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

Abstract

The invention provides application of oxysophocarpine in preparation of a medicine for treating non-alcoholic fatty liver disease. Experimental results show that oxysophocarpine can significantly reduce the liver visceral organ coefficient and the body fat rate of a non-alcoholic fatty liver model mouse, and regulate the glucose metabolism capability of the mouse and the levels of serum transaminase, serum high and low density fatty acids, serum total triglyceride and total cholesterol. In addition, oxysophocarpine can reduce pathological damage and lipid accumulation of liver tissues of diseased mice, and has a remarkable protective effect on the non-alcoholic fatty liver of the mice.

Description

Technical Field

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

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to a series of liver diseases that occur without excessive alcohol consumption (<20 g / day for women and <30 g / day for men). It is closely related to metabolic disorders such as obesity, diabetes, hyperlipidemia and hypertension, reflecting the multi-system metabolic dysfunction of the liver. If not prevented and treated in time, NAFLD may develop into fatty liver hepatitis (NASH), which is considered an important link in end-stage liver diseases such as cirrhosis, hepatocellular carcinoma and liver failure, and may become the main cause of liver transplantation in the future. NAFLD is a reversible disease. Early diagnosis and timely treatment can usually restore normal function, but once cirrhosis occurs, it is irreversible. In addition to increasing the risk of liver complications, NAFLD also increases the risk of type 2 diabetes, cardiovascular and cerebrovascular diseases and chronic kidney disease. In recent years, the prevalence of NAFLD has increased year by year, with a global prevalence estimated at 25.24%, placing tremendous pressure on the healthcare system. According to a meta-analysis, the prevalence in Asia is 27%, second only to the Middle East (32%) and South America (30%), and higher than North America (24%) and Europe (24%), so it should no longer be considered a Western disease.

[0003] At present, drug therapy is the main method for treating NAFLD. However, commonly used insulin sensitizers, metformin, antioxidants, lipid-lowering drugs and anti-inflammatory drugs have a single target, a narrow scope of application, and are accompanied by certain side effects and uncertainties. There is an urgent need to explore more effective treatment options. The US Food and Drug Administration (FDA) and the European Medicines Agency have not approved any drugs specifically for the treatment of NAFLD, so clinical treatment still mainly relies on lifestyle intervention, drug replacement therapy or surgery. The effect of lifestyle intervention is usually slow and patient compliance is poor; the effect of alternative drugs and their potential risks need to be evaluated; in this context, natural active ingredients have received 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 potential in the prevention and treatment of NAFLD.

[0004] After long-term clinical practice, traditional Chinese medicine, ethnic medicine and other traditional medicines have unique advantages in the treatment of NAFLD with their multi-target and multi-link characteristics. Studies have shown that "Thirteen-flavor safflower pills" can clear liver turbid heat, soothe the liver and regulate qi, remove rot and promote muscle regeneration, and improve liver microcirculation; "Twenty-five-flavor pine pills" improve liver cell fatty degeneration and fat accumulation by regulating flora disorders and the body's transportation and transformation functions. Systematic research on the mechanism of regulating the body's transportation and transformation functions by traditional Chinese medicine and ethnic medicine is expected to further improve the prevention and treatment effects of traditional Chinese medicine and ethnic medicine on NAFLD and achieve new breakthroughs in this field. Sophora moocroftiana (Benth.) Baker (Tibetan name: Juewei Zhewu) is the dried seed of the leguminous plant Sophora moocroftiana (Benth.) Baker. It is cool in nature and bitter in taste. It has significant antibacterial, anti-inflammatory, and anti-parasitic medicinal values. Oxysophocarpine is the main alkaloid active ingredient in Sophora moocroftiana, which has a wide range of pharmacological effects, including anti-inflammatory and antioxidant. Zhang et al. found that it has an improvement effect on acute alcoholic liver injury, indicating that it has good liver protection potential. Therefore, developing it as a drug for the treatment of non-alcoholic fatty liver disease has extremely high potential value and social significance. Summary of the invention

[0005] The present invention aims to study the pharmacological effects of oxidized sophocarpine and find that oxidized sophocarpine can significantly reduce the weight growth rate, body fat rate, liver lipid accumulation, serum transaminase, total triglyceride and total cholesterol content of mice with non-alcoholic fatty liver disease, thereby proposing the potential application of oxidized sophocarpine in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

[0006] In one aspect, the present invention provides the use of oxidized sophocarpine in the preparation of a drug for treating non-alcoholic fatty liver disease, wherein the structural formula of the oxidized sophocarpine is shown in formula (1):

[0007]

[0008] Furthermore, oxysophocarpine is the only active ingredient in the drug.

[0009] Furthermore, the non-alcoholic fatty liver disease is caused by damage to liver metabolic function.

[0010] Furthermore, the symptoms or indications of non-alcoholic fatty liver disease include but are not limited to increased liver index, liver fat accumulation, and increased serum transaminase and total cholesterol levels; the uses include lowering 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.

[0011] Furthermore, the drug is in the form of an injection, an oral dosage form or a transdermal dosage form.

[0012] Furthermore, the oral dosage form is tablets, capsules, and oral liquid; the injection dosage form is injection and powder injection.

[0013] In another aspect, the present invention provides a non-therapeutic method for reducing hepatic lipid accumulation in a subject, comprising administering oxidized sophocarpine to the subject.

[0014] Furthermore, the single application dose of oxysophocarpine is 20-40 mg / kg.

[0015] Furthermore, the single application dose of oxysophocarpine is 40-80 mg / kg.

[0016] Furthermore, the single application dose of oxysophocarpine is 80 mg / kg.

[0017] Furthermore, the subject may be a human or other mammal.

[0018] Furthermore, the mammal is, for example, a mouse, a rabbit, or a dog.

[0019] In another aspect, the present invention provides a composition for treating non-alcoholic fatty liver disease, comprising oxysophocarpine.

[0020] Furthermore, oxysophocarpine is the only active ingredient in the composition.

[0021] Furthermore, the composition is a medicine or a health product.

[0022] Furthermore, the composition is used in combination with Chinese and Western medicines or health products; or in combination with surgical treatment.

[0023] The chemical formula of oxysophocarpine in the present application is shown in Formula I above, and the CAS number is 26904-64-3, which can be used interchangeably with the English name Oxysophocarpine to represent the same meaning.

[0024] 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.

[0025] The symptoms or indications of non-alcoholic fatty liver disease in this application include, but are not limited to, increased weight gain rate, increased body fat percentage, increased liver coefficient, liver fat accumulation, increased serum transaminase, serum total triglyceride and total cholesterol levels.

[0026] 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.

[0027] In addition to oxidized sophoracarpine, the drug described in the present application may also include various pharmaceutically acceptable excipients / vegetarians, including but not limited to coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH regulators, flavoring agents, etc., and various excipients / vegetarians that can be used in oral dosage forms are particularly preferred.

[0028] 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.

[0029] The use of oxidized sophocarpine provided by the present invention in the preparation of a drug for treating non-alcoholic fatty liver disease has the following beneficial effects:

[0030] (1) Oxysophocarpine can significantly reduce lipid accumulation and pathological tissue damage in the liver;

[0031] (2) Oxysocarpine can reduce the weight growth rate, body fat percentage, serum transaminase, total triglyceride and total cholesterol levels in mice, and improve non-alcoholic fatty liver disease.

[0032] The present invention unexpectedly found that oxidized sophoracarpine 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

[0033] Figure 1 The figure shows the effect of oxidized sophoracarpine on the physical signs of mice with non-alcoholic fatty liver disease; A: body weight growth rate (%); B: average 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.

[0034] Figure 2 The effect of oxidized sophoracarpine 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.

[0035] Figure 3The effect of oxidized sophoracarpine on the liver phenotype of mice with non-alcoholic fatty liver disease; A: liver tissue morphology; B: liver coefficient; C: liver weight; Mean ± SEM (n = 8), *** p<0.001 compared with the normal group, ### p<0.001, compared with the model group.

[0036] Figure 4 The effect of oxidized sophoracarpine on serum biomarkers in mice with non-alcoholic fatty liver disease; A: serum ALT content; B: serum AST content; C: serum TC content; D: serum TG content; E: serum HDL-C content; F: serum LDL-C content; Mean ± SEM (n = 8), ** p<0.01, *** p<0.001 compared with the normal group, ## p<0.01, ### p<0.001, compared with the model group.

[0037] Figure 5 This figure shows the effect of oxidized sophorafoetida on liver tissue morphology and lipid accumulation in mice with non-alcoholic fatty liver disease. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below with reference to the examples. The oxysophocarpines used in the following examples are all compounds represented by the aforementioned formula (1) and can be obtained commercially.

[0039] Example 1

[0040] The invention discloses a use of oxidized sophoracarpine in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of oxidized sophoracarpine is 20 mg / kg for mice, and the dosage form of the drug is an oral dosage form.

[0041] Example 2

[0042] The invention discloses a use of oxidized sophoracarpine in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of oxidized sophoracarpine is 40 mg / kg for mice, and the dosage form of the drug is an oral dosage form.

[0043] Example 3

[0044] The invention discloses a use of oxidized sophoracarpine in preparing a drug for treating non-alcoholic fatty liver disease, wherein the single application dose of oxidized sophoracarpine is 80 mg / kg for mice, and the dosage form of the drug is an oral dosage form.

[0045] The following animal experiments further illustrate the effects of the above embodiments 1 to 3:

[0046] 1. Experimental Materials

[0047] 1.1 Animal Handling

[0048] 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.

[0049] 1.2 Experimental drugs and instruments

[0050] The main drugs and reagents involved in this experiment include: oxysophocarpine (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, TC, TG, HDL-C, LDL-C biochemical detection kits (purchased from Mindray Company).

[0051] The main instruments involved in this experiment include:

[0052] 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).

[0053] 1.3 Experimental Animal Grouping and Dosing

[0054] 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 into the control group, model group, silybin group (100 mg / kg) and oxidized sophocarpine group (20 mg / kg, 40 mg / kg, 80 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 treated with silybin (100 mg / kg) by gavage, and mice in the oxidized sophocarpine group were treated with oxidized sophocarpine (20 mg / kg, 40 mg / kg, 80 mg / kg) by gavage, with 15 mice in each group. Mice in the silybin group and oxidized sophocarpine groups were continuously administered for 4 weeks at a fixed time point every morning, 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.

[0055] 2. Experimental process

[0056] (I) Detection of changes in mouse physical signs

[0057] 1.1 Experimental methods:

[0058] Throughout the experiment, the weight and food intake of mice were recorded regularly, and 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 their body composition. 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.

[0059] 1.2 Experimental Results

[0060] like Figure 1 As shown in the results, compared with the normal group, the weight growth rate, Lee's index, fat content and free water content of the model group mice were significantly increased (p<0.001), while the muscle content was significantly decreased (p<0.001). However, after treatment with silybin and oxysphingomyelin, 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) compared with the model group, while the muscle content was significantly increased (p<0.05, p<0.001). At the same time, the average daily food intake did not show statistically significant differences among the groups.

[0061] (II) Determination of glucose tolerance in mice

[0062] 2.1 Experimental methods:

[0063] After 12 hours of fasting, the fasting blood glucose (FBG) levels of mice in different groups were measured by tail vein blood sampling. The test was performed using a blood glucose meter. 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 again at 0, 30, 60, 90 and 120 minutes after the start 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, a blood glucose change curve was drawn according to the blood glucose values ​​at each time point, and the area under the curve (AUC) was calculated. AUC is one of the key indicators for evaluating glucose tolerance, reflecting the ability of mice to clear glucose and their insulin sensitivity.

[0064] 2.2 Experimental results:

[0065] according to Figure 2 The results show that compared with the normal group, the area under the blood glucose curve of the model group mice was significantly increased (p<0.001). However, after treatment with oxidized sophocarpine and silybin, the area under the blood glucose curve of the mice showed a downward trend compared with the model group. This indicates that oxidized sophocarpine has a good improvement effect on the glucose clearance ability of mice with non-alcoholic fatty liver disease.

[0066] (III) Determination of mouse organ coefficients

[0067] 3.1 Experimental methods:

[0068] After the experiment, the mice were killed, and the chest cavity of the mice was quickly cut open using surgical scissors to fully expose the liver, remove the surrounding connective tissue, and take out the liver; then, the white fat was removed when the abdomen of the mice was cut open, accurately weighed using an electronic balance, and the blood on its surface was blotted out before taking pictures.

[0069] 3.2 Experimental results:

[0070] like Figure 3 As shown in the figure, compared with the normal group, the liver of the model group mice was enlarged, yellow in color, and pathological changes were visible to the naked eye. The liver weight and liver organ coefficient increased significantly (p<0.001); compared with the model group, after treatment with oxysphingomyelin and silybin, the liver pathological changes of the diseased mice were significantly alleviated, and the liver weight and liver organ coefficient decreased significantly (p<0.001).

[0071] (IV) Detection of mouse serum biomarkers

[0072] 4.1 Experimental methods:

[0073] During the experiment, the eyeball blood of each group of mice was collected into EP tubes by using the eyeball blood collection method, naturally coagulated at room temperature for 2 hours, centrifuged at 4000r / min for 15 minutes, and the supernatant was carefully collected. The serum was aliquoted and stored for later use. After that, the ALT, AST, TC, TG, HDL-C, and LDL-C levels in the mouse serum were determined using a fully automatic blood analyzer.

[0074] 4.2 Experimental results:

[0075] The results of transaminase and total cholesterol levels in mouse serum were as follows Figure 4 As shown in the results, the levels of ALT, AST, TC, TG, HDL-C, and LDL-C in the model group mice were significantly increased compared with those in the normal group (p<0.01, p<0.001); after treatment with silybin and oxysphingomyelin, the levels of ALT, AST, TC, TG, and LDL-C in the mice were significantly decreased compared with those in the model group (p<0.05). (V) Detection of liver pathological structure in mice

[0076] 5.1 Experimental methods:

[0077] After the dosing cycle, each group of mice was fasted for 24 hours, and samples were taken from the same part of the left lobe of their liver. After sampling, the liver tissue was washed with saline and placed in a universal tissue fixative for fixation. After fixation, it was dehydrated and embedded in paraffin. The paraffin-embedded liver tissue was cut into slices of approximately 5 μm thickness using a slicer, and routine hematoxylin-eosin (H&E) staining was performed. At the same time, another part of the liver tissue was washed with saline and immediately stored in a -80°C refrigerator for use in making frozen sections. Liver tissue sections were prepared using frozen sectioning technology, and the sections were stained with conventional Oil Red O staining. Finally, the pathological changes in the mouse liver were observed under an optical microscope and photographed.

[0078] 5.2 Experimental results:

[0079] according to Figure 5 The results shown in the figure show 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. Oil red O staining showed severe fat accumulation, and most of the area in the field of view was densely occupied by red, indicating that the fat content in the liver increased significantly and the lesions were more serious, proving that the NAFLD mouse model was successfully constructed. Compared with the model group, treatment with silybin and oxidized sophoracarpine effectively alleviated the pathological changes of liver fat accumulation, which was mainly manifested in a significant decrease in the number of lipid vacuoles in the liver cells of mice, and the average diameter was also reduced. The results of Oil Red O staining showed that the area of ​​the red area in the liver of the treated mice was significantly reduced.

Claims

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

2. The use according to claim 1, wherein oxysophocarpine 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 caused by damage to liver metabolic function.

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 in the form of an injection, oral dosage form or transdermal dosage form.

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

7. A non-therapeutic method for reducing lipid accumulation in the liver of a subject, characterized in that: The method comprises administering oxysophocarpine to a subject.

8. The method according to claim 7, wherein the single application dose of oxysophocarpine is 20-40 mg / kg.

9. The method according to claim 7, wherein the single application dose of oxysophocarpine is 40-80 mg / kg.

10. The method according to claim 7, wherein the single application dose of oxysophocarpine is 80 mg / kg.

11. The method according to any one of claims 7 to 10, wherein the subject is a human or other mammal.

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: The composition comprises oxysophocarpine.

14. The composition of claim 13, wherein oxysophocarpine is the only active ingredient in the composition.

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

16. The composition according to claim 13 or 14, which is used in combination with Chinese and Western medicines or health products; or in combination with surgical treatment.

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