Application of rupestonic acid in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention relates to application of rupestonic acid in preparation of drugs for treating non-alcoholic fatty liver disease, including non-alcoholic fatty liver disease and non-alcoholic steatohepatitis. In a high fat diet induced non-alcoholic fatty liver model, rupestonic acid can significantly reduce the levels of aspartate aminotransferase, alanine aminotransferase, triglyceride and total cholesterol in mouse serum, and effectively improve liver fatty degeneration and lipid droplet accumulation. In a non-alcoholic steatohepatitis model induced by methionine-choline deficiency diet, rupestonic acid can significantly reduce the levels of serum aspartate aminotransferase and alanine aminotransferase, and effectively improve the fatty degeneration, inflammatory infiltration and fibrosis degree of the liver. The results fully show that the rupestonic acid disclosed by the invention provides a solid foundation for the definite treatment effect on the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] This invention relates to the use of artemisinin in the preparation of drugs for treating non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD), also known as metabolic dysfunction-related fatty liver disease, is a multifactorial disease and is considered the most common cause of chronic liver disease. NAFLD includes nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NAFH, as a progressive form of NAF, is characterized by its progressive nature and is more likely to develop into liver fibrosis, cirrhosis, and hepatocellular carcinoma. NAF is characterized by the excessive accumulation of lipids (such as triglycerides, free fatty acids, and small amounts of cholesterol) within hepatocytes. The transformation from NAF to NAFH and liver fibrosis is closely related to a series of liver injuries, which occur sequentially in different hepatocytes due to lipotoxicity, oxidative stress, inflammation, and apoptosis. These injuries ultimately lead to the activation of liver regeneration and fibrosis processes, promoting the deposition of collagen and extracellular matrix, thereby exacerbating liver fibrosis. Currently, methods for preventing and treating non-alcoholic fatty liver disease (NAFLD) include strengthening early monitoring to prevent the disease from occurring, or using medications for other lipid metabolism-related diseases. However, these medications are not specifically targeted at NAFLD, and their treatment effects are often unsatisfactory, with many having certain side effects. Furthermore, the economic costs associated with NAFLD are extremely high. Therefore, there is an urgent need to study the pathogenesis of NAFLD and develop potential candidate drugs.

[0003] Artemisia annua ( Artemisia rupestris L. Artemisia annua is a widely used medicinal plant in Kazakh and Uyghur folk medicine, primarily for treating gastrointestinal and liver diseases. It is cold in nature, pungent and bitter in taste, and is mainly used to treat febrile, cholestatic, or hematologic diseases. It possesses pharmacological effects such as antimalarial, antioxidant, antitumor, anti-inflammatory, and antiviral properties. Artemisia annua extract has been shown to inhibit hepatocellular carcinoma by regulating the phosphatidylinositol 3-kinase / protein kinase B (PI3K / Akt) signaling pathway. Rupeestonic acid is a sesquiterpene compound extracted from Artemisia annua, with a content exceeding 1%. Previous studies have shown that rupeestonic acid has a protective effect against acute liver injury induced by carbon tetrachloride and D-galactosamine hydrochloride. Although the protective effect of rupeestonic acid against liver injury has been confirmed, its efficacy and mechanism of action in improving non-alcoholic fatty liver disease require further investigation. Summary of the Invention

[0004] The purpose of this invention is to provide the use of artemisinin in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD). NAFLD is a clinicopathological syndrome characterized by diffuse macrovesicular steatosis in hepatocellular cells, including NAFLD and its evolutionary progressions such as NAFLD steatohepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Experimental studies have shown that feeding mice with a high-fat diet and a methionine-choline-deficient diet induces NAFLD and NAFLD, followed by treatment with artemisinin at different starting times. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides, and total cholesterol levels were measured in mice with NAFLD and NAFLD. Subsequently, hepatic lipid deposition, collagen deposition, and the degree of liver fibrosis were detected using hematoxylin and eosin (H&E) staining, Oil Red O staining, and Masson's trichrome staining.

[0005] The use of artemisinin as described in this invention in the preparation of drugs for treating non-alcoholic fatty liver disease.

[0006] The use of artemisinin, a small molecule compound, in the preparation of a drug for treating non-alcoholic fatty liver disease is described in this invention. The artemisinin was purchased from Chengdu Prima Pharmaceutical Research Co., Ltd., with a purity > 98.0%.

[0007] The use of artemisinin, a small molecule compound, in the preparation of drugs for treating non-alcoholic fatty liver disease (NAFLD) is described in this invention. Intervention with artemisinin can delay weight gain in mice with NAFLD and reduce liver weight, fat weight, fat index, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), and total cholesterol (TC) levels. P <0.05.

[0008] Hematoxylin-eosin (H&E) and Oil Red O staining of liver tissue sections from mice with non-alcoholic fatty liver disease (NAFLD) showed that artemisinin-based ketones reduced lipid accumulation in the liver tissue of NAFLD mice and significantly decreased the levels of triglycerides (TG) and total cholesterol (TC) in the liver. P <0.05).

[0009] The intervention with artemisinin at various doses reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice with non-alcoholic steatohepatitis. P <0.05).

[0010] Hematoxylin-eosin (H&E) and Oil Red O staining of liver tissue sections from mice with non-alcoholic steatohepatitis (NAHH) showed that the small molecule compound artemisinin reduced lipid accumulation and fibrosis in the liver tissue of NHAH mice, and significantly reduced the levels of triglycerides (TG) and total cholesterol (TC) in the liver (P<0.05). < 0.05).

[0011] A mouse model of non-alcoholic fatty liver disease (NAFLD) was established by feeding the mouse with a high-fat diet. After 3 weeks of gavage treatment, the small molecule compound artemisinin effectively reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in NAFLD mice, while simultaneously improving blood triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Hepatic steatosis was also significantly reduced, thus effectively preventing the progression of NAFLD in mice. Furthermore, a mouse model of NAFLD was established by feeding the mouse with a methionine-choline deficient diet. After 3 weeks of gavage treatment, the small molecule compound artemisinin effectively reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in NAFLD mice, significantly improving liver inflammation and steatosis, and delaying liver fibrosis, thus effectively preventing the progression of NAFLD in mice. Attached Figure Description

[0012] Figure 1 This invention presents an image showing how artemisinin improves non-alcoholic fatty liver disease induced by a high-fat diet (HFD) in mice. (A) Artemisinin structure, (B) establishment of a mouse non-alcoholic fatty liver model and treatment with the small molecule compound artemisinin, (C) mouse liver morphology, (D) mouse weight gain curve, (E) mouse fat index, (F) mouse liver weight, and (G) mouse liver index. Note: Normal control group (Con) vs High-fat diet (HFD) model control group ( # p <0.05、 ## p <0.01、 ### p <0.001, treatment group (Rupestonic acid and Silibinin) vs High-fat diet (HFD) model control group ( * p <0.05、 ** p <0.01、 *** p <0.001).

[0013] Figure 2The present invention describes the effects of artemisinin on serum triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), and glucose (Glc) in mice with non-alcoholic fatty liver disease. Note: Normal control group (Con) vs High-fat diet (HFD) model control group ( # p <0.05、 ## p <0.01、 ### p <0.001, treatment group (Rupestonic acid and Silibinin) vs High-fat diet (HFD) model control group ( * p <0.05、 ** p <0.01、 *** p <0.001).

[0014] Figure 3 This invention provides an image of lipid accumulation in the liver of mice with non-alcoholic fatty liver disease improved by artemisinin-ketone acid. (A) Image of mouse liver tissue sections stained with hematoxylin-eosin (H&E) and Oil Red O (magnification: ×200, scale bar: 50 μm), (B) Oil Red O staining statistics, and (CD) the content of triglycerides (TG) and total cholesterol (TC) in the mouse liver. Normal control group (Con) vs High-fat diet (HFD) model control group ( # p <0.05、 ## p <0.01、 ### p <0.001, treatment group (Rupestonic acid and Silibinin) vs High-fat diet (HFD) model control group ( * p <0.05、 ** p <0.01、 *** p <0.001).

[0015] Figure 4The image shows the effect of artemisinin-based amoeboidic acid on methionine-choline deficiency diet (MCD) induced non-alcoholic steatohepatitis in mice according to the present invention; (A) establishment of a mouse non-alcoholic steatohepatitis model and treatment course of the small molecule compound artemisinin-based amoeboidic acid, (B) mouse liver morphology, (C) mouse weight gain curve, (D) mouse liver weight, and (E) mouse liver index. Note: Normal control group (Con) vs Methionine-choline deficiency diet (MCD) model control group ( # p <0.05、 ## p <0.01、 ### p <0.001), treatment group (Rupestonic acid and Silibinin) vs Methionine-choline deficiency diet (MCD) model control group ( * p <0.05、 ** p <0.01、 *** p <0.001).

[0016] Figure 5 This invention provides an image showing how artemisinin-based ketone acid improves lipid accumulation in the liver of mice with non-alcoholic steatohepatitis. (A) Images of mouse liver tissue sections stained with hematoxylin-eosin (H&E), Oil Red O, and Masson trichrome (magnification ×200, scale bar: 50 μm); (BC) Images of triglyceride (TG), total cholesterol (TC), and alanine aminotransferase (ALT) levels in the mouse liver. Note: Normal control group (Con) vs Methionine-choline deficiency diet (MCD) model control group ( # p <0.05、 ## p <0.01、 ### p <0.001), treatment group (Rupestonic acid and Silibinin) vs Methionine-choline deficiency diet (MCD) model control group ( * p <0.05、 ** p <0.01、 *** p <0.001). Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0018] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0019] Animal feed: Regular feed was purchased from the Animal Center of Xinjiang Medical University; 60% high-fat feed (Jiangsu Medison Biomedical Co., Ltd., MD12033); methionine-choline deficient feed (Jiangsu Medison Biomedical Co., Ltd., MD12052).

[0020] Triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (Glc), and alkaline phosphatase (ALP) test kits were all purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0021] The following experiments, where no temperature is specified, are reactions conducted under normal temperature conditions, which is room temperature, or 25±5℃.

[0022] This invention uses GraphPad Prism 10.0.1 to perform statistical analysis on the data, and employs the Shapiro-Wilk normality test to verify the normality of the data. For data groups conforming to a normal distribution, a parametric test is used: independent samples are applied for comparisons between two groups. t For comparisons among multiple groups, one-way ANOVA (Ordinary One-way ANOVA) is used. For data that do not conform to a normal distribution, the Mann-Whitney U rank-sum test (a nonparametric test) is employed. The statistical significance threshold is set at [value missing]. P <0.05, all experimental procedures were independently verified 6 times to ensure the reliability of the results. Example 1

[0023] The effect of artemisinin on high-fat diet (HFD)-induced non-alcoholic fatty liver disease: Laboratory animals: Specific pathogen-free (SPF) grade male C57BL / 6 mice (18-22 g, 4-6 weeks old) were purchased from the Animal Center of Xinjiang Medical University. During the rearing period, the temperature of the animal room was maintained at 20.8-24.3℃ with a daily temperature difference of <4℃. The mice were exposed to a 12-hour light / dark cycle, and the bedding was changed every 2-3 days. The mice were allowed free access to water and food. Animal grouping: Forty 4-6 week old specific pathogen-free (SPF) male C57BL / 6 mice were acclimatized for one week and then randomly divided into six groups: normal control (Con), high-fat diet model control (HFD), high-fat diet + low-dose rupestonic acid group (HFD + rupestonic acid-L, 15 mg / kg / d), high-fat diet + medium-dose rupestonic acid group (HFD + rupestonic acid-M, 30 mg / kg / d), high-fat diet + high-dose rupestonic acid group (HFD + rupestonicacid-H, 60 mg / kg / d), and high-fat diet + positive control drug silymarin capsules (HFD + silibinin, 20 mg / kg / d). The model control group (high-fat diet) consisted of 10 mice (4 of which were used for model validation), and the remaining groups each consisted of 6 mice. Before feeding the high-fat diet, mice were fasted for 8-12 hours to put them in a semi-starved state, and their water intake was kept normal during this period. The normal control group (Con) mice were fed a normal diet every day. The model group and the treatment group (artemisinin and silymarin capsule group) were fed the same diet as the high-fat diet model control group (HFD) every day, which consisted of casein 25.84%, maltodextrin 16.15%, sucrose 8.89%, cellulose 6.46%, soybean oil 3.23%, lard 31.66%, and feed 7.77%. The mice were weighed once a week and the model was established for 8 consecutive weeks. The same part of the liver was taken under fasting anesthesia for pathological sectioning to evaluate the success of the non-alcoholic fatty liver model. Intervention experiment: Silymarin is a commonly used hepatoprotective drug in clinical practice, possessing anti-inflammatory and antioxidant effects. In this study, it was used as a positive control. Mice in the treatment group were administered 10 mL / kg of silymarin via gavage. The mice in the low, medium, and high dose groups of artemisinin, as well as the positive control drug silymarin capsules, were given these drugs for 3 weeks. The normal control group continued to be fed a regular diet, while the remaining mice continued to be fed a high-fat diet. The mice were also administered the corresponding concentration of the drug and the control solution daily via gavage. During the daily administration period, their drinking water and feeding behavior were not restricted. Mice weight was recorded weekly. The specific procedures are detailed below. Figure 1 -B; Solution: Blood was collected under fasting anesthesia to separate serum. Liver, kidney, spleen, and adipose tissue were harvested. The levels of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), alanine aminotransferase (ALT), aspartate aminotransferase (AST), glucose (Glc), and alkaline phosphatase (ALP) in serum were measured according to the manufacturer's instructions. The levels of triglycerides (TG) and total cholesterol (TC) in liver tissue were also measured. Mouse liver samples were uniformly stored in 4% tissue fixative, and the remaining samples were aliquoted into cryovials. Mouse livers were embedded in paraffin, and the embedded sections were stained with hematoxylin and eosin. A portion of the frozen liver tissue was stained with Oil Red O to observe lipid accumulation in the liver. The livers of mice in the normal control group (Con) appeared smooth and reddish, while the livers of mice in the high-fat diet (HFD) model control group appeared yellowish. The livers of mice in the artemisinin group and the non-alcoholic fatty liver disease group were slightly redder and shinier than those in the non-alcoholic fatty liver disease group. (See attached table). Figure 1 -C; Compared with the Con group, the body weight, liver weight, and adipose tissue index of mice in the high-fat diet (HFD) model control group were significantly increased. Compared with the high-fat diet (HFD) model control group, the body weight, liver weight, and adipose tissue index of mice in each dose of artemisinic acid and the positive control group (silibinin capsules) were decreased, indicating that the small molecule compound artemisinic acid can improve body weight, liver weight, and adipose tissue index in mice with non-alcoholic fatty liver disease. Results are shown in […]. Figure 1 DG; In high-fat diet model control mice, liver injury-related markers alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly elevated compared to normal mice. p <0.05%, artemisinin treatment can significantly reduce the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). p <0.05, suggesting that the drug helps improve liver damage caused by a high-fat diet. Compared with Con mice, mice on a high-fat diet showed significantly abnormal levels of lipid metabolism-related indicators such as triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). p <0.05%, artemisinin treatment can significantly improve abnormal lipid metabolism indicators ( p <0.05), results are shown in Figure 2 AH; This invention involves staining mouse liver tissue with hematoxylin-eosin (H&E) and Oil Red O. The Oil Red O staining results reflect lipid accumulation in the liver, as shown in the following figures. Figure 3-A shows the results at a magnification of 200. The top six images are hematoxylin-eosin (H&E) staining, and the bottom six images are Oil Red O staining. The hematoxylin-eosin (H&E) staining results show that, compared with the high-fat diet (HFD) model control group, the hematoxylin-eosin (H&E) and Oil Red O staining of the liver significantly reduced lipid accumulation in the mouse liver, suggesting that the drug has the potential to treat non-alcoholic fatty liver disease. Example 2

[0024] Effects of artemisinin on methionine-choline deficient diet (MCD)-induced nonalcoholic steatohepatitis: Laboratory animals: Specific pathogen-free (SPF) grade male C57BL / 6 mice (18-22 g, 4-6 weeks old) were purchased from the Animal Center of Xinjiang Medical University. During the rearing period, the temperature of the animal room should be maintained at 20.8-24.3℃ with a daily temperature difference of <4℃. The mice were exposed to a 12-hour light / dark cycle, and the bedding was changed every 2-3 days. The mice were allowed free access to water and food. Animal grouping: Forty 4-6 week old specific pathogen-free (SPF) male C57BL / 6 mice were acclimatized for one week and then randomly divided into six groups: a normal control group (Con), a methionine-choline-deficient diet (MCD) model control group, a methionine-choline-deficient diet + low-dose rupestonic acid group (MCD + Rupestonic acid-L, 15 mg / kg / d), a methionine-choline-deficient diet + medium-dose rupestonic acid group (MCD + Rupestonic acid-M, 30 mg / kg / d), a methionine-choline-deficient diet + high-dose rupestonic acid group (MCD + Rupestonic acid-H, 60 mg / kg / d), and a methionine-choline-deficient diet + silymarin capsules (MCD + silibinin) group. The control group of mice in the methionine-choline deficiency diet (MCD) model consisted of 10 mice (4 of which were used for model validation), and the remaining mice were 6 mice in each group. Before feeding the methionine-choline deficiency diet (MCD), the mice were fasted for 8-12 hours to put them in a semi-starved state, and their water intake was kept normal during this period. The normal control group (Con) mice were fed with normal feed every day. The model group and the treatment group (artemisinin and silymarin capsule group) were fed the methionine-choline deficiency diet (MCD) every day, and their weight was measured once a week. The model was established for 4 consecutive weeks. The same part of the liver was taken under fasting anesthesia for pathological sectioning to evaluate the success of the non-alcoholic steatohepatitis model. Intervention experiment: Mice in the treatment group were administered the drug via gavage (10 mL / kg) in low, medium, and high doses of artemisinin and silymarin capsules for 3 weeks. The normal control group continued to be fed a normal diet, while the remaining mice continued to be fed a methionine-choline deficient diet (MCD) and were administered the corresponding concentration of the drug and control solvent via gavage daily. Drinking water and feeding behavior were not restricted during the daily administration period, and mouse weight was recorded weekly. Solution: Blood was collected under fasting anesthesia to separate serum. Liver, kidney, spleen, and adipose tissue were harvested. The levels of triglycerides (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) in serum were measured as required. The levels of TC and TG in liver tissue were also measured. Mouse liver samples were uniformly placed in 4% tissue fixative, and the remaining samples were aliquoted into cryovials. Mouse livers were embedded in paraffin, and the embedded sections were stained with hematoxylin-eosin and Masson's stain. A portion of frozen liver tissue was stained with Oil Red O to observe lipid accumulation in the liver. The results showed that compared with the normal control group (Con), the livers of mice in the methionine-choline deficiency diet (MCD) model control group were smaller in volume, more yellowish in color, slightly harder in texture, and generally smooth in surface, without erosion or nodular changes. The livers of mice in the artemisinin group were slightly redder and shinier than those in the methionine-choline deficiency diet (MCD) model control group. (See attached figures.) Figure 4 B; Compared with the normal control group (Con), the body weight and liver weight of mice in the methionine-choline deficiency diet (MCD) model control group were significantly decreased, while the liver weight-to-body weight ratio showed no significant change. Compared with the methionine-choline deficiency diet (MCD) model control group, the body weight, liver weight, and fat index of mice in each dose of rupestonic acid and the silibinin positive control group were slightly increased, but the differences were not statistically significant. See the results below. Figure 4 CE; Hematoxylin and eosin (H&E) staining showed that in the methionine-choline deficiency diet (MCD) model control group, hepatocytes were disordered, with a large amount of lipid deposition, ballooning degeneration, and interlobular inflammation. Artemisinin treatment significantly reduced hepatic lipid deposition and alleviated ballooning degeneration and interlobular inflammation. Oil Red O staining of mouse liver showed that 3 weeks of artemisinin treatment significantly reduced red lipid droplet deposition in the liver. Further Masson trichrome staining showed that blue collagen deposition was significantly reduced in the methionine-choline deficiency diet (MCD) model control group after artemisinin treatment. In conclusion, artemisinin significantly improved hepatic inflammatory infiltration induced by methionine-choline deficiency diet (MCD) in mice and inhibited the progression of liver fibrosis.

Claims

1. Use of artemisinic acid in the manufacture of a medicament for the treatment of nonalcoholic fatty liver disease.

Images