Application of ganoderic acid A in preparation of product for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention discloses an application of ganoderic acid A in preparation of a product for preventing and / or treating a non-alcoholic fatty liver disease. The ganoderic acid A can regulate bile acid reabsorption of C57BL / 6J mice induced by high fat diet and reduce absorption of triglyceride and cholesterol. The ganoderic acid A can also enhance expression of a key enzyme cholesterol 7alpha-hydroxylase gene for liver cholesterol decomposition, so that liver cholesterol is promoted to be converted into bile acid, and accumulation of liver cholesterol is relieved. The ganoderic acid A has remarkable prevention and improvement effects on the non-alcoholic fatty liver disease induced by high fat diet by regulating bile acid metabolism, inhibiting lipid absorption and relieving liver lipid accumulation. Therefore, the ganoderic acid A can be used as a health-care product, a functional product or a medicine for preventing and treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of ganoderic acid A in the preparation of products for the prevention and / or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide and the leading cause of disability and life expectancy impairment. It can lead to a series of complications, including liver inflammation and fibrosis, eventually developing into nonalcoholic steatohepatitis. Poor dietary habits, a sedentary lifestyle, and aging all increase the risk of NAFLD. According to literature, the global prevalence of NAFLD increased from 25.3% in 1990-2006 to 38.0% in 2016-2019. my country is also a high-incidence area for NAFLD, with an incidence rate between 23.8% and 29% as of 2018. NAFLD seriously threatens human health and quality of life, imposing a huge economic and medical burden on my country. Despite the persistently high and increasing incidence of NAFLD, safe and effective drugs for its treatment are still lacking. Targeting lipid metabolism is considered a major treatment strategy for NAFLD. Drugs that intervene in lipid metabolism disorders are often used to improve NAFLD, such as weight-loss drugs and lipid-lowering drugs. However, long-term use of these drugs can cause serious adverse reactions. Therefore, it is of great significance to develop safe, effective, and long-term usable drugs or health products for the prevention and treatment of NAFLD.

[0003] Ganoderma lucidum is widely used in Asia as a traditional Chinese medicine. Ganoderic acids are an important class of active substances in Ganoderma lucidum extracts. They are tetracyclic triterpenoids composed of 30 carbon atoms with a lanane skeleton. Based on the number and position of double bonds in the parent ring, ganoderic acids can be divided into different monomers. Among ganoderic acid extracts, ganoderic acid A is present in higher amounts than other ganoderic acid monomers and possesses various pharmacological effects, such as anti-inflammatory and antioxidant properties, and can also alleviate diseases caused by lipid metabolism disorders. However, the effect and mechanism of ganoderic acid A in improving NAFLD have not yet been reported. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides the application of ganoderic acid A in the preparation of products for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0005] The chemical structural formula of the ganoderic acid A is shown in Formula I:

[0006]

[0007] In the product, ganoderic acid A can be the sole active ingredient or one of the active ingredients.

[0008] The present invention also provides the use of compositions containing ganoderic acid A in the preparation of products for the prevention and / or treatment of non-alcoholic fatty liver disease.

[0009] In the above applications, the composition further comprises pharmaceutically acceptable excipients and / or drugs compatible with ganoderic acid A.

[0010] In this invention, the prevention and / or treatment of non-alcoholic fatty liver disease is manifested in at least one of the following aspects:

[0011] 1) Inhibit the increase in body weight, liver specific gravity and / or white fat specific gravity caused by non-alcoholic fatty liver disease;

[0012] 2) Improves liver damage caused by non-alcoholic fatty liver disease;

[0013] 3) Reduces lipid accumulation in the liver caused by non-alcoholic fatty liver disease;

[0014] 4) Regulates bile acid reabsorption and inhibits lipid (triglycerides and cholesterol) absorption;

[0015] 5) Enhance the expression of the gene for cholesterol 7α-hydroxylase (CYP7A1), a key enzyme in the breakdown of cholesterol in the liver.

[0016] Furthermore, the improvement of liver damage caused by non-alcoholic fatty liver disease is specifically reflected in the fact that ganoderic acid A can significantly reduce the elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) caused by non-alcoholic fatty liver disease.

[0017] In this invention, the non-alcoholic fatty liver disease is caused by a high-fat diet.

[0018] In this invention, the product may be a drug, health product, and / or functional food.

[0019] The drug contains an effective dose of ganoderic acid A. The effective dose is a unit dosage (e.g., the content in one tablet) or a unit dose for the treated patient (e.g., a dose per unit body weight). In this invention, the drug is used to treat humans or mammals (e.g., mice).

[0020] The dosage form of the drug described in this invention is not limited, as long as it enables the active ingredient to be effectively delivered into the body, including: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, lozenges, granules, powders, pills, powders, ointments, pills, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, patches, etc.

[0021] This invention utilizes a high-fat diet-induced C57BL / 6J mouse model to discover the alleviating effect of ganoderic acid A supplementation on non-alcoholic fatty liver disease. Compared with the control group, the high-fat group mice showed significantly increased body weight, liver index, and white adipose tissue index. Compared with the high-fat group, after daily oral administration of 20 mg / kg body weight of ganoderic acid to mice for 16 weeks, the mice showed significantly decreased body weight, liver index, and white adipose tissue index.

[0022] This invention measured blood biochemical indicators in mice in the blank group, high-fat group, and high-fat + ganoderic acid A group. The results showed that ganoderic acid A can significantly reduce the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice on a high-fat diet, suggesting that ganoderic acid A can improve liver damage induced by a high-fat diet.

[0023] This invention, through HE staining of liver tissue sections, observed significantly enlarged hepatocytes in mice in the high-fat group, with intracellular lipid accumulation and the formation of fat vesicles. After intervention with ganoderic acid A, the volume of fat vesicles within hepatocytes significantly decreased. Oil Red O staining of the liver showed that ganoderic acid A significantly reduced lipid deposition in the liver. Using a kit, this invention revealed that ganoderic acid A significantly reduced hepatic triglyceride and cholesterol levels. These findings suggest that ganoderic acid A can effectively alleviate the development and progression of non-alcoholic fatty liver disease.

[0024] Specifically, ganoderic acid A can reduce lipid accumulation in the liver of mice caused by a high-fat diet.

[0025] Specifically, ganoderic acid A can reduce the increase in triglyceride and cholesterol content in mouse liver tissue induced by a high-fat diet.

[0026] This invention, by detecting metabolites in the terminal ileum of mice in a blank group, a high-fat group, and a high-fat group + ganoderic acid A group, found that ganoderic acid A significantly affected cholesterol / bile acid metabolism in mice and reduced the levels of some long-chain fatty acids. Simultaneously, using a kit, it was found that ganoderic acid A significantly increased the content of bile acids in feces. Furthermore, the kit also showed that ganoderic acid A significantly increased the lipid content in feces. These findings suggest that ganoderic acid A can inhibit lipid absorption by regulating bile acid / cholesterol metabolism and bile acid reabsorption in mice.

[0027] Specifically, ganoderic acid A can lower levels of harmful bile acids such as taurocholic acid and glycochenodeoxycholic acid, inhibiting bile acid reabsorption. Ganoderic acid A also inhibits the absorption of triglycerides and cholesterol.

[0028] This invention uses real-time quantitative PCR technology to detect genes related to cholesterol breakdown in the livers of mice in the blank group, high-fat group, and high-fat + ganoderic acid A group.

[0029] Specifically, ganoderic acid A promotes cholesterol breakdown in the liver and reduces cholesterol accumulation in the liver by increasing the expression of the cholesterol 7α-hydroxylase (CYP7A1) gene.

[0030] The beneficial effects of this invention are as follows:

[0031] (1) This invention has discovered new natural active substances for the prevention and treatment of non-alcoholic fatty liver disease. It has the advantages of being safe, effective and suitable for long-term use, and has good application prospects.

[0032] (2) Ganoderma lucidum acid A has a significant effect in relieving non-alcoholic fatty liver disease.

[0033] (3) Ganoderma lucidum acid A can prevent and treat non-alcoholic fatty liver disease by inhibiting the reabsorption of bile acids and the expression of genes related to cholesterol accumulation in the liver. It can be used in a variety of ways to prevent and treat non-alcoholic fatty liver disease. Attached Figure Description

[0034] Figure 1 The effect of ganoderic acid A on mouse body weight;

[0035] Figure 2 The effects of ganoderic acid A on liver and fat indices;

[0036] Figure 3 The effect of ganoderic acid A on the concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in mouse blood;

[0037] Figure 4 Mouse liver sections were stained with hematoxylin and eosin (HE) and Oil Red O.

[0038] Figure 5 The effect of ganoderic acid A on triglyceride and cholesterol content in mouse liver tissue;

[0039] Figure 6 These are differentially expressed bile acid metabolites in mouse ileum tissue;

[0040] Figure 7 Differential long-chain fatty acids in mouse ileum tissue;

[0041] Figure 8 The effect of ganoderic acid A on triglycerides, cholesterol and total bile acids in mouse feces;

[0042] Figure 9 The effect of ganoderic acid A on the expression level of Cyp7a1 gene in mouse liver tissue. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0045] The experimental grouping and drug administration in the following examples are as follows:

[0046] Eighteen 8-week-old C57BL / 6J mice were acclimatized for one week and then randomly divided into three groups of six each: a control group, a high-fat group, and a high-fat + ganoderic acid A group. The control group was fed a normal diet, while the high-fat and high-fat + ganoderic acid A groups were fed a high-fat diet. The control and high-fat groups were administered 0.5% sodium carboxymethyl cellulose saline solution by gavage, while the high-fat + ganoderic acid A group was administered ganoderic acid A (20 mg / kg dissolved in 0.5% sodium carboxymethyl cellulose saline solution) by gavage once daily for 16 weeks. The normal and high-fat diets were purchased from Beijing Keao Xieli Feed Co., Ltd.

[0047] Example 1: The effect of ganoderic acid A in alleviating non-alcoholic fatty liver disease

[0048] Mice were weighed weekly, and the body weight of each group was recorded. After the last administration of the drug, the mice were dissected, and the liver and white fat were removed. The liver and white fat were weighed, and the specific gravity of the liver and the specific gravity of the white fat were calculated (the formulas are: specific gravity of liver = liver weight / body weight * 100%; specific gravity of white fat = weight of white fat / body weight * 100%).

[0049] Experimental results: see Figure 1 and Figure 2 Ganoderic acid A can significantly inhibit the increase in body weight, liver specific gravity, and white fat specific gravity induced by a high-fat diet.

[0050] Detection of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST): After the last administration experiment, mice were fasted for 12 hours, blood was collected, centrifuged at 3000 r / min for 10 min, serum was separated for testing, and serum ALT and AST levels were measured.

[0051] Experimental results: see Figure 3 Ganoderic acid A significantly reduced the elevated levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) induced by a high-fat diet, indicating that ganoderic acid A significantly improved liver damage caused by a high-fat diet.

[0052] HE staining and Oil Red O staining: After the last drug administration experiment, mice were dissected, liver tissue was collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. Pathological evaluation was performed using hematoxylin and eosin (H&E) and Oil Red O staining.

[0053] Experimental results: see Figure 4 Hematoxylin and eosin (HE) staining of liver tissue sections revealed significantly enlarged hepatocytes in the high-fat group, with intracellular lipid accumulation forming fat vesicles. Treatment with ganoderic acid A showed a significant alleviating effect, with a marked reduction in the volume of fat vesicles within hepatocytes. Oil Red O staining of the liver indicated that ganoderic acid A significantly reduced lipid deposition in the liver.

[0054] Triglyceride and cholesterol content detection: After the last administration experiment, liver tissue of mice was taken, and the triglyceride and cholesterol content of the liver was detected using the Triglyceride and Cholesterol Detection Kit of Prilex Company, in accordance with the instructions.

[0055] Experimental results: see Figure 5 Ganoderma lucidum acid A can significantly reduce the increase in triglyceride and cholesterol content in mouse liver tissue induced by a high-fat diet.

[0056] Example 2: Effect of ganoderic acid A on lipid absorption in mice on a high-fat diet

[0057] Non-targeted metabolomics assay: After the last administration experiment, the mice were dissected and the contents of the ileum were collected. Metabolomics analysis was performed using liquid chromatography-tandem mass spectrometry to detect the components of the mouse ileum contents.

[0058] Experimental results: see Figure 6 and Figure 7 Compared with the high-fat + ganoderic acid A group, the results showed that ganoderic acid A significantly affected the cholesterol / bile acid metabolism pathway in mice, reducing the levels of harmful bile acids such as taurocholic acid (leading to excessive lipid absorption) and glycochenodeoxycholic acid (which can cause glucose and lipid metabolism disorders). Simultaneously, ganoderic acid A reduced the levels of various long-chain fatty acids in the ileum. These results suggest that gavage administration of ganoderic acid A can affect bile acid / cholesterol metabolism in mice and influence lipid absorption by altering bile acid metabolism.

[0059] The levels of triglycerides, cholesterol, and bile acids in feces were measured. Feces were collected from mice after the last administration of the drug. Triglyceride and cholesterol levels were measured using a kit from Prilex Pharmaceuticals, and bile acid levels were measured using a kit from Nanjing Jiancheng Pharmaceutical Co., Ltd., following the manufacturer's instructions.

[0060] Experimental results: see Figure 8 Ganoderic acid A significantly increased the levels of triglycerides, cholesterol, and bile acids in feces, indicating that ganoderic acid A promotes the excretion of lipids and bile acids and reduces lipid absorption.

[0061] Example 3: Mechanism of action of ganoderic acid A in alleviating hepatic lipid accumulation

[0062] The expression level of Cyp7a1 gene in liver tissue was detected by real-time quantitative PCR.

[0063] Experimental results: see Figure 9 Ganoderic acid A significantly increased the expression level of Cyp7a1, which is responsible for converting excess liver cholesterol into bile acids and alleviating liver lipid accumulation.

[0064] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of ganoderic acid A in the preparation of a product for preventing and / or treating non-alcoholic fatty liver disease.

2. Use of a composition comprising ganoderic acid A in the preparation of a product for preventing and / or treating non-alcoholic fatty liver disease.

3. Use according to claim 2, characterized in that: The composition further comprises a pharmaceutically acceptable adjuvant and / or a drug compatible with ganoderic acid A.

4. Use according to any one of claims 1 to 3, characterized in that: The prevention and / or treatment of non-alcoholic fatty liver disease is manifested in at least one of the following aspects: 1) inhibiting the increase in body weight, liver specific gravity and / or white fat specific gravity caused by non-alcoholic fatty liver disease; 2) improving liver damage caused by non-alcoholic fatty liver disease; 3) reducing liver lipid accumulation caused by non-alcoholic fatty liver disease; 4) regulating bile acid reabsorption and inhibiting lipid absorption; 5) enhancing the expression of the key enzyme cholesterol 7a-hydroxylase (CYP7A1) gene for liver cholesterol decomposition.

5. Use according to claim 4, characterized in that: The improvement of liver damage caused by non-alcoholic fatty liver disease is specifically manifested in that ganoderic acid A can significantly reduce the increase in alanine aminotransferase and aspartate aminotransferase levels caused by non-alcoholic fatty liver disease.

6. Use according to any one of claims 1 to 5, characterized in that: The product can be a drug and / or a health product and / or a functional food.

7. Ganoderic acid A for use in preventing and / or treating non-alcoholic fatty liver disease.

8. A composition comprising ganoderic acid A for use in preventing and / or treating non-alcoholic fatty liver disease.

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