol, 1 minute; anhydrous ethanol, 1 minute; xylene, 1 minute; xylene, 1 minute. Neutral resin mounting medium (Sinopharm Chemical Reagent Co., Ltd., catalog number: E675007-00100) was used for mounting. All pathological slides were scanned and archived by a digital radiographer (LEICA, Germany, model: SCN400).

[0114] 2.3.2. Liver triglyceride (TG) detection

[0115] Before measuring liver TG, prepare liver tissue homogenate in advance: weigh 50 mg of liver tissue and place it in a 2 mL centrifuge tube containing 3 steel beads, add 375 μL of anhydrous ethanol and 375 μL of acetone, grind it with an automatic sample grinder at 65 Hz for 60 s*3 times to obtain liver tissue homogenate, let it stand in a 4°C refrigerator overnight, centrifuge it the next day for 15 minutes (4°C, 3000 rpm / min), take the supernatant for measuring TG content, and operate according to the instructions.

[0116] 2.3.3 Serum ALT detection

[0117] Serum ALT was tested using a kit according to the instructions.

[0118] 3. Experimental results

[0119] 3.1. Pathological changes of mouse liver tissue

[0120] After H&E staining of liver tissue, it was observed under the microscope that the hepatocytes of mice in the normal control group had normal morphology, clear liver lobule structure, and neatly arranged hepatic cords. The hepatocytes of mice in the model group showed varying degrees of swelling, with huge fat vacuoles in the cytoplasm, the hepatocyte nuclei were squeezed to the edge, accompanied by ballooning of hepatocytes, and inflammatory cell infiltration was visible in some portal areas and lobules. The fatty degeneration and ballooning of hepatocytes in mice in the medium- and high-dose groups of gentisic acid, and the medium- and high-dose groups of magnolia officinalis phenol were significantly reduced, and the infiltration of inflammatory cells was reduced. The fatty degeneration and ballooning of hepatocytes in mice in the obeticholic acid control group were also significantly reduced, and inflammatory cells were rare (such as Figure 3 A).

[0121] 3.2. TG content in mouse liver

[0122] The results of liver tissue TG content determination also showed that the TG content of liver tissue of mice in the model group was significantly higher than that in the normal control group, and the TG content of liver tissue of mice in the medium and high dose groups of gentisic acid, medium and high dose groups of honokiol, and obeticholic acid groups was significantly lower than that in the model group. There was no significant difference among the drug groups (such as Figure 3 B).

[0123] 3.3. Mouse serum ALT

[0124] Compared with the normal group, the serum ALT activity of the model group mice was significantly increased, and the serum ALT activity of the mice in the medium and high dose groups of gentisic acid, low, medium and high dose groups of honokiol and obeticholic acid intervention was significantly lower than that in the model group. There was no significant difference among the drug groups (such as Figure 3 C).

[0125] Example 3 Pharmacodynamic screening experiment of the combination of magnolol-gentisic acid

[0126] 1. Experimental Materials

[0127] 1.1 Experimental Animals

[0128] Male SPF grade C57BL / 6 mice, 6-8 weeks old, were housed in an environment of 22-25°C, 55-60% relative indoor humidity, natural light, and free access to drinking water.

[0129] 1.2 Drugs

[0130] Gentisic acid and magnolol.

[0131] 1.3 Reagents

[0132] Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD, catalog number: A06071302, Research Diets Inc), triglyceride kit (catalog number: A010017, Zhejiang Dongou Diagnostic Products Co., Ltd.).

[0133] 2. Experimental methods

[0134] 2.1 Experimental Design Method

[0135] Different proportions of gentisic acid and magnolia officinalis phenol were calculated according to the uniform design table U6*(6 4) table (as shown in Table 1), two monomer components: gentisic acid (represented by "G") and magnolia officinalis phenol (represented by "O") were used as the investigation factors, and each factor took 6 dose levels, with the upper limit of G dose being 200 mg / kg and the upper limit of O dose being 2 mg / kg, decreasing at a rate of 0.5, according to U6*(6 4 ) table uses the table (as shown in Table 2) to select the 1st and 3rd columns, as shown in Table 3.

[0136] Table 1 Uniform design table U6*(6 4 )surface

[0137] Group 1 2 3 4 1 1 2 3 6 2 2 4 6 5 3 3 6 2 4 4 4 1 5 3 5 5 3 1 2 6 6 5 4 1

[0138] Table 2U6*(6 4 ) Table usage table

[0139] s Column number D 2 1,3 0.1875 3 1,2,3 0.2656 4 1,2,3,4 0.299

[0140] Table 3 Uniform design experimental plan

[0141] Group G (mg / kg) O(mg / kg) UA 6.25 0.25 UB 12.5 2 UC 25 0.125 UD 50 1 UE 100 0.5 UF 200 0.0625

[0142] In addition, the GO group (gentisic acid = 100 mg / kg, honokiol = 0.13 mg / kg) was added on this basis.

[0143] 2.2 Animal model preparation and drug administration

[0144] After 1 week of adaptive feeding, the mice were randomly divided into a normal control group, a model group, a GO formula group, a GO different ratio group, namely a uniformly designed UA-UF group, and an obeticholic acid control group, with 8 mice in each group. The normal control group was fed with ordinary feed for 6 weeks, and the other groups were fed with CDAHFD feed for 6 weeks. From the third week, the GO formula group, UA-UF group, and obeticholic acid control group were given corresponding drugs, and the normal control group and the model group were gavaged with an equal amount of sterilized drinking water, and the samples were collected at the end of the sixth week.

[0145] 2.3 Sample Collection

[0146] 2.3.1 Serum

[0147] The samples were collected at the end of the 6th week. The mice were fasted for 12 hours, and their eyeballs were removed to collect blood. The mice were then killed by cervical dislocation. The blood was kept at 4°C for 4 hours and centrifuged at 3000 r / min for 15 minutes. 200 μL of serum was collected in a 1.5 ml epoxide tube and stored at -80°C.

[0148] 2.3.2 Liver

[0149] The mouse liver was removed from the abdominal cavity, rinsed with saline, dried on filter paper, photographed and weighed, and then two pieces of liver tissue with a size of about 1cm*1cm*1cm were taken from the central part of the liver lobe. One piece was placed in a pathology box and stored in formalin. The remaining liver tissue was quickly frozen in liquid nitrogen and stored in a -80℃ refrigerator.

[0150] 2.4、Indicator detection

[0151] 2.4.1. Hematoxylin and eosin (H&E) staining of liver tissue was the same as in Example 2.

[0152] 2.4.2. Liver triglyceride (TG) detection

[0153] Same as Example 2.

[0154] 2.4.3 Serum ALT detection

[0155] Same as Example 2.

[0156] 3. Experimental results

[0157] 3.1. Pathological changes of mouse liver tissue

[0158] After H&E staining of liver tissue, it was observed under the microscope that the hepatocytes of mice in the normal control group had normal morphology, clear liver lobule structure, and neatly arranged hepatic cords. The hepatocytes of mice in the model group showed varying degrees of swelling, with huge fat vacuoles in the cytoplasm, the hepatocyte nuclei were squeezed to the edge, accompanied by ballooning of hepatocytes, and inflammatory cell infiltration was visible in some portal areas and lobules. The fatty degeneration and ballooning of hepatocytes in mice in the GO prescription group (gentisic acid = 100 mg / kg, magnolia officinalis phenol = 0.13 mg / kg), UC, UD, UE, and UF groups were significantly reduced, and the infiltration of inflammatory cells was reduced. The fatty degeneration and ballooning of hepatocytes in mice in the obeticholic acid control group were also significantly reduced, and inflammatory cells were rare (such as Figure 4 A).

[0159] 3.2. TG content in mouse liver

[0160] The results of liver tissue TG content determination also showed that the TG content of liver tissue of mice in the model group was significantly higher than that in the normal control group, and the TG content of liver tissue of mice in the GO prescription group, UC group, and UD group obeticholic acid group was significantly lower than that in the model group. There was no significant difference among the drug groups (such as Figure 4 B).

[0161] 3.3. Mouse serum ALT

[0162] Compared with the normal group, the serum ALT activity of the mice in the model group was significantly increased, and the serum ALT activity of the mice in the GO prescription group, UC, UD, UE, UF group and after the intervention of obeticholic acid was significantly lower than that in the model group. Figure 4 C).

[0163] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method of the present invention and its core idea. At the same time, changes or deformations made by those skilled in the art based on the ideas of the present invention, the specific implementation methods and application scope of the present invention, all belong to the scope of protection of the present invention. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Use of a compound of formula I or its salt, stereoisomer, tautomer, deuterated product, solvate, metabolite, prodrug or mixture thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease, in, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C8 alkyl group, a C2 to C8 alkenyl group, a C2 to C8 alkynyl group, a C1 to C8 fluoroalkyl group, a C2 to C8 fluoroalkenyl group, a C2 to C8 fluoroalkynyl group, a C1 to C8 chloroalkyl group, a C2 to C8 chloroalkenyl group, a C2 to C8 chloroalkynyl group, a C1 to C8 bromoalkyl group, a C2 to C8 bromoalkenyl group, a C2 to C8 bromoalkynyl group, a C1 to C8 iodoalkyl group, a C2 to C8 iodoalkenyl group, a C2 to C8 iodoalkynyl group, a C6-C 10 Aryl, substituted C6-C 10 Aryl, 3-8 membered heteroaryl, substituted 3-8 membered heteroaryl, monosaccharide, disaccharide or polysaccharide.

2. The compound according to claim 1, characterized in that R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl, a C1 to C3 alkyl group, a C2 to C3 alkenyl group, a C2 to C3 alkynyl group, a C1 to C3 fluoroalkyl group, a C2 to C3 fluoroalkenyl group or a C2 to C3 fluoroalkynyl group.

3. The compound according to claim 1, characterized in that R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, hydroxy, methyl, ethyl or propyl.

4. The compound according to claim 1, characterized in that The compound of formula I is the honokiol shown in formula II, 5. A pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease comprising a compound of formula I according to any one of claims 1 to 4 or a salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or a mixture thereof and gentisic acid or a salt thereof, characterized in that: The mass ratio of the gentisic acid or its salt to the compound of formula I or its salt, stereoisomer, tautomer, deuterated product, solvate, metabolite, prodrug or mixture thereof is (45-800):about 1.

6. The pharmaceutical composition according to claim 2, characterized in that The mass ratio between the gentisic acid or its salt and the compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof is about 769.2: about 1, or about 200: about 1, or about 50: about 1.

7. Use of a pharmaceutical composition comprising a compound of formula I as described in any one of claims 1 to 4 or a salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof and gentisic acid or a salt thereof in the preparation of a medicament for preventing and treating non-alcoholic fatty liver disease.

8. The use according to claim 1, characterized in that The compound of formula I or its salt, stereoisomer, tautomer, deuterated substance, solvate, metabolite, prodrug or mixture thereof prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, reducing serum alanine aminotransferase levels and inhibiting lipid deposition in AML12 cells; Preferably, the liver tissue pathological abnormalities include one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

9. The use according to claim 7, characterized in that: The pharmaceutical composition prevents and treats non-alcoholic fatty liver disease in one or more of the following ways: improving liver tissue pathological abnormalities, reducing liver tissue triglyceride levels, and reducing serum alanine aminotransferase levels; Preferably, the liver tissue pathological abnormalities include one or more of the following: fatty degeneration of hepatocytes, ballooning degeneration of hepatocytes and infiltration of inflammatory cells.

10. The use according to any one of claims 1 to 9, 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 fatty liver fibrosis and non-alcoholic fatty liver cirrhosis.

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