Application of cryptochlorogenic acid in preparation of medicine for preventing and treating alcoholic liver injury

Abstract

The invention belongs to the technical field of pharmacotherapeutics, and particularly relates to application of cryptochlorogenic acid in preparation of a medicine for preventing and treating alcoholic liver injury. The cryptochlorogenic acid has a structural formula as shown in a formula A: # imgabs0 # experiments show that the cryptochlorogenic acid can reduce liver tissue lipid metabolism disorder and reduce cell inflammation in a dose-dependent manner, reduce the levels of ALT, AST, TG, TG and LPS in animal serum, remarkably improve alcohol-induced deletion of intestinal tight junction protein, reduce intestinal leakage, and improve the bioavailability of the liver tissue. Therefore, the compound has a certain capability of treating or preventing the alcoholic liver injury, and has the potential of being developed into a medicine for treating or preventing the alcoholic liver injury.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug therapy, and specifically relates to the application of cryptochlorogenic acid in the preparation of drugs for preventing and treating alcoholic liver damage. Background Art

[0002] In the spectrum of alcoholic liver disease, alcoholic liver injury (ALI) is the earliest and most common form. It often manifests as acute or chronic liver inflammation, which may lead to fatty liver, cirrhosis and even liver cancer as the disease progresses. Acute alcoholic liver injury mainly occurs after drinking a lot of alcohol, and is often manifested by a significant increase in liver function indicators, accompanied by clinical symptoms such as jaundice and pain in the liver area. Epidemiological data show that alcoholic liver disease is one of the main causes of liver disease-related deaths worldwide.

[0003] The gut-liver axis plays a key role in the occurrence and development of alcoholic liver injury (ALI). The gut-liver axis refers to the complex interaction between the intestinal microbiota, intestinal barrier function and the liver. The disorder of this system is considered to be one of the important pathogenic mechanisms of alcoholic liver disease (ALBILLOS A, DE GOTTARDI A, RESCIGNOM. The gut-liver axis in liver disease: Pathophysiological basis for therapy [J]. Journal of hepatology, 2020, 72 (3): 558-77, DUBINKINAV B, TYAKHT AV, ODINTSOVAVY, et al. Links of gut microbiota composition with alcohol dependence syndrome and alcoholic liver disease [J]. Microbiome, 2017, 5 (1): 141.). Excessive drinking can directly damage the intestinal barrier function, leading to increased intestinal permeability, allowing intestinal endotoxins (such as lipopolysaccharide, LPS) to enter the portal vein system. These endotoxins enter the liver through the portal vein blood flow, triggering an inflammatory response in hepatocytes, leading to hepatocyte damage and fibrosis. In recent years, more and more studies have shown that the occurrence of alcoholic liver damage can be significantly reduced by regulating intestinal flora, enhancing intestinal barrier function, and inhibiting the transfer of endotoxins to the liver (CHEN G, RAN X, LI B, et al. Sodium Butyrate Inhibits Inflammation and Maintains Epithelium Barrier Integrity in a TNBS-induced Inflammatory Bowel Disease Mice Model [J]. EBioMedicine, 2018, 30: 317-25, OKUMURA ​​R, TAKEDA K. Roles of intestinal epithelial cells in the maintenance of gut homeostasis [J]. Experimental & molecular medicine, 2017, 49 (5): e338.).

[0004] So far, drugs for treating alcoholic liver injury through the liver-gut axis are still in the exploratory stage, and there is no recognized effective treatment. Therefore, it is extremely important to develop innovative drugs that can regulate and enhance intestinal barrier function and reduce liver inflammatory response. Summary of the invention

[0005] In order to solve the above technical problems, the present invention first provides the use of cryptochlorogenic acid in the preparation of a drug for preventing and / or treating alcoholic liver damage. The structural formula of the cryptochlorogenic acid is shown in Formula A:

[0006]

[0007] As a further technical solution, the alcoholic liver injury is acute alcoholic liver injury.

[0008] The present invention further provides a drug for preventing and / or treating alcoholic liver damage, which contains a pharmaceutically effective dose of cryptochlorogenic acid, and the structural formula of the cryptochlorogenic acid is shown in Formula A:

[0009]

[0010] As a further technical solution, the alcoholic liver injury is acute alcoholic liver injury.

[0011] As a further technical solution, the drug also contains a pharmaceutically acceptable carrier, which includes one or more functional auxiliary materials selected from the group consisting of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents; such as starch, lipids, wax, dextrin, sucrose, lactose, microcrystalline cellulose, gelatin, citric acid, inorganic salts, hydroxypropyl methylcellulose, hydroxyethyl cellulose, and the like.

[0012] As a further technical solution, the drug is any one of an injection, tablet, pill, capsule, suspension or emulsion.

[0013] The beneficial effects of the present invention are:

[0014] Cryptochlorogenic acid (CCGA) is a phenolic acid compound that is widely present in many plants, especially in coffee beans, Asteraceae plants, potatoes and Solanaceae plants. Different from chlorogenic acid, there are few studies on cryptochlorogenic acid. It is generally believed that the effects of cryptochlorogenic acid are in the direction of anti-platelet aggregation, antibacterial and anti-inflammatory. For example, by inhibiting the expression of inflammatory factors, it can reduce inflammatory responses and has potential therapeutic effects on various inflammatory diseases. Patent CN117599036A discloses the use of cryptochlorogenic acid in the preparation of products for the treatment and / or prevention of non-alcoholic fatty liver disease, which also achieves the treatment and / or prevention of non-alcoholic fatty liver disease by reducing the level of inflammatory factors.

[0015] The inventor of this patent accidentally discovered in the experiment that cryptochlorogenic acid can significantly improve the lipid metabolism disorder in the liver tissue of ALI mice, reduce the levels of ALT, AST, TC and TG in the serum, and reduce the level of lipopolysaccharide (LPS) in the serum. More importantly, CCGA can significantly restore the expression of alcohol-induced tight junction proteins (such as ZO-1, Occludin, Claudin-1) in the mouse intestine, and repair the alcohol-induced loss of tight junction proteins. The restoration of this tight junction protein reduces the permeability between intestinal epithelial cells and prevents the occurrence of intestinal leakage, thereby effectively maintaining the integrity of the intestinal barrier. By reducing intestinal leakage, preventing enteric toxins from entering the blood, and then reducing liver damage, CCGA shows the potential to treat and / or prevent alcoholic liver injury. Accordingly, cryptochlorogenic acid has the potential to develop into a drug for the treatment or prevention of alcoholic liver injury.

[0016] The present invention further explores the application of chlorogenic acid in the treatment and prevention of liver disease. The related experiments provide support for the application of chlorogenic acid in the treatment and prevention of alcoholic liver injury, which is of great significance to the application of chlorogenic acid and the treatment and prevention of alcoholic liver injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the H&E, Oil Red, and Immunohistochemical F4 / 80 staining images of mouse liver in Example 1.

[0018] Figure 2 The figure shows the effect of different concentrations of chlorogenic acid on ALT in serum in Example 2.

[0019] Figure 3 This is the effect of different concentrations of chlorogenic acid on AST in serum in Example 2.

[0020] Figure 4 The figure shows the effect of different concentrations of chlorogenic acid on TC in serum in Example 2.

[0021] Figure 5 The effect of different concentrations of chlorogenic acid on serum TG in Example 2.

[0022] Figure 6 The figure shows the effect of different concentrations of chlorogenic acid on serum lipopolysaccharide (LPS) in Example 3.

[0023] Figure 7 These are the H&E staining and immunohistochemical ZO-1 staining images of mouse ileum in Example 3. DETAILED DESCRIPTION

[0024] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0025] The technical solution of the present invention is described in more detail below in conjunction with the examples. The cryptochlorogenic acid used in the experiment was purchased from Chengdu Ruifensidan Biological Company, CAS: 905-99-7.

[0026] Example 1

[0027] Effects of cryptochlorogenic acid on alcohol-induced liver damage in mice

[0028] Thirty C57BL / 6J male mice aged 8-10 weeks and weighing more than 20 g were selected and first given normal feed and tap water, and allowed to eat and drink freely to adapt to the environment for one week before the experiment.

[0029] In order to study the preventive effect of chlorogenic acid in alcohol-induced acute liver injury (ALI), a mouse ALI model was first established. After one week of adaptive feeding, the mice were randomly divided into 5 groups (n=6): control group (Vehicle), alcohol model group (ETOH), high-dose chlorogenic acid prevention group (ETOH+CCGA-90mg / kg), medium-dose chlorogenic acid prevention group (ETOH+CCGA-60mg / kg), and low-dose chlorogenic acid prevention group (ETOH+CCGA-30mg / kg). Each group of mice was raised in a cage of 3 mice.

[0030] In the process of model establishment, all mice were first fed adaptively. The control group (Vehicle) was fed with ordinary liquid feed, and the alcohol model group (ETOH) was fed with ethanol liquid feed only. The ethanol feed was fed for 13 days, including 1 / 3 of the alcohol dose on the 4th day and 2 / 3 of the alcohol dose on the 5th day, and then gradually increased to full ethanol feed; the high, medium and low dose prevention groups of chlorogenic acid (ETOH+CCGA-90mg / kg, ETOH+CCGA-60mg / kg, ETOH+CCGA-30mg / kg) received gavage of chlorogenic acid from the 5th day at the same time for 10 days to observe the preventive effect of chlorogenic acid on alcoholic liver injury. All feeds were prepared with 0.9% saline.

[0031] On the last day, all the alcohol groups (ETOH), alcohol group + high-dose chlorogenic acid group (ETOH + CCGA-90mg / kg), alcohol group + medium-dose chlorogenic acid group (ETOH + CCGA-60mg / kg), and alcohol group + low-dose chlorogenic acid group (ETOH + CCGA-30mg / kg) were gavaged with 33% ethanol solution (5g / kg). All mice were anesthetized and killed by inhalation of 5% isoflurane 9 hours after gavage, and the liver tissue, intestinal tissue and blood specimens of the mice were collected and the liver tissue was frozen or fixed for the preparation of paraffin sections and frozen sections and the preparation of proteins and RNA.

[0032] Take fresh mouse liver tissue blocks (no larger than 0.5 cm in size) and place them in tissue fixative (4% paraformaldehyde) for 24-48 hours; use the alcohol gradient dehydration method, use alcohol as a dehydrating agent, and dehydrate the liver tissue from low to high concentrations. After removing the water in the liver tissue, place the tissue block in xylene to make it transparent, and replace the alcohol in the liver tissue with xylene; place the liver tissue block after dehydration and transparency in melted paraffin, and embed it after the paraffin is fully immersed in the liver tissue block; fix the embedded wax block on a slicer, cut it into 4-8 μm slices, and stick the cut slices on a slide (the smooth surface of the slide can be placed on a 40°C warm water surface to heat it to stretch the tissue slices, and then place the slide in a 40°C oven overnight; according to Place the slide in xylene-xylene-100% alcohol-100% alcohol-95% alcohol-90% alcohol-80% alcohol-70% alcohol-distilled water (10 minutes each), which can be reduced or increased according to the temperature change; put the slice into the hematoxylin aqueous solution for staining (3-5 minutes), colorize with acid water and ammonia, and rinse with double distilled water after color separation. Dehydrate in 70% and 90% alcohol for 10 minutes respectively, and then stain with alcohol eosin staining solution for several minutes (3-4 minutes is appropriate). Finally, repeat the dehydration and transparent treatment in step 2; drop neutral gum or glycerol gelatin next to the tissue, seal the slide and dry it (be careful not to produce bubbles when sealing the slide). After the gum is slightly dry, you can stick the label on it, and then the slice specimen can be imaged.

[0033] The results are as follows Figure 1 As shown, HE staining detected the liver pathology of mice and found that compared with the ALI mice induced by alcohol, the fat vacuoles in the drug-treated group were reduced and the intercellular space was reduced. Oil red staining showed that there were a large number of fat droplets in the liver of the model group, and as the concentration of chlorogenic acid increased, the fat vacuoles gradually decreased, indicating that chlorogenic acid administration can reduce liver fat droplets. The results of the F4 / 80 staining experiment showed that as the concentration of chlorogenic acid increased, the number and state of inflammatory cells in the high-concentration chlorogenic acid group were significantly better than those in the alcohol group. This shows that chlorogenic acid can reduce lipid metabolism disorders in liver tissue and reduce cellular inflammation in a concentration-dependent manner, and chlorogenic acid has a significant preventive effect on alcohol-induced liver inflammation, liver fat deposition and damage.

[0034] Example 2

[0035] Effects of cryptochlorogenic acid on ALT and AST in mice induced by alcohol

[0036] Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) are one of the indicators of liver function test. Elevated ALT and AST mean liver function is impaired.

[0037] The mouse blood and serum obtained in Example 1 were taken as samples and operated according to the instructions of the alanine aminotransferase (ALT) kit, aspartate aminotransferase (AST), total cholesterol (TC) and triglyceride (TG) detection kit (microwell 96T microplate method) (Nanjing Jiancheng Bioengineering Institute).

[0038] Test results such as Figure 2-Figure 5 As shown, it can be seen that compared with alcohol-induced ALI mice, cryptochlorogenic acid (90 mg / kg, 60 mg / kg, 30 mg / kg) can significantly reduce the levels of serum ALT, AST, TG and TG (p<0.05), indicating that cryptochlorogenic acid has a certain protective effect on alcohol-induced ALI.

[0039] Example 3

[0040] Effects of cryptochlorogenic acid on alcohol-induced leaky gut

[0041] Excessive drinking can damage the intestinal barrier and lead to increased intestinal permeability, i.e., leaky gut. The resulting intestinal bacteria and endotoxins (such as lipopolysaccharide, LPS) pass through the damaged intestinal wall into the blood and enter the liver through the portal vein, triggering an inflammatory response and immune activation in the liver. This inflammatory response exacerbates alcoholic liver damage, promotes the progression of the disease, and may eventually lead to liver fibrosis, cirrhosis, and even liver cancer. Therefore, leaky gut is one of the important mechanisms for the occurrence and development of alcoholic liver disease.

[0042] The intestinal tissue in Example 1 was fixed with 4% paraformaldehyde, immersed in wax, etc. (see Example 2 for specific operations); the dewaxed sections were rinsed with clean water, added with 3% H2O2 and soaked for 10 minutes to remove endogenous catalase, rinsed twice in double distilled water, added with citric acid buffer and boiled for 3 minutes, cooled to room temperature, and then boiled again to expose antigen sites; after the citric acid buffer was poured out, the slide was washed with distilled water and then placed in PBS for washing twice (5 minutes each time), the residual solution PBS was sucked dry and serum was added to the sections to block nonspecific sites in the tissue, and then dried at 37°C for 30 minutes. Serum was diluted 10 times before use (900μl PBS: 100μl serum blocking solution); the residual serum was wiped off, and after adding the primary antibody, the slide was placed in a 4℃ refrigerator overnight (preferably 12-24h); after the primary antibody incubation, the slide was taken out, washed 3 times in PBS (5min each time), the residual PBS solution was wiped off, the secondary antibody was added, and then incubated at 37℃ for 30min; after the secondary antibody incubation, the slide was taken out, washed 3 times in PBS (5min each time), SABC (streptavidin + horseradish enzyme labeled biotin) was added, and then placed in a 37℃ oven for 30min. SABC was diluted 100 times before use (990μl PBS: 10μl SABC); the slide was washed 3 times with PBS (5min each time) and the color developer was added. (Preparation of color developer: add 1 drop of color developer A (phosphate buffer), 1 drop of color developer B (H2O2), and 1 drop of color developer C (DAB) to 1 ml of water and shake well); rinse the colored tissue sections with distilled water for a period of time, soak them in hematoxylin for half a minute, rinse the restained slides with double distilled water, and then put the slides into 70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% alcohol-100% alcohol-xylene-xylene (each for 2 minutes) in sequence, ventilate and dry, use glycerol gelatin or neutral gum as a sealing medium to solidify overnight, and after labeling, you can image under a microscope.

[0043] The LPS content in mouse serum was detected using a mouse lipopolysaccharide / endotoxin (LPS) quantitative detection kit (ELISA). The results are shown in Figure 6 It can be seen that chlorogenic acid can reduce the level of LPS in serum, suggesting that chlorogenic acid has a certain protective effect on alcohol-induced intestinal leakage.

[0044] The results of mouse ileum H&E staining and immunohistochemical ZO-1 staining can be found in Figure 7, immunohistochemistry results showed that compared with alcohol-induced ALI mice, cryptochlorogenic acid (90mg / kg, 60mg / kg, 30mg / kg) can significantly improve and reduce the loss of alcohol-induced intestinal tight junction proteins. The restoration of this tight junction protein reduces the permeability between intestinal epithelial cells and prevents the occurrence of intestinal leakage, thereby effectively maintaining the integrity of the intestinal barrier. The integrity of the intestinal barrier is crucial to prevent harmful substances (such as LPS) from entering the portal vein system. When the intestinal barrier function is impaired, enteric endotoxins such as LPS enter the liver through intestinal leakage, activate Kupffer cells in the liver, trigger liver inflammation and oxidative stress, and aggravate liver damage. Therefore, the experiment shows that cryptochlorogenic acid repairs intestinal tight junction proteins, reduces intestinal leakage, and prevents enteric toxins from entering the blood, thereby reducing liver damage. This shows that cryptochlorogenic acid has a certain protective effect on the intestinal barrier and can prevent and treat alcohol-induced ALI.

[0045] The above experiments show that chlorogenic acid can reduce liver fat, ALT, AST, TC and TG levels, and reduce the level of LPS in serum, and has a significant effect in treating and / or preventing alcoholic liver damage.

[0046] The above are only preferred practical examples of the present invention and are not intended to limit the invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of cryptochlorogenic acid in the preparation of a drug for preventing and / or treating alcoholic liver damage, wherein the structural formula of the cryptochlorogenic acid is shown in formula (A):

2. The use according to claim 1, characterized in that The alcoholic liver injury is acute alcoholic liver injury.

3. A drug for preventing and / or treating alcoholic liver damage, comprising a pharmaceutically effective dose of chlorogenic acid, wherein the structural formula of the chlorogenic acid is as shown in formula (A):

4. The drug according to claim 3, characterized in that The alcoholic liver injury is acute alcoholic liver injury.

5. The drug according to claim 3, characterized in that The drug also contains a pharmaceutically acceptable carrier.

6. The drug according to claim 5, characterized in that The pharmaceutically acceptable carrier includes one or more functional carriers selected from the group consisting of excipients, stabilizers, antioxidants, colorants, diluents, and sustained-release agents.

7. The drug according to claim 6, characterized in that The medicine is any one of injection, tablet, pill, capsule, suspension or emulsion.

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