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

Abstract

The invention provides application of farrerol in preparation of a medicine for treating non-alcoholic fatty liver disease. Experimental results show that the farrerol can significantly reduce the liver visceral organ coefficient, the body fat rate, the serum transaminase and the serum total cholesterol level of a non-alcoholic fatty liver model mouse and reduce the accumulation of liver fat and tissue pathological damage, and has a significant protective effect on the non-alcoholic fatty liver of the mouse.

Description

Technical Field

[0001] The present application belongs to the medical and health fields such as medicines, health products, and foods. Specifically, the present application relates to the field of digestive system diseases, and in particular to the use of rhododendron in the preparation of drugs for the treatment of non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a common chronic metabolic disease characterized by abnormal accumulation of lipids in the liver and is not associated with heavy drinking. NAFLD usually starts with simple fatty liver in the early stage and gradually develops into nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma (HCC). As one of the most important chronic liver diseases in the world, NAFLD has affected about 20%-30% of the world's population, especially in obese people, with an incidence rate of up to 75%. In recent years, the association between NAFLD and metabolic syndrome, insulin resistance, type 2 diabetes, and cardiovascular disease has become increasingly clear. Although the exact pathogenesis of NAFLD has not yet been fully clarified, the original "double hit hypothesis" proposed excessive accumulation of lipids in the liver ("first hit") and subsequent inflammation and oxidative stress ("second hit"). However, modern studies have shown that its pathological mechanism is more complex, and a "multiple parallel hit hypothesis" has been proposed, in which the interaction between the liver, multiple organ systems, and intestinal flora is jointly involved in the development of the disease. In particular, the role of the gut-liver axis has attracted widespread attention. Intestinal flora imbalance not only affects liver metabolism, but also activates liver inflammatory response and promotes fibrosis by increasing the transport of bacterial endotoxins such as lipopolysaccharide (LPS). In addition, intestinal metabolites such as short-chain fatty acids and bile acids also play an important role in regulating liver metabolism and immune response.

[0003] The high incidence of NAFLD is closely related to the prevalence of obesity and metabolic syndrome, and the economic burden it brings is increasing. At present, the US Food and Drug Administration (FDA) and the European Medicines Agency have not approved any drugs specifically for the treatment of NAFLD, and clinical treatment is still mainly lifestyle intervention, drug replacement therapy or surgery. However, lifestyle intervention has a slow effect and poor patient compliance; the effect and potential risks of alternative drugs still need to be evaluated; and surgical intervention is limited by its trauma and postoperative complications. Therefore, the development of safe and effective treatment methods has become a hot topic in international research. In this context, natural active ingredients have attracted widespread attention as an auxiliary or alternative treatment for NAFLD. Studies have shown that natural ingredients such as polyphenols, saponins and polysaccharides can effectively regulate lipid metabolism and improve liver function, showing the potential for the prevention and treatment of NAFLD.

[0004] Sophora moocroftiana (Benth.) Baker (Tibetan name: Juewei Zhewu) is the dried seed of Sophora moocroftiana (Benth.) Baker, a plant of the Leguminosae family. It is cool in nature and bitter in taste, and has significant antibacterial, anti-inflammatory, and anti-parasitic medicinal values. According to the Tibetan medicine classic "Zangyao Zhi", Sophora moocroftiana has anti-inflammatory and detoxifying effects, and is often used to treat insect diseases, as well as damp-heat jaundice, icteric hepatitis and other diseases. Modern pharmacological studies have shown that the total alkaloids in Sophora moocroftiana have a significant improvement effect on carbon tetrachloride-induced rat liver fibrosis. At the same time, Sophora moocroftiana has a significant therapeutic effect on hepatitis B virus (HBV) unique to Tibet, showing its potential in antiviral treatment. Farrerol is the main bioactive component of flavonoids in Sophora moocroftiana. Farrerol has a wide range of pharmacological activities, including anti-inflammatory, antioxidant, antibacterial and neuroprotective effects. Li Dong et al. found that farrerol can improve diabetic liver disease. Therefore, developing it into a drug for the treatment of non-alcoholic fatty liver disease has extremely high potential value and social significance.

[0005] The invention patent with application number "CN201110303137.0" and invention name "Application of rhododendron and its derivatives and pharmaceutically acceptable salts in the preparation of drugs for cardiovascular and cerebrovascular diseases caused by vasoconstriction" discloses new medical uses of the dihydroflavonoid compound rhododendron and its pharmaceutically acceptable salts and three derivatives of rhododendron and their pharmaceutically acceptable salts, and discloses the application in the preparation of drugs for cardiovascular and cerebrovascular diseases caused by vasoconstriction. The application shows that it has a significant vasodilating effect on blood vessels through experiments on isolated rat vascular rings, and has the characteristics of low toxicity and small side effects. It can be synthesized industrially and is inexpensive. It can also be formulated with pharmaceutically acceptable pharmaceutical excipients to prepare common preparations such as injections, tablets, pills or capsules, which are used for clinical medication for the prevention and treatment of cardiovascular and cerebrovascular diseases caused by vasoconstriction, providing new methods and means for the clinical treatment of cardiovascular and cerebrovascular diseases.

[0006] The invention patent with application number "CN202211164826.2" and invention name "Application and method of rhododendron to promote the efficiency of somatic cell nuclear transfer" discloses an application and method of rhododendron to promote the efficiency of somatic cell nuclear transfer. In this invention, rhododendron is used to prepare drugs for improving the development rate and birth rate of somatic cell nuclear transfer embryos. Compared with the current methods of promoting the development efficiency of somatic cell nuclear transfer embryos by various small molecule compounds, the principles involved are different from the previous methods of regulating epigenetic differences in the process of somatic cell nuclear transfer. Rhododendron can effectively promote the genomic stability of somatic cell nuclear transfer embryos and significantly improve the embryonic development rate and birth rate, so there is the possibility of combining with other drugs to achieve better results. Summary of the invention

[0007] The present invention aims to study the pharmacological effects of rhododendron, find that it can significantly reduce liver lipid accumulation, serum transaminase and total cholesterol content in mice with non-alcoholic fatty liver disease, and thus propose the potential application of rhododendron in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

[0008] In one aspect, the present invention provides the use of rhododendron in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the structural formula of rhododendron is shown in formula (1):

[0009]

[0010] Furthermore, rhododendron tinctorius is the only active ingredient in the drug.

[0011] Furthermore, the non-alcoholic fatty liver disease is caused by damage to liver metabolic function.

[0012] Furthermore, the non-alcoholic fatty liver disease is characterized by liver fat accumulation and elevated serum transaminase and total cholesterol levels.

[0013] Furthermore, the drug is in the form of an injection or oral dosage form.

[0014] Furthermore, the drug is in oral dosage form.

[0015] In another aspect, the present invention provides a non-therapeutic method for enhancing liver fat metabolism comprising administering rhododendrin.

[0016] Furthermore, the single application dose of rhododendronine is 20-30 mg / kg.

[0017] Furthermore, the single application dose of rhododendronine is 30-45 mg / kg.

[0018] Furthermore, the single application dose of rhododendron tinctorius is 45 mg / kg.

[0019] The non-therapeutic methods in this application include but are not limited to health care methods and scientific research methods, and the subjects of implementation can be humans or other mammals such as mice, rabbits, dogs, etc.

[0020] The symptoms or indications of non-alcoholic fatty liver disease in this application include but are not limited to increased liver index, liver fat accumulation, and increased serum transaminase and total cholesterol levels.

[0021] The uses include lowering the liver organ coefficient, serum transaminase, and serum total cholesterol levels of non-alcoholic fatty liver disease, and reducing the accumulation of liver fat and tissue pathological damage, having a protective effect on non-alcoholic fatty liver disease and reducing body weight and body fat percentage.

[0022] The available pharmaceutical dosage forms of the present application include various injection and oral dosage forms, including but not limited to tablets, capsules, oral solutions, injections, powder injections, and transdermal preparations, with oral dosage forms being particularly preferred.

[0023] In addition to rhododendron, the drug described in the present application may also include various pharmaceutically acceptable excipients / excipients, including but not limited to coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH adjusters, flavoring agents, etc., and various excipients / excipients that can be used in oral dosage forms are particularly preferred.

[0024] The drug of the present invention may contain other Chinese and Western medicines or health products for treating non-alcoholic fatty liver disease, or the drug of the present invention may be used in combination with these Chinese and Western medicines or health products or surgical treatments. The Chinese and Western medicines or health products include but are not limited to anti-infective drugs, antioxidant drugs, and energy metabolism and blood circulation improvers.

[0025] The experimental results show that rhododendron can significantly reduce the liver organ coefficient, body fat percentage, serum transaminase, and serum total cholesterol levels of non-alcoholic fatty liver model mice, and reduce the accumulation of liver fat and tissue pathological damage, and has a significant protective effect on non-alcoholic fatty liver in mice.

[0026] The use of rhododendronine provided by the present invention in the preparation of a drug for treating non-alcoholic fatty liver disease has the following beneficial effects:

[0027] (1) Rhododendron can significantly reduce lipid accumulation in the liver;

[0028] (2) Rhododendron can reduce the body weight, serum transaminase levels and total cholesterol content of mice and improve non-alcoholic fatty liver disease.

[0029] The present invention unexpectedly discovered that rhododendron has the effect of treating non-alcoholic fatty liver disease and can be used to prepare a drug for treating non-alcoholic fatty liver disease, providing a new and better choice for such drugs, health products, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The effect of rhododendronine on the physical signs of mice with non-alcoholic fatty liver disease; A: body weight growth rate (%); B: daily food intake (g); C: Lee's index; D: fat content (%); E: free water content (%); F: muscle content (%); the results are expressed as Mean ± SEM (n = 8), *** p<0.001 compared with the normal group, # p<0.05, ## p<0.01, ### p<0.001, compared with the model group.

[0031] Figure 2 The effect of rhododendron on glucose tolerance in mice with non-alcoholic fatty liver disease; A: blood glucose change curve; B: area under the curve; Mean ± SEM (n = 3), *** p<0.001 compared with the normal group.

[0032] Figure 3 The effect of rhododendrin on the disease phenotype of non-alcoholic fatty liver disease in mice; A: liver tissue morphology; B: liver coefficient; C: liver weight; D: serum ALT content; E: serum AST content; F: serum TC content; Mean ± SEM (n = 8), * p<0.05, *** p<0.001 compared with the normal group, # p<0.05, ### p<0.001, compared with the model group.

[0033] Figure 4 This is a diagram showing the effect of rhododendron on tissue morphology in mice with non-alcoholic fatty liver disease.

[0034] Figure 5 This is a diagram showing the effect of rhododendron on lipid accumulation in tissues of mice with non-alcoholic fatty liver disease. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to the examples. The rhododendrons used in the following examples are all compounds represented by the aforementioned formula (1) and can be obtained commercially.

[0036] Example 1

[0037] Use of rhododendron in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of rhododendron is 20 mg / kg for mice, and the drug is in an oral dosage form.

[0038] Example 2

[0039] Use of rhododendron in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of rhododendron is 30 mg / kg for mice, and the drug is in an oral dosage form.

[0040] Example 3

[0041] Use of rhododendron in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of rhododendron is 45 mg / kg for mice, and the drug is in an oral dosage form.

[0042] The following animal experiments further illustrate the effects of the above embodiments 1 to 3:

[0043] 1. Experimental Materials

[0044] 1.1 Animal Handling

[0045] SPF-grade C57BL / 6 adult male mice weighing between 20 and 25 grams were selected and bred by the Experimental Animal Center of Xi'an Jiaotong University (Animal Certificate No.: SYXK(Shaanxi)2020-005). The mice were raised according to the standards for raising experimental animals, and the raising environment was a 12-hour artificially controlled day and night alternation, and the mice had free access to water.

[0046] 1.2 Experimental drugs and instruments

[0047] The main drugs and reagents involved in this experiment include: rhododendron (purchased from Shanghai Bid Pharmaceutical Co., Ltd., purity ≥ 98%); silybin (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., purity ≥ 98%); high-fat feed (purchased from Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.); physiological saline (purchased from Tianjin Damao Chemical Reagent Factory); universal tissue fixative (purchased from Servicebio Co., Ltd.); ALT, AST and TC biochemical detection kits (purchased from Mindray Company).

[0048] The main instruments involved in this experiment include:

[0049] Optical microscope (OLYMPUS); small animal ultrasound / photoacoustic multimodal imaging system (Fujifilm); fully automatic blood biochemical analyzer (Mindray); RWD tissue cryostat (Rayward); electronic balance (purchased from Sartorius).

[0050] 1.3 Experimental Animal Grouping and Dosing

[0051] Male adult C57BL / 6 mice weighing 20-25 grams were fed adaptively in a clean-grade feeding environment for 7 days, and then they were evenly distributed to the control group, model group, silybin group (100 mg / kg) and rhododendron group (20 mg / kg, 30 mg / kg, 45 mg / kg, respectively) according to the weight gradient sampling method. In addition to normal feeding, mice in all groups, except the control group, were fed with a high-fat diet for 9 weeks, and the changes in body weight and food intake were recorded to construct a non-alcoholic fatty liver mouse model. Mice in the silybin group were given silybin (100 mg / kg) by gavage, and rhododendron group were given rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg) by gavage, with 15 mice in each group. Mice in the silybin group and rhododendron group were given drugs at fixed time points every morning for 4 consecutive weeks, while mice in the control group and model group were given the same volume of normal saline, and the rest of the experimental conditions were the same.

[0052] 2. Experimental process

[0053] (I) Detection of changes in mouse physical signs

[0054] 1.1 Experimental methods:

[0055] The weight and food intake of mice were recorded regularly throughout the experiment. Body composition analysis was performed on mice 24 hours after the last administration. Magnetic resonance imaging (MRI) was used to accurately measure parameters such as fat mass, muscle and free water content of mice to observe changes in body composition of mice. Before dissection, the length from mouth to anus (nose-to-anus length, NAL) of each mouse was measured, and Lee's index was calculated based on its weight.

[0056] 1.2 Experimental Results

[0057] The changes of mouse physical signs Figure 1 As shown, compared with the normal group, the weight growth rate, Lee's index, fat content, and free water content of the mice in the model group were significantly increased (p<0.001), and the muscle content was significantly decreased (p<0.001); after treatment with silybin (100 mg / kg) and rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg), compared with the model group, the weight growth rate, Lee's index, fat content, and free water content of the mice were significantly decreased (p<0.05, p<0.01, p<0.001), and the muscle content was significantly increased (p<0.05, p<0.001); there was no significant statistical difference in food intake.

[0058] (II) Glucose tolerance test

[0059] 2.1 Experimental methods:

[0060] After 12 hours of fasting, the fasting blood glucose (FBG) levels of mice in different groups were measured by blood sampling from the tail vein. Blood glucose was detected using a blood glucose meter to ensure the accuracy and repeatability of the results. Subsequently, a glucose solution was gavaged at a dose of 2.0 g / kg per mouse to simulate an oral glucose tolerance test (OGTT). After gavage, tail vein blood samples were collected from mice again at 0, 30, 60, 90 and 120 min of metabolism, and the blood glucose values ​​at the corresponding time points were recorded using a blood glucose meter. In order to evaluate the overall blood glucose metabolism of mice after glucose loading, blood glucose change curves were drawn based on the blood glucose values ​​at each time point, and the area under the curve (AUC) was calculated. AUC is the key to evaluating glucose tolerance, reflecting the overall level of blood glucose changes over time, and can effectively characterize the glucose clearance ability and insulin sensitivity of mice.

[0061] 2.2 Experimental results:

[0062] The results are as follows Figure 2 The results showed that compared with the normal group, the area under the blood glucose curve of the mice in the model group was significantly increased (p<0.001). After treatment with rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg) and silybin (100 mg / kg), the area under the blood glucose curve of the mice showed a downward trend compared with the model group, indicating that rhododendron has a good improvement effect on the glucose clearance ability of mice with non-alcoholic fatty liver disease.

[0063] (III) Organ coefficient determination

[0064] 3.1 Experimental methods:

[0065] The weight of mice in each group was weighed and recorded 24 hours after the last administration. After the experiment, the mice were humanely killed, the chest cavity of the mice was quickly cut open with surgical scissors, the liver was fully exposed, the surrounding connective tissue was removed, and the liver was taken out; then the abdomen of the mice was cut open, the white fat in the abdomen was taken out, the blood on the surface was sucked dry, and its weight was accurately weighed with an electronic balance and photographed.

[0066] 3.2 Experimental results:

[0067] result Figure 3 As shown in (AC), the liver weight and liver organ coefficient of the model group mice were significantly increased compared with the normal group (p<0.001). The liver weight and liver organ coefficient of the mice treated with rhododendron (20mg / kg, 30mg / kg, 45mg / kg) were significantly reduced (p<0.001), and silybin treatment significantly reduced the liver weight and liver organ coefficient of the model mice (p<0.001).

[0068] (IV) Detection of transaminase and total cholesterol in mouse serum

[0069] 4.1 Experimental methods:

[0070] During the experiment, blood was collected from each group of mice by eyeball blood sampling into EP tubes, naturally coagulated at room temperature for 2 hours, centrifuged at 4000r / min for 15 minutes, and the supernatant was carefully collected, and the serum was aliquoted and stored for later use. Afterwards, the ALT, AST, and TC levels in the mouse serum were determined using a fully automatic blood analyzer.

[0071] 4.2 Experimental results:

[0072] The results of transaminase and total cholesterol levels in mouse serum were as follows Figure 3(DF) showed that the ALT, AST, and TC levels of mice in the model group were significantly increased compared with those in the normal group (p<0.05, p<0.001); after treatment with silybin (100 mg / kg) and rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg), the ALT, AST, and TC levels of mice were significantly decreased compared with those in the model group (p<0.05).

[0073] (V) Detection of liver pathological structure

[0074] 5.1 Experimental methods:

[0075] After the 13-week dosing cycle, each group of mice was fasted for 24 hours, and samples were taken from the same part of the left lobe of the liver of each group of mice. After washing with normal saline, the liver tissue was placed in a universal tissue fixative for fixation. The fixed liver tissue was dehydrated and then embedded in paraffin. The paraffin-embedded liver tissue was cut into slices with a thickness of about 5 μm using a slicer, and conventional hematoxylin-eosin (HE) staining was performed. The slices were placed under an optical microscope for observation, and the pathological changes in the mouse liver were evaluated and photographed.

[0076] 5.2 Experimental results:

[0077] result Figure 4 As shown in the figure, the results of hematoxylin-eosin (HE) staining showed that compared with the normal group, the number of lipid vacuoles and the size of lipid droplets in the liver cells of the model group mice increased significantly, proving that the NAFLD mouse model was successfully constructed. After treatment with silybin (100 mg / kg) and rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg), compared with the model group, although there were still more lipid vacuoles in the liver cells of the mice in the low-dose group, their number was significantly reduced, and the average diameter of the lipid vacuoles was reduced. With the increase of the dosage, the lipid vacuolation phenomenon was further improved in the medium-dose and high-dose groups, especially in the high-dose group, the pathological damage of the mouse liver tissue was almost restored.

[0078] (VI) Detection of lipid accumulation in liver tissue

[0079] 6.1 Experimental methods:

[0080] After the 13-week dosing cycle, each group of mice was fasted for 24 hours, and samples were taken from the same part of the left lobe of the liver of each group of mice. The removed liver tissue was washed with saline and immediately placed in a -80°C refrigerator for subsequent preparation of frozen sections. Liver tissue sections were prepared using frozen sectioning technology and stained according to the conventional Oil Red O staining process. After staining, the sections were sealed and observed and photographed under an optical microscope.

[0081] 6.2 Experimental results:

[0082] result Figure 5 As shown in the figure, the results of Oil Red O staining showed that the liver tissue of mice in the normal group was light blue as a whole, and the fat content in the hepatocytes was normal, with no significant increase. Compared with the normal group, the liver tissue of mice in the model group showed more serious fat accumulation, and the fat droplets stained red occupied most of the field of vision, showing a dense red area, indicating that the fat content in the liver increased significantly and the lesions were more serious. Intervention with silybin (100 mg / kg) and rhododendron (20 mg / kg, 30 mg / kg, 45 mg / kg) significantly alleviated the pathological changes of liver fat accumulation, the fat content was significantly reduced, and the red area was also reduced.

Claims

1. Use of rhododendron in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the structural formula of rhododendron is shown in formula (1):

2. The use according to claim 1, wherein rhododendron quinone is the only active ingredient in the drug.

3. The use according to claim 1 or 2, wherein the non-alcoholic fatty liver disease is liver metabolic function damage.

4. The use according to claim 1 or 2, wherein the symptoms or indications of non-alcoholic fatty liver disease include but are not limited to increased liver index, liver fat accumulation, increased serum transaminase and total cholesterol levels; the use includes reducing the liver organ index, serum transaminase, and serum total cholesterol levels of non-alcoholic fatty liver disease, and reducing liver fat accumulation and tissue pathological damage, having a protective effect on non-alcoholic fatty liver disease and reducing body weight and body fat percentage.

5. The use according to any one of claims 1 to 4, wherein the drug is an injection or oral dosage form, or a transdermal preparation.

6. The use according to claim 5, wherein the oral dosage form is tablets, capsules, oral liquid; the injection dosage form is injection, powder injection, transdermal preparation.

7. A non-therapeutic method for reducing lipid accumulation in the liver of a subject, comprising administering rhododendron to the subject.

8. The method according to claim 7, wherein the single application dose of rhododendronine is 20-30 mg / kg.

9. The method according to claim 7, wherein the single application dose of rhododendronine is 30-45 mg / kg.

10. The method according to claim 7, wherein the single application dose of rhododendron is 45 mg / kg.

11. The method according to claim 7, wherein the subject of the method can be a human or other mammals.

12. The method according to claim 11, wherein the mammal is a mouse, a rabbit or a dog.

13. A composition for treating non-alcoholic fatty liver disease, characterized in that: Contains rhododendron.

14. The composition according to claim 13, characterized in that Rhododendron is the only active ingredient.

15. The composition according to claim 13 or 14, characterized in that The composition is a medicine or a health product.

16. The composition according to claim 13 or 14, characterized in that Rhododendron tinctorius is used in combination with Chinese and Western medicines or health products; or in combination with surgical treatments.

Images