Application of alnustone and its analogues in the prevention and treatment of fatty liver disease

Abstract

The present invention belongs to the field of biomedical technology and provides an application of betulinone and its analogs in the prevention and treatment of fatty liver disease and adjuvant lipid-lowering. The present invention also provides a composition for treating fatty liver disease or adjuvant lipid-lowering, which comprises an effective dose of betulinone and its analogs. The betulinone and its analogs provided by the present invention have the effects of reducing blood triglyceride and liver triglyceride in vivo, reversing liver steatosis, and can improve liver fibrosis and inflammatory symptoms of mice with fatty hepatitis, and have good safety and no toxic side effects, and can be used for preparing preventive and therapeutic drugs for fatty liver disease, fatty hepatitis and related diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of alnustone in the prevention and treatment of fatty liver disease. Background Art

[0002] Disclosing the information of this background art is intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] With the great changes in modern people's eating habits and lifestyles, metabolic dysfunction-associated fatty liver disease (MASLD), also known as non-alcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease (MAFLD), has become the most common chronic liver disease and a worldwide medical and health problem. Its prevalence and incidence are continuously increasing, especially in China. This type of fatty liver disease includes a series of liver diseases such as simple steatosis, steatohepatitis, and its related liver fibrosis and cirrhosis. Metabolic dysfunction-associated steatohepatitis (MASH), also known as non-alcoholic steatohepatitis (NASH) or metabolic-associated steatohepatitis, is a severe form of fatty liver disease, which can further develop into advanced fibrosis, cirrhosis, hepatocellular carcinoma, and ultimately lead to liver-related death. In fact, MASLD is becoming the main cause of liver failure and liver transplantation in the next few decades, resulting in huge medical costs every year. With the continuous increase in its incidence and severe impact, fatty liver disease will continue to impose a heavy health and socioeconomic burden globally.

[0004] However, there are very few drugs for the treatment of the above-mentioned fatty liver disease, especially the effective drugs for reversing liver fibrosis are extremely scarce. In existing clinical studies, ursodeoxycholic acid can only improve the biochemical indexes of liver function, but cannot relieve steatohepatitis. Another commonly used drug, obeticholic acid, a farnesoid X receptor agonist, has the effect of relieving liver fibrosis, but adverse reactions such as itching and dyslipidemia have hindered its clinical approval. So far, only one drug, a liver-selective thyroid hormone receptor beta agonist, has been approved for the clinical treatment of MASH complicated with fibrosis. Therefore, it is urgent to discover more effective and safe drugs to cope with the increasing burden of fatty liver disease.

[0005] Natural products have many advantages in the prevention and treatment of various diseases due to their wide sources, broad treatment spectra, high safety, etc. Alpinone is a non-phenolic diarylheptane and a natural active product derived from the traditional Chinese medicine Alpinia katsumadai. Previous studies have found that alpinone has activities such as anti-tumor and antibacterial. In the prior art, the related medical uses of alpinone have also been disclosed: CN116019792A discloses the use of alpinone in inhibiting and treating gastric cancer, and it is disclosed that alpinone inhibits the proliferation of undifferentiated gastric cancer cells HGC-27. CN115364080A discloses the application of alpinone in the preparation of drugs for the prevention and treatment of disorders of carbohydrate metabolism, and it is disclosed that alpinone is used in the preparation of drugs for disorders of carbohydrate metabolism. CN117159514A discloses the application of alpinone in the preparation of drugs for preventing or treating atopic dermatitis, and it is disclosed that alpinone is used in the preparation of drugs for atopic dermatitis, as well as the combined application of alpinone and other drugs for atopic dermatitis. CN118217267A discloses the use of alpinone or its derivatives in the preparation of drugs for treating thrombocytopenia, and it is disclosed that alpinone or its derivatives are used in the preparation of drugs for treating thrombocytopenia. Alpinone has no effect on the cell viability of megakaryocytes in vitro, is safer to use, and has a certain activity in promoting the differentiation and maturation of megakaryocytes and the production of proplatelets in vitro. However, whether this compound has a preventive and therapeutic effect on fatty liver disease, non-alcoholic steatohepatitis and related liver diseases has not been reported previously. Summary of the Invention

[0006] In order to find agents or drug lead compounds that can be used for metabolic liver diseases such as fatty liver disease and non-alcoholic steatohepatitis, the present invention provides a new medical application of alpinone, which can improve hepatic steatosis, fibrosis and inflammation, and is used for the prevention and treatment of fatty liver disease.

[0007] To achieve the above object, the present invention adopts the following technical solutions.

[0008] Use of a linear diarylheptane compound and its derivatives in the preparation of drugs for treating and preventing fatty liver disease.

[0009] Use of a linear diarylheptane compound and its derivatives in the preparation of health products for assisting in reducing blood lipid.

[0010] The structural formula of the linear diarylheptane compound is:

[0011] ,

[0012] wherein, R1, R2, R3, and R4 are each independently selected from -H, -OH, and -OCH3.

[0013] Preferably, the compound is selected from:

[0014] Compound 1: (Alnusone);

[0015] Compound 2: ;

[0016] Compound 3: ; or,

[0017] Compound 4: (Tsaokoarylone).

[0018] The derivatives of the linear diarylheptane compounds are physiologically acceptable salts formed by the linear diarylheptane compounds and inorganic bases or organic bases; including but not limited to alkali metal salts, alkaline earth metal salts, ammonium salts, and salts formed with nitrogen-containing organic bases; such as sodium salts, potassium salts, calcium salts, and magnesium salts. The nitrogen-containing organic bases include but are not limited to procaine, trimethylamine, triethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, diethylamine, dicyclohexylamine, dibenzylamine, N-benzyl-β-phenethylamine, N,N'-dibenzylethylenediamine.

[0019] The derivatives are physiologically acceptable esters formed by the above linear diarylheptane compounds and inorganic acids or organic acids; the inorganic acids are selected from sulfuric acid, phosphoric acid, carbonic acid, and hydrochloric acid; the organic acids are selected from formic acid, acetic acid, propionic acid, butyric acid, maleic acid, oxalic acid, methanesulfonic acid, succinic acid, or fatty acids.

[0020] The linear diarylheptane compounds can be obtained by chemical synthesis or biological extraction. Preferably, the biological extraction sources are selected from Alpinia katsumadai, Alnus cremastogyne, Curcuma longa, Amomum tsaoko, etc.

[0021] The diseases include but are not limited to metabolic dysfunction-related fatty liver disease, metabolic-related fatty liver disease, non-alcoholic fatty liver disease, alcoholic fatty liver disease, metabolic dysfunction-related steatohepatitis, metabolic-related steatohepatitis, non-alcoholic steatohepatitis, alcoholic steatohepatitis, liver fibrosis, and cirrhosis caused by it.

[0022] A composition for treating fatty liver diseases or assisting in reducing blood lipids, comprising an effective dose of linear diarylheptane compounds and their derivatives, plants containing linear diarylheptane compounds and their derivatives, or plant extracts containing linear diarylheptane compounds and their derivatives.

[0023] Preferably, the plants containing linear diarylheptane compounds are selected from Alpinia katsumadai, Alnus cremastogyne, Curcuma longa, and Amomum tsaoko.

[0024] The above composition can be prepared according to methods known in the art or disclosed in the prior art.

[0025] The above composition further comprises pharmaceutically or food-acceptable excipients for preparing the active ingredient into any dosage form suitable for human or animal use. Preferably, the intake form of the composition can be an oral preparation or an injection preparation. For the convenience of taking medicine, the composition can also be prepared into a sustained-release preparation or a controlled-release preparation. The above-mentioned oral preparations include tablets, capsules, granules, oral liquids or syrups.

[0026] The present invention has the following advantages:

[0027] The present invention has confirmed through experiments that alnustone has the effects of reducing blood triglyceride and liver triglyceride in vivo, reversing liver steatosis, and can improve liver fibrosis and inflammatory symptoms in mice with fatty hepatitis. Moreover, it has good safety, no toxic and side effects, and can be used to prepare preventive and therapeutic drugs for fatty liver disease, fatty hepatitis and related diseases. Description of the Drawings

[0028] Figure 1 Serum triglyceride levels in fatty liver disease mice after treatment with control solvent and alnustone;

[0029] Figure 2 Liver triglyceride levels in fatty liver disease mice after treatment with control solvent and alnustone;

[0030] Figure 3 Liver histological HE staining morphology, Oil Red O staining morphology and Masson staining morphology in fatty liver disease mice after treatment with control solvent and alnustone;

[0031] Figure 4 Liver histological lipid droplet vacuole quantification level, Oil Red O staining lipid quantification level and Masson staining fibrosis quantification level in fatty liver disease mice after treatment with control solvent and alnustone;

[0032] Figure 5 Serum triglyceride levels in fatty hepatitis mice after treatment with control solvent and alnustone;

[0033] Figure 6 Liver triglyceride levels in fatty hepatitis mice after treatment with control solvent and alnustone;

[0034] Figure 7 Liver histological HE staining morphology, Oil Red O staining morphology and Masson staining morphology in fatty hepatitis mice after treatment with control solvent and alnustone;

[0035] Figure 8 Liver histological lipid droplet vacuole quantification level, Oil Red O staining lipid quantification level and Masson staining fibrosis quantification level in fatty hepatitis mice after treatment with control solvent and alnustone;

[0036] Figure 9 The expression levels of liver inflammatory factors in mice with non-alcoholic steatohepatitis after treatment with control solvent and betulone

[0037] Figure 10 The body weights of mice after treatment with control solvent and betulone Specific embodiments

[0038] The present invention will be further described below in conjunction with the embodiments and the drawings, but the present invention is not limited by the following embodiments. It can be confirmed by experiments that compounds 1-4 can improve the blood lipid and liver lipid levels of mice with non-alcoholic fatty liver disease, reverse hepatic steatosis and alleviate liver fibrosis in mice with non-alcoholic steatohepatitis. Taking compound 1 (betulone) as an example, the research process will be specifically described. Compound 1 (betulone) used in the examples was purchased commercially, and the HPLC purity was not less than 98%.

[0039] Example 1 Betulone improves blood lipid and liver lipid levels of mice with non-alcoholic fatty liver disease and reverses hepatic steatosis

[0040] 1. Construction of non-alcoholic fatty liver disease animal model

[0041] Wild-type C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed a high-fat diet (60% fat, Research Diet) starting at 8 weeks of age for 12 consecutive weeks. Mice fed with this high-fat diet can exhibit metabolic injury phenotypes such as obesity, elevated blood lipid levels, hepatic lipid accumulation and steatosis, which is a commonly used non-alcoholic fatty liver disease mouse model.

[0042] 2. Betulone administration protocol

[0043] The non-alcoholic fatty liver disease mice were randomly divided into two groups. Betulone group: Betulone powder (purchased from Shanghai TargetMol Co., Ltd.) was dissolved in corn oil containing 5% DMSO and intraperitoneally injected into C57 mice at a dose of 10 mg / kg daily. Control group: The same model mice were intraperitoneally injected with the same dose of corn oil solvent containing 5% DMSO daily. After two weeks of administration, the samples were taken to observe the therapeutic effect of betulone on non-alcoholic fatty liver disease mice.

[0044] 3. Detection of triglyceride content

[0045] (1) Serum: After non-alcoholic fatty liver disease mice were continuously intraperitoneally injected with betulone for 14 days, blood was collected after fasting for 6 hours. Serum was collected by centrifuging at 3000 rpm for 15 minutes at 4°C, and the triglyceride content in the serum was detected according to the enzymatic determination kit for liquid sample triglyceride (TG) content (purchased from Beijing Pulilai Gene Technology Co., Ltd.). The absorbance was measured by the colorimetric method using an enzyme-linked immunosorbent assay reader.

[0046] (2) Liver: When sampling, cut an equal amount of liver samples and freeze them in liquid nitrogen. Before testing, add lysis buffer to the samples for fragmentation, then centrifuge at 4°C 12000g for 10 minutes to obtain the supernatant. The triglyceride content in the supernatant was detected using the tissue cell triglyceride (TG) content enzymatic assay kit (purchased from Beijing Pulilai Gene Technology Co., Ltd.), and the protein content in the supernatant was detected using the BCA protein quantification kit (purchased from Thermo Fisher Scientific). The absorbance was measured by colorimetry using an enzyme-labeled instrument. The triglyceride content of each sample was corrected using the protein content.

[0047] 4. Liver histology HE staining, Oil Red O staining, Masson staining and quantification

[0048] (1) HE staining: Fresh liver tissue was fixed in 4% paraformaldehyde fixative for at least 24 hours, dehydrated and embedded, and 5 μm tissue sections were cut. The sections were routinely dewaxed into distilled water. Hematoxylin staining was performed for 2 minutes, and the tissues were washed with tap water to turn blue. After washing with distilled water, the tissues were stained with eosin for 1 minute, dehydrated with gradient alcohol, and sealed with neutral gum. Images were collected under a microscope, and the vacuolar area was quantitatively analyzed using Image J software.

[0049] (2) Oil Red O staining: Mouse liver frozen sections were rewarmed and fixed in 4% paraformaldehyde fixative for 20 minutes. After washing with distilled water, they were stained with 60% isopropanol for 2 minutes, then directly stained in Oil Red O staining solution in the dark for 20 minutes, washed with distilled water, stained with hematoxylin for 2 minutes, and then washed with distilled water and sealed with glycerol gelatin. Images were collected under a microscope, and the positive area of Oil Red staining was quantitatively analyzed using Image J software.

[0050] (3) Masson staining: The sections were routinely dewaxed to distilled water. The sections were stained with mordant solution in a 60°C incubator for 1 hour, and then rinsed with distilled water 3 times, 3 minutes each time. The sections were dripped with lapis lazuli blue stain for 2 minutes, and washed with distilled water 2 times, 10 seconds each time. The sections were dripped with Mayer's hematoxylin stain for 2 minutes, and washed with distilled water 2 times, 10 seconds each time. The sections were differentiated with acidic differentiation solution and washed with water. The sections were rinsed with tap water for 10 minutes to return to blue. The sections were dripped with Ponceau fuchsin stain for 5 minutes, and washed with distilled water 2 times, 10 seconds each time. The sections were differentiated with phosphomolybdic acid solution for 5 minutes. The upper solution was discarded and aniline blue stain was directly added for 3 minutes. The sections were washed with weak acid solution for 2 minutes. The sections were rapidly dehydrated with 95% ethanol for 3 seconds. The sections were dehydrated with anhydrous ethanol 2 times, 5 seconds each time. The sections were transparentized with xylene 2 times, 2 minutes each time. The sections were sealed with neutral gum. Images were collected under a microscope, and the positive area of Masson staining was quantitatively analyzed using Image J software.

[0051] The data analysis results after the above experiments showed that in the diet-induced fatty liver disease mouse model, the serum triglyceride level of mice was significantly reduced after treatment with alder ketone ( Figure 1 ), triglyceride levels in the liver decreased significantly (Figure 2 ). More importantly, the HE staining results of liver tissues showed that the lipid droplets and vacuoles in hepatocytes were significantly reduced after treatment with betulinone, and there were no other adverse effects on the morphology of hepatocytes ( Figure 3 ). In addition, Oil Red O staining of the liver showed that the lipid levels in hepatocytes were significantly reduced after treatment with betulinone; Masson staining of the liver showed that betulinone could significantly alleviate the already formed liver fibrosis ( Figure 3 and Figure 4 ).

[0052] The experimental data of the above examples show that betulinone can effectively improve the blood lipid and liver lipid levels in the animal model of fatty liver disease, reverse and treat liver steatosis, and has a significant improvement effect on liver fibrosis, providing sufficient in vivo biological evidence for the application of betulinone in fatty liver disease.

[0053] Example 2 Betulinone alleviates liver fibrosis and inflammation levels in mice with non-alcoholic steatohepatitis

[0054] 1. Construction of the non-alcoholic steatohepatitis animal model

[0055] Wild-type C57BL / 6J mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) were fed a methionine-choline-deficient diet (A02082002B, Research Diet) starting at 8 weeks of age for 4 consecutive weeks. Mice fed a methionine-choline-deficient diet developed significant fatty liver within 2 weeks and then rapidly evolved into hepatitis and fibrosis, which can mimic the pathophysiological process of non-alcoholic steatohepatitis and is a commonly used preclinical non-alcoholic steatohepatitis mouse model.

[0056] 2. Betulinone administration protocol

[0057] The non-alcoholic steatohepatitis mice were randomly divided into two groups. Betulinone group: Betulinone powder (purchased from Shanghai TargetMol Co., Ltd.) was dissolved in corn oil containing 5% DMSO and intraperitoneally injected into C57 mice at a dose of 10 mg / kg daily. Control group: The same model mice were intraperitoneally injected with the same dose of corn oil containing 5% DMSO daily. After two weeks of administration, samples were taken to observe the therapeutic effect of betulinone on non-alcoholic steatohepatitis mice.

[0058] 3. Detection of serum and liver triglycerides and histological staining of the liver

[0059] (1) Triglyceride: After 14 consecutive days of intraperitoneal injection of betulinone to non-alcoholic steatohepatitis mice, serum was collected according to the method described in Example 1, and the content of triglyceride in the serum was detected according to the instructions of the assay kit. Liver samples were processed according to the method described in Example 1, and the triglyceride content in the supernatant of the lysate was detected according to the instructions of the assay kit, and corrected with the protein content.

[0060] (2) HE staining: Fresh liver tissues were fixed in 4% paraformaldehyde fixative for at least 24 hours. After dehydration and embedding, 5-μm tissue sections were cut. The sections were routinely dewaxed to distilled water. They were stained with hematoxylin for 2 minutes, rinsed with tap water to blue back, stained with eosin for 1 minute after distilled water washing, dehydrated with gradient ethanol, and sealed with neutral gum. Images were collected under a microscope, and the vacuole area was quantitatively analyzed using Image J software.

[0061] (3) Oil red O staining: Frozen sections of mouse liver were rewarmed and fixed in 4% paraformaldehyde fixative for 20 minutes. After distilled water washing, they were stained with 60% isopropanol for 2 minutes, and then directly stained with oil red O stain in the dark for 20 minutes. After distilled water washing, they were stained with hematoxylin for 2 minutes, and sealed with glycerol gelatin after distilled water washing. Images were collected under a microscope, and the positive area of oil red staining was quantitatively analyzed using Image J software.

[0062] (3) Masson staining: The sections were routinely dewaxed to distilled water. They were stained with mordant solution in an incubator at 60 °C for 1 hour, and then washed with distilled water 3 times, 3 minutes each time. Celestine blue staining solution was dropped and stained for 2 minutes, washed with distilled water 2 times, 10 seconds each time. Mayer hematoxylin staining solution was dropped and stained for 2 minutes, washed with distilled water 2 times, 10 seconds each time. Acidic differentiation solution was used for differentiation and washing. They were rinsed with tap water for 10 minutes to blue back. Ponceau fuchsin staining solution was dropped and stained for 5 minutes, washed with distilled water 2 times, 10 seconds each time. Phosphomolybdic acid solution was used for differentiation treatment for 5 minutes. The upper solution was poured off, and aniline blue stain was directly dropped and stained for 3 minutes. They were washed with weak acid solution for 2 minutes. They were quickly dehydrated with 95% ethanol for 3 seconds. Dehydrated with absolute ethanol 2 times, 5 seconds each time. Transparentized with xylene 2 times, 2 minutes each time. Sealed with neutral gum. Images were collected under a microscope, and the positive area of Masson staining was quantitatively analyzed using Image J software.

[0063] 4. Detection of the expression levels of liver inflammatory factors

[0064] Total RNA of liver samples stored in liquid nitrogen after sampling was extracted using the TRIZOL method, reverse transcribed into cDNA, and then the expression changes of inflammatory factor ( Tnfα, Adgre1, Ccl2 ) mRNA in liver tissues were detected by real-time quantitative fluorescence PCR.

[0065] From the above experimental results, it can be seen that after induction with a methionine-choline-deficient diet, the lipid accumulation in the livers of mice increased significantly, and obvious liver fibrosis was visible by Masson staining, indicating that the mouse models of steatohepatitis and liver fibrosis were successfully constructed. After treatment with alnustone in these steatohepatitis mice, the serum triglyceride level of the mice decreased significantly ( Figure 5 ) and the triglyceride content in the liver decreased significantly ( Figure 6). Moreover, the histological staining results of the liver showed that after treatment with betulin, the lipid droplets and neutral lipid content in hepatocytes were significantly reduced, and there were no other adverse effects on the morphology of hepatocytes ( Figure 7 ). More importantly, Masson staining of the liver after treatment with betulin showed a significant reduction in liver fibrosis ( Figure 8 ), and Tnfα Ccl2 the expression levels of multiple inflammatory factors such as Figure 9 ) were significantly decreased.

[0066] In addition, after treatment with betulin in mice with fatty liver disease, the body weight of the mice did not decrease significantly ( Figure 10 ), indicating that betulin treatment has good safety and no obvious toxic and side effects on mice.

[0067] The above experimental data all confirm that betulin can effectively relieve the symptoms of animal models of fatty liver disease, steatohepatitis, and liver fibrosis, especially reduce blood lipid and liver lipid levels, reverse and treat liver steatosis, and can effectively reduce the already formed liver fibrosis and reduce the liver inflammation level. These results provide sufficient in vivo biological evidence for the application of betulin in fatty liver disease.

[0068] The above research constructed an animal model of fatty liver disease to simulate the pathological pathogenesis of human fatty liver disease, revealing the new function of betulin in significantly improving body lipid metabolism, treating fatty liver disease, steatohepatitis, and liver fibrosis, and having no obvious toxic and side effects. It shows that betulin can be used as a new and effective drug for clinical prevention and treatment of fatty liver diseases.

[0069] The above is only the embodiment of the present application and is not used to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. Use of a linear diarylheptane compound in the preparation of a medicament for treating and preventing fatty liver diseases, characterized in that, The structural formula of the linear diarylheptane compound is as follows: 。 2. Use of a linear diarylheptane compound in the preparation of a health food for assisting in reducing blood lipid, characterized in that, The structural formula of the linear diarylheptane compound is as follows: 。 3. The application according to claim 1 or 2, characterized in that, The linear diarylheptane compound is obtained by chemical synthesis or biological extraction.

4. The application according to claim 3, characterized in that, The source of biological extraction is selected from Alpinia katsumadai, Alnus cremastogyne, Curcuma longa or Amomum tsaoko.

5. Use of a composition in the preparation of a drug for treating fatty liver disease or a health product for assisting in reducing blood lipid, characterized in that, It includes an effective dose of a linear diarylheptane compound, a plant containing a linear diarylheptane compound or a plant extract containing a linear diarylheptane compound; The structural formula of the linear diarylheptane compound is as follows: 。 6. The application according to claim 5, wherein The plant containing a linear diarylheptane compound is selected from Alpinia katsumadai, Alnus cremastogyne, Curcuma longa or Amomum tsaoko.

7. The application according to claim 5, characterized in that, The composition further includes a pharmaceutically or food-acceptable excipient; the intake form of the composition is oral or injection; the preparation of the composition is a sustained-release preparation or a controlled-release preparation; the oral preparation is selected from tablets, capsules, granules, oral liquids or syrups.

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