Application of Dioscorea opposita in the preparation of drugs for treating liver fibrosis

Abstract

This invention relates to the field of pharmaceutical technology, specifically the application of *Dioscorea opposita* (a type of medicinal herb) in the preparation of drugs for treating liver fibrosis. This invention provides an extract of *Dioscorea opposita* with anti-liver fibrosis effects, which can effectively reduce serum AST and ALT levels in mice with liver fibrosis models, and also reduce HYP, HA, PCIII, and COLⅣ levels in liver tissue, indicating that *Dioscorea opposita* has a protective effect on the liver against damage and reduces liver fibrosis. Furthermore, pathological changes in liver tissue also demonstrate that the extract of *Dioscorea opposita* has anti-liver fibrosis effects. This application provides a novel, safe, and effective traditional Chinese medicine drug, *Dioscorea opposita*, capable of preventing or reversing liver fibrosis, laying the foundation for the clinical application of *Dioscorea opposita* in anti-liver fibrosis drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of *Gnaphalium affine* in the preparation of drugs for treating liver fibrosis. Background Technology

[0002] Liver fibrosis is a common pathological basis for many chronic liver diseases. It is generally believed that liver fibrosis begins with the recruitment of inflammatory immune cells caused by cell damage, which in turn activates hepatic stellate cells, ultimately leading to excessive extracellular matrix deposition and functional impairment. The occurrence of liver fibrosis is often associated with factors such as viral infection, non-alcoholic hepatitis, excessive alcohol intake, metabolic abnormalities, obesity, and cholestasis. Among these, liver damage caused by obesity and excessive alcohol consumption is gradually becoming the main cause of liver fibrosis. Further development of liver fibrosis can lead to cirrhosis and even liver cancer, seriously impacting the patient's health and life. Therefore, early control of hepatitis and the progression of liver fibrosis has become a key focus of clinical treatment, and the search for effective traditional Chinese medicine drugs to prevent or reverse liver fibrosis is gaining increasing attention.

[0003] Therefore, finding safer and more effective traditional Chinese medicines to prevent or reverse liver fibrosis is an urgent problem to be solved.

[0004] Herbal medicines are an important source for finding safer and more effective traditional Chinese medicines to prevent or reverse liver fibrosis. *Ficus tikoua* Bur., a vine belonging to the genus *Ficus* of the Moraceae family, is also known as ground vine, ground loquat, and ground melon, and is mainly distributed in Hubei, Yunnan, Guizhou, Guangxi, Tibet, Sichuan, and Gansu provinces of my country. The entire plant can be used medicinally; it is cold in nature and bitter in taste, possessing the functions of clearing heat and dampness, detoxifying and reducing swelling. It is mainly used to treat sore throat, cough with hemoptysis, dysentery, and indigestion. Modern pharmacological studies have shown that *Ficus tikoua* extract has hypoglycemic, antibacterial, antioxidant, anti-inflammatory, and analgesic activities. Its chemical components are mainly flavonoids, coumarins, steroids, and terpenes. However, there are no reports of using *Ficus tikoua* to prepare drugs for treating liver fibrosis. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides *Gynostemma pentaphyllum* and its extracts, which have the effect of inhibiting or reversing liver fibrosis, thus providing an effective drug for the treatment of liver fibrosis, as detailed below:

[0006] Application of *Gnaphalium affine* in the preparation of drugs for treating liver fibrosis. This invention, through establishing a CCl4-induced mouse liver fibrosis model, found that *Gnaphalium affine* extract can produce good resistance to CCl4-induced mouse liver fibrosis, laying the foundation for the clinical application of *Gnaphalium affine* in anti-liver fibrosis drugs.

[0007] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that lowers AST and ALT levels.

[0008] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that lowers HYP levels.

[0009] Furthermore, the aforementioned drug for treating liver fibrosis is a drug that reduces the levels of PCⅢ, COLⅣ, and HA.

[0010] This application demonstrates through research and experiments that *Gnaphalium affine* can effectively reduce serum AST and ALT levels in mice with liver fibrosis, and also reduces HYP, HA, PCIII, and COLⅣ levels in liver tissue, indicating that *Gnaphalium affine* has the effect of protecting the liver from damage and reducing liver fibrosis.

[0011] Furthermore, the aforementioned *Ficus tikoua* refers to the vine *Ficus tikoua*, belonging to the genus *Ficus* of the Moraceae family. *Ficus tikoua* Bur. is also known as ground vine, ground loquat, and ground melon, and is mainly distributed in Hubei, Yunnan, Guizhou, Guangxi, Tibet, Sichuan, and Gansu provinces of my country. The entire plant can be used medicinally; it is cold in nature and bitter in taste, possessing the functions of clearing heat and dampness, detoxifying and reducing swelling. It is mainly used to treat sore throat, cough with hemoptysis, dysentery, and indigestion. Modern pharmacological studies have shown that *Ficus tikoua* extract has hypoglycemic, antibacterial, antioxidant, anti-inflammatory, and analgesic activities. Its chemical components are mainly flavonoids, coumarins, steroids, and terpenes.

[0012] Furthermore, the aforementioned *Gnaphalium affine* is an extract of *Gnaphalium affine*, especially an extract of the whole plant.

[0013] Furthermore, the *Dihuang* fruit and its extract are alcohol extracts of *Dihuang* fruit, primarily low-grade alcohol extracts (especially ethanol extracts). The *Dihuang* fruit can be in the form of raw *Dihuang* fruit, *Dihuang* fruit extract, or effective parts of *Dihuang* fruit; it can also be in the form of a composition of *Dihuang* fruit with other traditional Chinese medicines. All forms of *Dihuang* fruit contain the characteristics of *Dihuang* fruit and should be considered equivalent to it.

[0014] Furthermore, the alcohol extraction method involves 2-3 extractions, with the first reflux extraction time being 1-3 hours and the second-to-third reflux extraction time being 1-2 hours. Specifically, the extraction is achieved through the following steps: Take dried *Gnaphalium affine* plants, crush them, add low-grade alcohol and reflux extract for 1-3 hours, filter to obtain filtrate 1 and residue; add low-grade alcohol to the residue and reflux extract for 1-2 hours, filter to obtain filtrate 2 and residue; add low-grade alcohol to the residue and reflux extract for 1 hour to obtain filtrate 3 and residue; combine filtrate 1, filtrate 2, and filtrate 3, and concentrate to obtain an extract.

[0015] Furthermore, the aforementioned drugs for treating liver fibrosis include oral formulations, injectable formulations, and various other dosage forms. During the formulation preparation process, the fruit or its extracts may also be combined with pharmaceutically acceptable excipients to create the corresponding formulation.

[0016] Aspartate aminotransferase (AST), also known as aspartate aminotransferase, and alanine aminotransferase (ALT), also known as alanine aminotransferase, are commonly used enzymes in liver function indicators. AST and ALT are abundant in liver cells; however, liver damage leads to hepatocyte necrosis, increased cell membrane permeability, and elevated levels of transaminases released into the bloodstream. Therefore, measuring serum AST and ALT concentrations can assess the degree of liver damage. Hydroxyproline (HYP) is a non-essential amino acid. In liver fibrosis, the main component increasing in the liver is collagen fibers. Hydroxyproline is unique to collagen fibers; measuring liver hydroxyproline content can be converted into liver collagen content. Detecting HYP content in liver tissue can objectively reflect the degree of liver fibrosis. Hyaluronic acid (HA) levels in tissues are a good marker of the degree of liver fibrosis. HA is mainly synthesized and secreted by hepatic interstitial fibroblasts. During liver fibrosis, the HA synthesis level of hepatic stellate cells increases significantly, while the HA clearance capacity of endothelial cells decreases, leading to elevated HA levels due to these dual factors. Collagen ions (COLIV) can sensitively reflect the collagen turnover rate of the basement membrane. During the progression from hepatitis to cirrhosis, the basement membrane forms in the Disse space, and the COLIV content in liver tissue and blood increases accordingly, making it one of the early markers of liver fibrosis. Polycystic fibrosis (PCIII) is a major component of extracellular matrix collagen in hepatocytes, and its level can reflect the intrahepatic collagen synthesis. As liver fibrosis develops, collagen is deposited extracellularly, thus serum PCIII levels gradually increase. Therefore, detecting the levels of HYP, HA, PCIII, and COLIV in tissue homogenates can accurately and sensitively measure the intrahepatic inflammatory activity and the degree of liver fibrosis, making it an important indicator for clinical diagnosis of liver fibrosis development and treatment efficacy. The results showed that *Gnaphalium affine* effectively reduced serum AST and ALT levels in mice with liver fibrosis, and also reduced HYP, HA, PCIII, and COLⅣ levels in liver tissue, indicating that *Gnaphalium affine* has a protective effect on the liver against damage and reduces liver fibrosis. Furthermore, pathological changes in liver tissue also demonstrate that *Gnaphalium affine* extract has an anti-liver fibrosis effect.

[0017] Compared with the prior art, the technical effects of this invention are reflected in:

[0018] This invention provides an extract of *Dioscorea opposita* (a type of medicinal herb) with anti-hepatic fibrosis effects. *Dioscorea opposita* effectively reduces serum AST and ALT levels in mice with hepatic fibrosis models, and also lowers HYP, HA, PCIII, and COLⅣ levels in liver tissue, indicating that it protects the liver from damage and reduces hepatic fibrosis. Furthermore, pathological changes in liver tissue also demonstrate the anti-hepatic fibrosis effect of the *Dioscorea opposita* extract. This application provides a novel, safe, and effective traditional Chinese medicine, *Dioscorea opposita*, capable of preventing or reversing hepatic fibrosis, laying the foundation for its clinical application as an anti-hepatic fibrosis drug. Attached Figure Description

[0019] Figure 1 A statistical graph showing the effect of the fruit of this application on serum AST levels in mice with liver fibrosis.

[0020] Figure 2 A statistical graph showing the effect of the fruit of this application on serum ALT levels in mice with liver fibrosis.

[0021] Figure 3 A statistical graph showing the effect of the fruit of this application on HYP levels in liver tissue of mice with liver fibrosis.

[0022] Figure 4 A statistical graph showing the effect of the fruit of this application on the PCⅢ level in liver tissue of mice with liver fibrosis.

[0023] Figure 5 A statistical graph showing the effect of the fruit of this application on the COLⅣ level in liver tissue of mice with liver fibrosis.

[0024] Figure 6 A statistical graph showing the effect of the fruit of this application on the HA level in the liver tissue of mice with liver fibrosis.

[0025] Figure 7 The effects of the results of this application on the liver pathology and histology of mice with liver fibrosis;

[0026] also, Figures 1 to 7 "D12" in this context is an abbreviation for Dihuang. ***p<0.001, comparison between normal control group and model group; #p<0.05, ##p<0.01, ###p<0.001, comparison between treatment group and model group. Detailed Implementation

[0027] The technical solution of the present invention will be further defined below with reference to specific embodiments, but the scope of protection is not limited to the description made.

[0028] Example 1

[0029] I. Preparation of Earth Fruit Extract

[0030] Weigh 500g of dried *Gnaphalium affine* powder, first add 75% ethanol and reflux for 2 hours, filter to obtain filtrate 1 and residue; then add 75% ethanol to the residue and reflux for 1 hour, filter to obtain filtrate 2 and residue; then add 75% ethanol to the residue and reflux for 1 hour, filter to obtain filtrate 3 and residue. Combine filtrate 1, filtrate 2 and filtrate 3, and concentrate to obtain an extract.

[0031] II. Effects of Dioscorea opposita extract on a CCl4-induced mouse model of liver fibrosis

[0032] Seventy-two male Kunming mice were randomly divided into a normal control group, a model group, a colchicine group, and low, medium, and high dose groups of Dioscorea opposita extract, with 12 mice in each group. The dosage of Dioscorea opposita extract was set as follows: low dose group: 37.5 mg / kg, medium dose group: 75 mg / kg, and high dose group: 150 mg / kg.

[0033] Except for the normal control group, mice in the other groups were injected intraperitoneally with a 20% CCl4 solution diluted with olive oil to induce liver fibrosis, twice a week for 8 consecutive weeks.

[0034] During the fifth week of the experiment, the colchicine group was administered 0.1 mg / kg by gavage, while the low, medium, and high dose groups of Dioscorea opposita extract were administered 37.5, 75, and 150 mg / kg of Dioscorea opposita extract by gavage, respectively. The normal control group and the liver fibrosis model group were administered an equal volume of physiological saline by gavage. The treatment was repeated once a day until the end of the eighth week. During this period, the appearance and activity status of the mice were closely observed.

[0035] Results of the effect of *Gnaphalium affine* extract on the general condition of mice with liver fibrosis: Before the experiment, mice in all groups were in good spirits, with normal water intake and appetite, and smooth, glossy fur. At the end of the experiment, the normal control group had glossy fur, normal dietary metabolism, and normal activity; the model group mice showed significant decreased appetite, reduced metabolic function, slightly darker fur, and slightly sluggish reactions, exhibiting a disease state; mice in the colchicine group and the low, medium, and high dose *Gnaphalium affine* groups had normal activity levels and normal food intake. Upon examination of liver tissue, the normal control group showed a smooth, translucent surface without abnormalities; the model group showed significant adhesion between the liver and surrounding tissues, rounded edges, hard texture, and diffuse fine nodules on the liver surface accompanied by pale yellow ascites; the conditions in the low, medium, and high dose *Gnaphalium affine* extract groups and the colchicine group were less severe compared to the model group.

[0036] Example 2

[0037] Effects of Dioscorea opposita on serum AST and ALT levels in mice with liver fibrosis

[0038] Based on Example 1, after the second oral administration at the end of the eighth week, mice were fasted but allowed free access to water for 24 hours. Blood was collected from the orbital cavity of the mice. After settling at room temperature for 2 hours, the mice were centrifuged at 3000 r / min for 15 min to separate the serum. The serum was stored at -20℃ and frozen. When ready for testing, the serum was taken out and brought to room temperature. The effects of the fruit on serum ALT and AST levels in mice with liver fibrosis were detected according to the instructions of the kit, and statistical analysis was performed.

[0039] The results are as follows: Figure 1 , Figure 2As shown, compared with normal control mice, the serum AST and ALT levels of liver fibrosis model mice were significantly increased; the extract of *Gnaphalium affine* reduced the serum AST and ALT levels of liver fibrosis model mice, indicating that the extract of *Gnaphalium affine* can alleviate liver fibrosis and improve liver function.

[0040] Example 3

[0041] Effects of Dioscorea opposita on HYP levels in liver tissue of mice with liver fibrosis

[0042] Based on Example 1, after the second gavage administration at the end of the eighth week, mice were fasted but allowed free access to water for 24 hours. Blood was collected from the orbital rim of the mice, and they were euthanized. Liver tissue was quickly removed from an ice tray, and 80 mg of wet tissue was accurately weighed and placed in a test tube. 1 mL of hydrolysis buffer was added and mixed thoroughly. The mixture was incubated in a 95°C water bath for 20 minutes, with mixing every 10 minutes. Following the instructions in the kit, the supernatant was carefully collected to detect the effect of the drug on the HYP level in the liver tissue of mice with liver fibrosis, and statistical analysis was performed.

[0043] The results are as follows Figure 3 As shown, compared with normal control mice, the HYP level in the liver tissue of the liver fibrosis model mice was significantly increased; the extract of Dioscorea opposita significantly reduced the HYP level in the liver tissue homogenate of mice and had an alleviating effect on liver fibrosis.

[0044] Example 4

[0045] Effects of Dioscorea opposita on PCⅢ, COLⅣ, and HA levels in liver tissue of mice with liver fibrosis

[0046] Based on Example 1, after the second gavage administration at the end of the eighth week, mice were fasted but allowed free access to water for 24 hours. Blood was collected from the orbital rim of the mice, and they were euthanized. Liver tissue was quickly removed from the frozen area, and 40 mg of liver tissue was weighed from a fixed location. The tissue was rinsed with pre-cooled physiological saline to remove surface blood and wiped dry with filter paper. PBS buffer was added, and the mixture was homogenized using a homogenizer. The homogenate was then centrifuged at -4°C, and the supernatant was collected. The effects of the drug on serum PCⅢ, COLⅣ, and HA levels in mice with liver fibrosis were detected according to the kit instructions, and statistical analysis was performed.

[0047] The results are as follows Figure 4 , Figure 5 and Figure 6 As shown, compared with normal control mice, the levels of PCⅢ, COLⅣ and HA in the liver tissue of liver fibrosis model mice were increased; the extract of Dioscorea opposita reduced the levels of PCⅢ, COLⅣ and HA in the serum of liver fibrosis model mice, indicating that the extract of Dioscorea opposita has an effect in alleviating liver fibrosis.

[0048] Example 5

[0049] Observation of the effect of Ficus tikoua Bureau on the liver pathological histology of liver fibrosis mice by HE staining method

[0050] On the basis of Example 3, during the experiment, liver tissues were taken and placed in paraformaldehyde with a concentration of 4%. After conventional steps such as ethanol dehydration and paraffin embedding, serial sections were made and then sealed. HE staining was performed by a conventional method, and the pathological morphology of liver tissues was observed under a microscope.

[0051] The results are as Figure 7 shown. Control group: Under the microscope, the liver tissue morphology was intact, the hepatocytes were arranged regularly, no obvious inflammatory cell infiltration was seen, and no other obvious abnormalities were found. Model group: Under the microscope, the liver tissue morphology was intact, the arrangement of hepatocytes was disordered, a large amount of connective tissue hyperplasia accompanied by aggregated bleeding points (black arrows) could be seen in the portal area, a large number of hepatocytes were damaged with vacuolar degeneration (blue arrows), and a large number of inflammatory cells infiltrated in the stroma (yellow arrows). Positive group: Under the microscope, the liver tissue morphology was intact, the arrangement of hepatocytes was irregular, some hepatic sinusoids were slightly dilated (green arrows), local hepatocytes were damaged and the nuclei were pyknosis and disintegrated (blue arrows), and a small amount of connective tissue hyperplasia (black arrows) could be seen around the peripheral of the lobular vein blood vessels and accompanied by obvious inflammatory cell infiltration (yellow arrows). Ficus tikoua Bureau extract administration group (37.5 mg / kg): Under the microscope, the liver tissue morphology was intact, the arrangement of hepatocytes was irregular, connective tissue hyperplasia (black arrows) could be seen in the portal area, local hepatocytes were damaged with vacuolar degeneration, and obvious inflammatory cell infiltration could be seen in the stroma. Ficus tikoua Bureau extract administration group (75 mg / kg): Under the microscope, the liver tissue morphology was intact, the arrangement of hepatocytes was disordered, the hepatic sinusoids were slightly dilated, obvious connective tissue hyperplasia could be seen in the portal area, some hepatocytes were damaged and the nuclei were pyknosis and disintegrated, and obvious inflammatory cell infiltration could be seen in the stroma. Ficus tikoua Bureau extract administration group (150 mg / kg): Under the microscope, the liver tissue morphology was intact, the arrangement of hepatocytes was irregular, connective tissue hyperplasia could be seen in the portal area, some hepatocytes were damaged with vacuolar degeneration, and a small amount of inflammatory cells infiltrated in the stroma. The results show that the Ficus tikoua Bureau extract has an anti-liver fibrosis effect on liver fibrosis mice

[0052] Finally, it should be pointed out that the above examples are only relatively representative examples of the present invention. Obviously, the technical solution of the present invention is not limited to the above examples, and there can be many variations. All variations that can be directly derived or联想到 from the content disclosed in the present invention by those of ordinary skill in the art should be considered as the protection scope of the present invention.

Claims

1. The application of *Gnaphalium affine* extract in the preparation of drugs for treating liver fibrosis, characterized in that, The *Dihuang* refers to the vine *Dihuang* of the genus *Ficus* in the family Moraceae. The *Dihuang* extract is obtained by the following steps: Weigh the dried *Dihuang* powder, first add 75% ethanol and reflux for 2 hours, filter to obtain filtrate 1 and residue; then add 75% ethanol to the residue and reflux for 1 hour, filter to obtain filtrate 2 and residue; then add 75% ethanol to the residue and reflux for 1 hour, filter to obtain filtrate 3 and residue; combine filtrate 1, filtrate 2 and filtrate 3, and concentrate to obtain an extract.

2. The application according to claim 1, characterized in that, The medication mentioned for treating liver fibrosis is one that lowers AST and ALT levels.

3. The application according to claim 1, characterized in that, The medication mentioned for treating liver fibrosis is one that lowers HYP levels.

4. The application according to claim 1, characterized in that, The aforementioned medication for treating liver fibrosis is a drug that lowers the levels of PCIII, COLIV, and HA.

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