Application of eriodictyol in preparation of medicine for preventing or treating cholestasis

Abstract

The invention belongs to the technical field of medicine, and relates to application of eriodictyol in preparation of a medicine for preventing or treating cholestasis. According to test verification, the eriodictyol can remarkably reduce the content of total bilirubin and total bile acid in serum of a cholestasis mouse constructed by bile duct ligation, and the content of total bile acid in the cholestasis mouse is reduced; the activity of alkaline phosphatase, glutamyl transferase, hyaluronidase and laminin is inhibited, the liver index is reduced, and a new thought is provided for treating cholestasis.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to the application of eriodictyol in the preparation of drugs for preventing or treating cholestasis. Background Art

[0002] Cholestasis is a pathological state in which bile abnormally accumulates in the liver or blood due to disorders in bile synthesis, secretion, or excretion. Typical clinical symptoms of cholestasis include jaundice, pruritus, darkening of urine color, etc. If not intervened in time, it can cause hepatocyte necrosis or liver fibrosis, and ultimately develop into cirrhosis, portal hypertension, or even liver failure. Some patients may develop cardiovascular or nervous system damage due to the toxicity of bile acids, leading to systemic complications. The existing therapeutic drug, ursodeoxycholic acid (UDCA), can promote bile excretion, but has problems such as side effects (such as gastrointestinal reactions), drug resistance, or invasiveness, and UDCA is expensive. Therefore, there is an urgent need to develop new drugs with low price and high safety in clinical practice.

[0003] Eriodictyol is a naturally occurring dihydroflavonol compound, widely distributed in plants such as citrus fruits (such as lemons, grapefruits), mint, and rosemary. Its chemical structure has a benzopyranone core skeleton and contains multiple hydroxyl substituents (such as 4'-OH, 5-OH), endowing it with significant antioxidant and anti-inflammatory activities. Eriodictyol has attracted much attention due to its natural source, low toxicity, and pleiotropic properties. Studies have shown that eriodictyol has various activities such as antioxidant, anti-inflammatory, and improvement of lipid metabolism disorders. In recent years, the preparation technology of eriodictyol has been extended from traditional plant extraction to microbial synthesis (such as fermentation of genetically engineered bacteria), providing a new way for large-scale production.

[0004] Currently, there is no report on the treatment of cholestasis with eriodictyol. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies existing in the prior art, and provides the application of eriodictyol in the preparation of drugs for preventing or treating cholestasis.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is: The application of eriodictyol in the preparation of drugs for preventing or treating cholestasis.

[0007] Furthermore, the application of eriodictyol as the sole active ingredient in the preparation of drugs for preventing or treating cholestasis.

[0008] The beneficial effects produced by adopting the above technical solution are as follows: The present invention provides an application of eriocitrin in the preparation of a drug for preventing or treating cholestasis. Through experiments, it is verified that eriocitrin can significantly reduce the contents of total bilirubin and total bile acid in the serum of cholestatic mice constructed by bile duct ligation, inhibit the activities of alkaline phosphatase, glutamyl transferase, hyaluronidase, and laminin, and reduce the liver index, showing a certain therapeutic effect on cholestasis.

[0009] In the present invention, eriocitrin is a natural product with high safety and no obvious side effects, providing a new idea for the preparation of drugs for treating cholestasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is a view of the appearance of the liver of the mice in the present invention; Figure 2 is a statistical chart of the liver index (liver weight / body weight) of the mice in the present invention (n = 4), where * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; Figure 3 is a statistical chart of the serum biochemical indexes of the mice in the present invention (n = 4), * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; A is total bile acid, B is total bilirubin, C is alkaline phosphatase, and D is γ-glutamyl transferase; Figure 4 is a statistical chart of the serum Elisa indexes of the mice in the present invention (n = 4), * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001; A is hyaluronidase and B is laminin. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0011] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be clearly and completely described below in conjunction with specific embodiments.

[0012] Unless otherwise specified or stated, the equipment and materials used in this embodiment are commercially available. For a more detailed description of the present invention, the sources of the test drugs, experimental animals, and reagents used in this embodiment are described, but this is not a limitation to this application: Eriocitrin was purchased from Wuhan Tianzhi Biotechnology Co., Ltd.; male C57BL / 6J mice (20 ± 2 g, 7 weeks, SPF grade) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.; kits for total bilirubin (TBIL), total bile acid (TBA), alkaline phosphatase (ALP), and glutamyl transferase (GGT) were purchased from Nanjing Jiancheng Bioengineering Institute; HA (hyaluronidase) and LN (laminin) Elisa kits were purchased from Wuhan Huamei Bioengineering Co., Ltd.

[0013] Example 1 1. Experimental grouping A total of 12 male C57BL / 6J mice (20±2 g, 7 weeks old, SPF grade) were used. The breeding conditions were as follows: humidity 55%±10%, temperature 20±2 °C, and a 12-hour day-night cycle under SPF conditions. After 1 week of adaptive feeding, the mice were randomly divided into 3 groups: the sham operation group (Sham group), the bile duct ligation model group (BDL group), and the eriocitrin administration group (BDL+Eriodictyol group).

[0014] In the bile duct ligation model group and the eriocitrin administration group, a bile duct ligation procedure was performed to establish a mouse cholestasis model: On the day of the experiment, the hair on the abdomen of the mice was shaved to expose the skin. The mice were anesthetized with isoflurane using a gas anesthesia machine. The mice were fixed in the supine position and the surgical site was disinfected with an iodophor cotton ball. An abdominal incision of about 2 cm was made along the midline. A cotton swab dipped in normal saline was used to expose the bile duct. The bile duct was carefully separated from the lateral portal vein and hepatic artery using forceps, and the bile duct was ligated doubly with a suture. The abdomen was closed layer by layer, and the surgical incision was disinfected.

[0015] In the sham operation group, the bile duct was only separated without ligation, and the other procedures were the same as those in the bile duct ligation model group.

[0016] 2. Administration and treatment plan The eriocitrin administration group was given an eriocitrin solution at a dose of 80 μg / kg by gavage, and the other groups were given the same volume of distilled water by gavage once a day for 2 weeks. 24 hours after the last administration, the mice were weighed and anesthetized with isoflurane. Blood was collected from the abdominal aorta, and whole blood and liver were collected. The whole blood was allowed to stand at room temperature for 2 h, centrifuged at 4 °C and 1000 x g for 15 min to separate the serum; the liver was weighed, photographed, and stored in a -80 °C refrigerator.

[0017] 3. Index detection Biochemical kits were used to detect indicators such as total bilirubin (TBIL), total bile acid (TBA), alkaline phosphatase (ALP), and glutamyl transferase (GGT) in the serum of mice, and the operations were performed according to the kit instructions. An ELISA kit was used to detect the activities of hyaluronic acid (HA) and laminin (LN) in the serum of mice, and the operations were performed according to the kit instructions.

[0018] 4. Data statistics The experimental data were expressed as mean ± standard deviation (x±s). GraphPad Prism 10.0 (GraphPad Software, San Diego, CA) software was used for statistical analysis. One-way ANOVA was used for comparison among multiple groups. If P<0.05, it indicated that the difference was statistically significant.

[0019] 5. Experimental results 5.1 Morphological study After bile duct ligation, bile acids accumulate in the liver, and cholestasis can lead to pathological damage to the liver. The livers of mice were photographed and the differential changes between different groups were observed.

[0020] The results are shown in Figure 1 , in the figure, it shows that the liver of the Sham group has a normal morphology, is shiny, presents a dark red color, is elastic in texture, the gallbladder is very small, and the bile is light yellow; the liver of the BDL group is enlarged due to cholestasis, the texture becomes hard, the color is brown, the surface is rough with a granular feeling, the gallbladder is significantly dilated and the color of the contents becomes darker; the color and morphology of the liver in the BDL+Eriodictyol group are somewhat restored, the surface restores luster, the dilation of the gallbladder is reduced, and the color of the contents becomes lighter.

[0021] 5.2 Liver specific gravity Cholestasis causes the dilation of intrahepatic bile ducts, the accumulation of substances such as bile acids and bilirubin in hepatocytes, an increase in the weight of hepatocytes, and at the same time, the deposition of collagen substances in the liver, resulting in an increase in the ratio of liver weight to body weight. The results of the mouse liver index (liver weight / body weight) are shown in Figure 2 , compared with the Sham group, the liver index in the BDL group was significantly increased and decreased after administration of eriodictyol.

[0022] 5.3 Serum biochemical indicators Bile acids are the main components of bile. The increase in total serum bile acids (TBA) is one of the main indicators of cholestasis, reflecting the dysfunction of the liver in the synthesis, uptake, and excretion of bile acids. It is significantly increased in cholestatic liver diseases (such as primary biliary cholangitis, biliary obstruction) and cirrhosis. Bilirubin is a product of hemoglobin decomposition, which is taken up by the liver, conjugated, and excreted through bile. The increase in total bilirubin (TBIL) is the direct cause of jaundice and is used to evaluate bile excretion ability and the severity of liver injury. Alkaline phosphatase (ALP) is widely present in the liver (bile duct epithelium), bone, intestine, and placenta; gamma-glutamyl transferase (GGT) is mainly present in the liver cell membrane and bile duct epithelial cells and participates in glutathione metabolism. The synchronous increase of GGT and ALP is a specific indicator of hepatobiliary diseases, indicating pathological conditions such as cholestasis and biliary obstruction.

[0023] Figure 3The results showed that, compared with the Sham group, in the BDL group, bile could not be normally discharged into the intestine, and bile components flowed back into the blood, resulting in a significant increase in serum TAB and TBIL. In addition, after ligation, the pressure in the bile duct increased, stimulating the proliferation and activation of bile duct epithelial cells and releasing ALP and GGT into the blood, so the levels of the two in the serum increased. After administration of eriocitrin, the levels of TAB, TBIL, ALP, and GGT decreased significantly, indicating that eriocitrin could relieve bile stasis caused by bile duct ligation.

[0024] 5.4 Detection of Elisa indicators HA (hyaluronic acid) is mainly synthesized by liver endothelial cells and interstitial cells and is taken up and degraded by the liver. It is an important component of the extracellular matrix. Laminin (LN) is the main glycoprotein component of the basement membrane (such as the hepatic sinusoid basement membrane), which is synthesized by hepatocytes, endothelial cells, and tumor cells, etc., and participates in cell adhesion and the maintenance of basement membrane structure. After bile duct ligation, bile excretion is blocked, flowing back into the intrahepatic bile ducts and blood. The deposition of bile acids causes damage to hepatocytes and bile duct epithelial cells. Hepatic stellate cells are activated and transformed into myofibroblasts, which synthesize a large amount of extracellular matrix such as HA and LN, leading to liver fibrosis.

[0025] The results were as Figure 4 shown. Compared with the Sham group, after bile duct ligation in the BDL group, bile stasis induced liver injury, inflammatory response, and stellate cell activation, resulting in an increase in the synthesis of ECM and a decrease in its clearance, ultimately manifested as an increase in serum HA and LN. After administration of eriocitrin, HA and LN decreased significantly, indicating that eriocitrin significantly improved liver fibrosis injury caused by bile stasis.

[0026] In summary, eriocitrin can significantly reduce the levels of serum total bilirubin (TBIL) and total bile acid (TBA) in mice with bile stasis, inhibit the activities of alkaline phosphatase (ALP), glutamyl transferase (GGT), HA (hyaluronidase), and LN (laminin), and decrease the liver index (liver mass / body mass), showing a significant therapeutic effect on bile stasis.

[0027] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of eriodictyol in the preparation of drugs for preventing or treating cholestasis.

2. Use of eriodictyol as the sole active ingredient in the preparation of drugs for preventing or treating cholestasis.

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