Application of formononetin in preparing medicine for preventing or treating gallstones

Abstract

The present invention discloses the use of formononetin in the preparation of a drug for preventing or treating gallstones. The present invention firstly finds that formononetin can be used for preventing or treating gallstones, with significant effects, and studies have confirmed that formononetin can reduce gallbladder wall hyperplasia and inflammatory damage in gallstone model mice induced by a high cholesterol diet, relieve gallstone accumulation, and improve fat accumulation and tissue damage in the liver in the model, thus having good prospects for drug development.

Description

Technical Field

[0001] The invention relates to a new application of a compound, and in particular to an application of formononetin in preparing a medicine for preventing or treating gallstones. Background Art

[0002] Gallstones, also known as cholelithiasis, refer to a disease in which stones form in the biliary system, including the gallbladder or bile duct. The main causes of gallstones may be: 1. Prefer quietness and less movement. Some people do less exercise and physical labor. Over time, the contraction force of their gallbladder muscles will inevitably decrease, and bile emptying will be delayed, which can easily cause bile stasis and cholesterol crystallization, creating conditions for the formation of gallstones; 2. Obesity. Usually, people like to eat high-fat, high-sugar, high-cholesterol drinks or snacks, and obesity is an important basis for gallstones; 3. Not eating breakfast. Many people nowadays do not eat breakfast, and not eating breakfast for a long time will increase the concentration of bile, which is conducive to bacterial reproduction and easily promotes the formation of gallstones. If you insist on eating breakfast, it can promote the outflow of some bile, reduce the viscosity of bile stored overnight, and reduce the risk of gallstones; 4. Post-meal posture. When a person is in a curled-up position, the intra-abdominal pressure increases, gastrointestinal motility is restricted, which is not conducive to the digestion and absorption of food and the excretion of bile. Sitting for a long time after a meal affects the reabsorption of bile acid, resulting in an imbalance in the ratio of cholesterol to bile acid in bile, and cholesterol is easily deposited; 5. Cirrhosis of the liver. This is related to the reduced estrogen inactivation function in the body of patients with cirrhosis. If the estrogen inactivation function in the body is reduced, the estrogen level is higher. In addition, multiple factors such as low gallbladder contraction function, poor gallbladder emptying, varicosity of the bile duct, and increased bilirubin in the blood in patients with cirrhosis can cause gallstones; 6. Genetic factors. Genetic factors obviously play an important role in determining the risk of gallstones. Gallstones occur more frequently in close relatives of patients with cholesterol cholelithiasis.

[0003] At present, the treatment methods for gallstones include drug treatment and surgical treatment. Among them, in terms of drug treatment, the treatment effect of gallstones is not good. Long-term use of gallstone treatment drugs is difficult to completely remove gallstones, and there are side effects. Although surgical treatment can effectively remove gallstones in the human body, it causes great trauma to patients.

[0004] Formononetin is an active ingredient in the traditional Chinese medicine licorice, and its activity in preventing and treating cardiovascular diseases has been reported in the literature. However, no studies have shown whether formononetin has a therapeutic effect on gallstones. Summary of the invention

[0005] Purpose of the invention: The purpose of the present invention is to provide the use of formononetin in the preparation of a method for preventing or treating gallstones.

[0006] Technical solution: Use of formononetin in the preparation of medicines for preventing or treating gallstones.

[0007] The gallstones are high cholesterol gallstones.

[0008] The high cholesterol gallstones are caused by a high cholesterol and high bile acid diet.

[0009] The pharmaceutical composition is prepared by using formononetin as the only active ingredient and adding pharmaceutically acceptable excipients or carriers.

[0010] The dosage form of the pharmaceutical composition includes tablets, capsules, granules, powders, powder injections, pills, emulsions, suspensions, sprays, patches or sublingual tablets.

[0011] The pharmaceutical composition is used in preparing medicine for preventing or treating gallstones.

[0012] The formononetin can alleviate the abnormal accumulation of liver lipids and reduce oxidative stress.

[0013] The therapeutic effect of the formononetin on gallstones is that the dosage range of the formononetin is 40 mg / kg-80 mg / kg.

[0014] The pharmaceutically acceptable excipients described in the present invention refer to various conventional excipients required for the preparation of different dosage forms, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbents, etc., which are prepared into any commonly used oral preparations by conventional preparation methods, such as granules, powders, tablets, capsules, pills, oral liquids, decoctions, and drop pills.

[0015] The present invention first discovered through pharmacological experiments that formononetin has a good therapeutic effect on gallstone model mice similar to humans induced by a high cholesterol and high bile acid (HC) diet. A systematic study of this animal experiment found that formononetin successfully weakened the development of gallstones by reducing gallstone accumulation and alleviating liver damage.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention first discovered that formononetin can be used to prevent or treat gallstones, with significant effects. Studies have shown that formononetin can reduce gallbladder wall hyperplasia and inflammatory damage in gallstone model mice induced by a high cholesterol diet, relieve gallstone accumulation, and improve fat accumulation and tissue damage in the liver in the model. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of formononetin on the liver and gallbladder tissues of mice in each group;

[0018] Figure 2 The effect of formononetin on the liver weight of mice in each group;

[0019] Figure 3 The effect of formononetin on the staining of liver and gallbladder and microscopic examination of gallstones in mice of each group;

[0020] Figure 4 The effect of formononetin on H&E staining of heart, muscle, spleen, lung and kidney of each group of mice;

[0021] Figure 5 The effect of formononetin on the gallstone formation rate of each group of mice;

[0022] Figure 6 The effect of formononetin on the serum total bile acid level of mice in each group;

[0023] Figure 7 The effect of formononetin on the serum total bilirubin level of each group of mice;

[0024] Figure 8 The figure is the effect of formononetin on the serum alkaline phosphatase level of mice in each group;

[0025] Fig. 9 The effect of formononetin on serum interleukin-1β level in each group of mice;

[0026] Fig.10 The effect of formononetin on serum alanine aminotransferase in each group of mice;

[0027] Fig.11 The effect of formononetin on serum aspartate aminotransferase in each group of mice;

[0028] Fig.12 The effect of formononetin on the total cholesterol level in the serum of each group of mice;

[0029] Fig.13 The effect of formononetin on the level of low-density lipoprotein cholesterol in the serum of each group of mice;

[0030] Fig.14 The effect of formononetin on the total cholesterol level in the liver of each group of mice;

[0031] Fig.15 The effect of formononetin on the level of alanine aminotransferase in the liver of each group of mice;

[0032] Fig.16 The effect of formononetin on the level of serum aspartate aminotransferase in each group of mice;

[0033] Fig.17 The effect of formononetin on the total cholesterol level in the feces of each group of mice;

[0034] Fig.18 The effect of formononetin on the total cholesterol level in the gallbladder of each group of mice;

[0035] Fig.19 The effect of formononetin on the total bile acid level in the feces of mice in each group;

[0036] Fig. 20 This is the effect of formononetin on the total bile acid level in the gallbladder of each group of mice. DETAILED DESCRIPTION

[0037] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0038] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0039] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0040] Example 1

[0041] Study on the effect of formononetin on the histology of cholelithiasis model in mice

[0042] Experimental animals and methods: C57BL mice, SPF grade, male, 6 weeks old, weighing (22±2) g. After 2 weeks of adaptive feeding, the mice were randomly divided into five groups: 10 blank control groups, given normal control feed; 15 model groups, given HC (high cholesterol, high bile acid) diet; 10 low-dose groups, high-dose groups and positive drug groups, these three groups were given HC diet and formononetin 40mg / kg / day, formononetin 80mg / kg / day, lovastatin 60mg / kg / day. Each drug group was gavaged daily for 10 weeks. During the experiment, the light was on for 12 hours and the night was dark for 12 hours, and the mice were free to eat and drink. After 10 weeks of continuous feeding, 0.2% sodium pentobarbital (0.2ml / 10g) was injected intraperitoneally for anesthesia, the eyeballs were removed for blood collection, and then the mice were killed by cervical dislocation. Then, the mouse tissues were obtained by dissection for subsequent experimental analysis.

[0043] Drug preparation method: The drugs in each administration group were suspended in 0.5% CMC-Na solution to ensure the uniformity of the system.

[0044] Observation indicators:

[0045] aTissue morphology and weight: liver and gallbladder tissue morphology, liver weight statistics;

[0046] b Pathological examination: HE staining, Oil red staining, Masson staining, Philippine staining of liver, Philippine staining of intestine, HE staining, Masson staining of gallbladder, polarized light examination of gallstones, HE staining of heart, muscle, spleen, lung, and kidney;

[0047] c. Obvious particles found in the gallbladder wall or bile by naked eye observation through the gallbladder wall or after opening the gallbladder were determined to be stones formed. The incidence of stones in each group of mice was calculated.

[0048] Results Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 ;

[0049] Results: In the analysis of various tissues of mice, the liver weight and gallbladder of mice in each drug-treated group showed a significant reduction compared with the model group; the liver of the model group mice showed whitening and white spots caused by fatty degeneration, and the edges were blunted. The gallbladder of the model group was significantly thickened, with a large number of inflammatory spots, a significant increase in columnar cells and matrix layers, and a significant loosening of connective tissue, indicating that the HC diet successfully induced a cholelithiasis model, accompanied by obvious fatty degeneration and damage of the liver. In the drug-treated group, the whitening of the liver was significantly reduced, and the pathological changes of the gallbladder were significantly alleviated. In the staining analysis, H&E staining of the heart, spleen, lung, kidney, and muscle showed no physiological toxicity of formononetin; HE staining and oil red staining of the liver showed that the lipid accumulation in the liver of the drug-treated group was significantly reduced compared with the model group, and HE staining and Masson staining of the gallbladder showed that the inflammation and fibrosis of the gallbladder were significantly reduced after the administration of formononetin; Philippine staining showed that the cholesterol accumulation in the liver of the model group was significantly upregulated, while the cholesterol accumulation in each drug-treated group was significantly downregulated, and the intestinal cholesterol absorption was also significantly inhibited. The incidence of stones in each group was Figure 5 As shown, the incidence of stones in each drug-treated group was significantly lower than that in the model group. All the above results indicate that formononetin has significant activity in the treatment of the HC diet-induced gallstone model in mice.

[0050] Example 2

[0051] Study on the effect of formononetin on biochemical indexes of cholelithiasis model in mice

[0052] Experimental animals and methods: C57BL mice, SPF grade, male, 6 weeks old, weighing (22±2) g. After 2 weeks of adaptive feeding, the mice were randomly divided into five groups: 10 blank control groups, given normal control feed; 15 model groups, given HC (high cholesterol, high bile acid) diet; 10 low-dose groups, high-dose groups and positive drug groups, these three groups were given HC diet and formononetin 40mg / kg / day, formononetin 80mg / kg / day, ursodeoxycholic acid 60mg / kg / day. Each drug group was gavaged daily for 8 weeks. During the experiment, the light was on for 12 hours and the night was dark for 12 hours, and the mice were free to eat and drink. After 10 weeks of continuous feeding, 0.2% sodium pentobarbital (0.2ml / 10g) was injected intraperitoneally for anesthesia, the eyeballs were removed for blood collection, and then the mice were killed by cervical dislocation. Then, the mouse tissues were obtained by dissection and subsequent experimental analysis was completed.

[0053] Drug preparation method: The drugs in each administration group were suspended in 0.5% CMC-Na solution to ensure the uniformity of the system.

[0054] Observation indicators:

[0055] aCholelithiasis-related indicators: serum TBA, TBIL, AKP, IL-1β

[0056] bIndicators related to liver lipid accumulation: liver TC;

[0057] c Liver function related indicators: liver ALT, AST, serum ALT, AST;

[0058] d Blood lipid related indicators: serum TC, LDL-c;

[0059] e Indicators related to fecal and bile lipid accumulation: fecal TC, fecal TBA; bile TC, bile TBA;

[0060] Detection method:

[0061] The eyeballs were removed to obtain whole blood from the mice, and the blood was kept at room temperature for 4-6 hours. The blood was then ultracentrifuged at 3000 r / min for 10 minutes and the serum was collected. Kits were used to detect changes in serum total bile acid (TBA), total bilirubin (TBIL), alkaline phosphatase (AKP), interleukin 1-β (IL-1β), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (T-CHO), and low-density lipoprotein cholesterol (LDL-c) levels in each group of mice.

[0062] After obtaining the mouse liver tissue, a certain amount of liver tissue was accurately weighed, and an appropriate amount of anhydrous ethanol and magnetic beads were added. After magnetic disruption at 60 MHz, 4 min, ultracentrifugation was performed at 12000 r / min for 10 min. The supernatant was aspirated and the kit was used to detect the changes in the total alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total cholesterol (T-CHO) levels in the liver tissue of each group of mice.

[0063] Results Figure 6 , Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 , Fig.15 , Fig.16 , Fig.17 , Fig.18 , Fig.19 , Fig. 20 ;

[0064] Analysis of experimental results: In the analysis of biochemical indicators of serum, liver, feces and bile in each group, it can be seen that after the administration of formononetin, the levels of total bile acid (TBA), total bilirubin (TBIL), and alkaline phosphatase (AKP) in the serum of NASH model mice induced by HC diet were significantly downregulated, and the levels of interleukin 1-β (IL-1β), an inflammatory indicator, were significantly downregulated, and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), an indicator of liver damage, were downregulated. It also had a significant inhibitory effect on the levels of total cholesterol and LDL-c in serum. And it also achieved a significant downregulation of TC, TBA, AST, and ALT in the liver. In the analysis of fecal samples, it was found that TC and TBA in the feces were significantly upregulated after administration compared with the model group, while TC and TBA in the bile were significantly downregulated after administration. The above results suggest that formononetin has a good down-regulating activity on the abnormal increase of cholesterol in serum, liver and bile in the HC-induced cholelithiasis mouse model, and can significantly improve the liver damage associated with cholelithiasis and other clinical indicators of cholelithiasis.

[0065] In summary: Based on the HC diet-induced C57BL / 6J mouse gallstone model, it was found that formononetin can significantly improve various pathological indicators, including total bile acid, total bilirubin, total alkaline phosphatase, total cholesterol, alanine aminotransferase, and aspartate aminotransferase levels in serum, total cholesterol, alanine aminotransferase, and aspartate aminotransferase levels in the liver, total cholesterol and total bile acid levels in the gallbladder, and upregulate total cholesterol and total bile acid levels in feces.

[0066] These data indicate that this compound has good activity in treating and preventing gallstones, and can also be used as a potential drug for the treatment or control of NAFLD and NASH. However, its specific mechanism of action requires further study. The above conclusions provide a certain reference basis for its future related research and development.

Claims

1. The application of formononetin in the preparation of medicines for preventing or treating gallstones.

2. The use according to claim 1, characterized in that: The gallstones are high cholesterol gallstones.

3. The use according to claim 2, characterized in that: The high cholesterol gallstones are caused by a high cholesterol and high bile acid diet.

4. Use of a pharmaceutical composition prepared by adding formononetin as the sole active ingredient and a pharmaceutically acceptable excipient or carrier in the preparation of a drug for preventing or treating gallstones.

5. The use according to claim 4, characterized in that: The dosage form of the pharmaceutical composition includes tablets, capsules, granules, powders, powder injections, pills, emulsions, suspensions, sprays or patches.

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