Use of extract of raphiolepidium reevesii in preparation of medicine for preventing and / or treating cholestatic liver injury

Abstract

The application provides application of an extract of Radix Lysimachiae in preparation of medicines for preventing and / or treating cholestatic liver injury, relates to the technical field of biological medicine, and first verifies definite drug efficacy of the extract of Radix Lysimachiae in treating cholestatic liver injury, finds that the extract of Radix Lysimachiae relieves cholestasis and liver injury caused by cholestasis through comprehensive action of multiple pathways, improves serum biochemical indexes on one hand, and reduces pathological changes on the tissue level and inhibits expression of a pathological bile duct hyperplasia marker CK19 on the molecular level on the other hand. The extract of Radix Lysimachiae is revealed to have the functions of protecting liver and benefiting gallbladder from multiple aspects of serum biochemistry, histopathology and molecular marker (CK19), which indicates that the extract can comprehensively improve liver cell injury, promote bile excretion and inhibit pathological bile duct reaction; and the application provides modern pharmacological evidence for traditional efficacy and lays a solid foundation for modern development of the traditional medicinal material.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of an extract of *Pterocarya radiata* in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury. Background Technology

[0002] Cholestatic liver injury is a common pathological process in many liver diseases, leading to hepatocyte necrosis, bile duct hyperplasia, and fibrosis, eventually progressing to cirrhosis. Currently, first-line clinical drugs (such as ursodeoxycholic acid) have limited response rates and insufficient efficacy in some patients. Therefore, developing novel, highly effective, and safe therapeutic agents is of significant clinical importance.

[0003] The traditional Mongolian medicinal plant *Lomatogonium rotatum* has been used to treat "liver and gallbladder heat syndrome" and jaundice, suggesting its potential for choleretic and hepatoprotective effects. However, this traditional understanding lacks systematic validation through modern pharmacology, particularly in the areas of efficacy evaluation in standardized animal models of cholestasis, dose-response analysis, elucidation of its mechanism of action, and direct comparative studies with standard clinical drugs. Therefore, there is an urgent need in this field to utilize modern research methods to scientifically verify the anti-choleretic effect of this herb, determine its effective dosage, and elucidate its mechanism of action, thereby transforming it from a traditional empirical treatment into a candidate drug with modern medical evidence, filling the gaps in existing treatment options.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide the application of the extract of *Pterocarya radiata* in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury. For the first time, the definite efficacy of the extract of *Pterocarya radiata* in treating cholestatic liver injury has been systematically verified, providing modern pharmacological evidence for its traditional efficacy and laying a solid foundation for the modern development of this traditional medicinal material.

[0006] A second objective of this invention is to provide the use of the extract of *Pterocarya radiata* in the preparation of drugs that improve liver function and / or reduce pathological damage to liver tissue.

[0007] A third objective of this invention is to provide a pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: In a first aspect, the present invention provides the use of the extract of *Pterocarya radiata* in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury.

[0009] Furthermore, the cholestatic liver injury includes at least one of hepatocyte necrosis, inflammatory cell infiltration, or bile duct hyperplasia; Preferably, the cholestatic liver injury is induced by bile duct ligation surgery.

[0010] Furthermore, the prevention and / or treatment of cholestatic liver injury includes reducing the level of at least one of the A1-A7 markers in serum: A1, alanine aminotransferase; A2, aspartate aminotransferase; A3, Total bilirubin; A4. Direct bilirubin; A5, alkaline phosphatase; A6, γ-glutamyltransferase; A7. Total bile acids.

[0011] Furthermore, the prevention and / or treatment of cholestatic liver injury includes inhibiting the expression of CK19 in the bile ducts.

[0012] Secondly, the present invention provides the use of the extract of *Pterocarya radiata* in the preparation of medicaments for improving liver function and / or reducing pathological damage to liver tissue.

[0013] Furthermore, the extract of *Radiata ribbed* flower is obtained by extraction from a traditional Chinese medicine raw material, which is *Radiata ribbed* flower; Preferably, the extraction solvent includes water, ethanol, or an aqueous solution of ethanol.

[0014] Furthermore, the application of the Radial Ribaceous Flower Extract increases the efficacy of prevention and / or treatment of cholestatic liver injury in a dose-dependent manner; and / or increases the efficacy of improving liver function and / or alleviating pathological damage to liver tissue in a dose-dependent manner.

[0015] Thirdly, the present invention provides a pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury, wherein the active ingredient of the pharmaceutical composition comprises an extract of *Pterocarya radiata*.

[0016] Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable excipients; Preferably, the excipients include at least one of diluent, wetting agent, binder, disintegrant, flow aid, lubricant, flavoring agent, coating material, suspending agent, and antioxidant; Preferably, the dosage form of the pharmaceutical composition includes an oral formulation or an injectable formulation; Preferably, the oral preparation includes at least one of powder, granules, tablets, capsules, or oral liquid; Preferably, the injectable formulation includes an injection solution or a lyophilized powder for injection.

[0017] Furthermore, the active ingredient also includes at least one of ursodeoxycholic acid, S-adenosylmethionine, obeticholic acid, fenofibrate or glycyrrhizic acid compounds and their pharmaceutically acceptable salts.

[0018] The application of *Pterocarya radiata* extract in the preparation of drugs for the prevention and / or treatment of cholestatic liver injury, provided by this invention, is the first to systematically verify the definite efficacy of *Pterocarya radiata* extract in treating cholestatic liver injury using a standardized bile duct ligation (BDL) animal model. It was found that *Pterocarya radiata* extract alleviates cholestasis and its resulting liver damage through a multi-pathway comprehensive effect. Specifically, on the one hand, it improves serum biochemical indicators, directly reflecting the reduction of hepatocyte damage and improvement of bile excretion function; on the other hand, it alleviates pathological changes at the tissue level and inhibits the expression of the pathological bile duct hyperplasia marker CK19 at the molecular level. The hepatoprotective and choleretic effects of *Pterocarya radiata* extract are revealed from multiple levels, including serum biochemistry, histopathology, and molecular marker (CK19), indicating that it can comprehensively improve hepatocyte damage, promote bile excretion, and inhibit pathological bile duct reactions. This provides modern pharmacological evidence for its traditional efficacy and lays a solid foundation for the modern development of this traditional medicinal material. Attached Figure Description

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0020] Figure 1 Comparative photographs of liver morphology in mice with different treatments in cholestasis models provided in embodiments of the present invention; Figure 2 The bar chart shows the statistical histograms of serum hepatocyte damage indicators in cholestasis model mice with different treatments provided in the embodiments of the present invention, where A is ALT and B is AST; Figure 3 The following is a pathological analysis of liver tissue from cholestasis model mice with different treatments provided in the embodiments of the present invention. In this paper, A is a representative photomicrograph of liver tissue stained with H&E, with a scale bar of 100µm, and B is a semi-quantitative score statistical chart of liver tissue pathology based on H&E staining. Figure 4 Immunohistochemical analysis of CK19, a marker of bile duct reaction, in liver tissue of cholestasis model mice under different treatments provided in this embodiment of the invention. In this paper, A is a representative micrograph of immunohistochemical staining, with the first row scale bar at 100µm and the second row scale bar at 25µm. B is the quantitative statistical result of CK19. Figure 5 The bar chart shows the statistical indicators of serum cholestasis and bile duct injury in mice with different treatments provided in the embodiments of the present invention. In the bar chart, A is TBL, B is DBIL, C is ALP, and D is γ-GT. Figure 6 A statistical bar chart showing the comprehensive bile acid metabolism index of cholestasis model mice under different treatments provided in the embodiments of the present invention. Detailed Implementation

[0021] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0022] Generally, the nomenclature and techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization, together with those described herein, are those well-known and commonly used in the art. Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well-known in the art and described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, together with those described herein, are those well-known and commonly used in the art.

[0023] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] In this invention, "treatment" includes preventative (e.g., medication during a period of risk), therapeutic, or alleviating treatment. Specifically, it refers to the application of commercially available extracts or pharmaceutical compositions of the present invention of *Pterocarya radiata* to individuals who have or may have cholestatic liver injury and related symptoms to partially or completely alleviate, improve, or reduce one or more symptoms and signs of the condition, or to delay its onset, prevent its progression, or reduce its severity.

[0025] In this invention, "individual" refers to a mammal, including humans, that is capable of receiving the extracts or pharmaceutical compositions described in this invention.

[0026] In this invention, "effective amount" refers to the amount of the Radial Ribaceous Flower Extract or Pharmaceutical Composition of this invention sufficient to produce the expected preventive or therapeutic effect. This amount can be determined based on factors such as the specific condition to be treated, individual physiological conditions (e.g., weight, age, sex), duration of treatment, concurrent treatments, and the specific dosage form used, and can be expressed as total weight (e.g., mg, g) or dose per unit body weight (e.g., mg / kg).

[0027] This invention provides, in one aspect, the use of the extract of *Pterocarya radiata* in the preparation of medicaments for the prevention and / or treatment of cholestatic liver injury.

[0028] The core characteristic of cholestatic liver injury is the obstruction and accumulation of bile acids in the liver, directly causing damage to hepatocytes and bile duct epithelial cells, leading to inflammation, necrosis, and reactive bile duct hyperplasia. This invention, for the first time in a standardized bile duct ligation (BDL) animal model, systematically verified the definite efficacy of *Pterocarya radiata* extract in treating cholestatic liver injury. It was found that *Pterocarya radiata* extract alleviates cholestasis and its resulting liver damage through a multi-pathway comprehensive effect. Specifically, on the one hand, it improves serum biochemical indicators, directly reflecting the reduction of hepatocyte damage and improved bile excretion function; on the other hand, it alleviates pathological changes at the tissue level and inhibits the expression of the pathological bile duct hyperplasia marker CK19 at the molecular level. The hepatoprotective and choleretic effects of *Pterocarya radiata* extract were revealed from multiple levels, including serum biochemistry, histopathology, and molecular marker (CK19), indicating that it can comprehensively improve hepatocyte damage, promote bile excretion, and inhibit pathological bile duct reactions. This provides modern pharmacological evidence for its traditional efficacy and lays a solid foundation for the modern development of this traditional medicinal material.

[0029] In some specific embodiments, the cholestatic liver injury includes at least one of hepatocyte necrosis, inflammatory cell infiltration, or bile duct hyperplasia; in some specific embodiments, the cholestatic liver injury is induced by bile duct ligation surgery.

[0030] In some specific embodiments, the prevention and / or treatment of cholestatic liver injury includes reducing the level of at least one of the following in serum: alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), alkaline phosphatase (ALP), gamma-glutamyl transferase (γ-GT), or total bile acids (TBA).

[0031] In some specific implementations, the prevention and / or treatment of cholestatic liver injury includes inhibiting the expression of CK19 in the bile ducts.

[0032] According to another aspect of the invention, the use of the extract of *Pterocarya radiata* in the preparation of medicaments for improving liver function and / or alleviating pathological damage to liver tissue is also provided.

[0033] In some specific implementations, pathological damage to liver tissue includes at least one of hepatocyte necrosis, inflammatory cell infiltration, or bile duct hyperplasia.

[0034] In some specific embodiments, the extract of *Pterocarya radiata* is obtained by extracting from a traditional Chinese medicine raw material, which is *Pterocarya radiata*. In some specific embodiments, the preparation method of the radially brachiated flower extract includes mixing the radially brachiated flower with an extraction solvent for extraction. In some specific embodiments, the extraction solvent includes water, ethanol, or an aqueous solution of ethanol, preferably water. In some specific embodiments, after extraction, the extract is further subjected to at least one of filtration, concentration, drying, or purification to obtain the radially brachiated flower extract. In some specific embodiments, the extraction method includes at least one of heating reflux extraction, percolation extraction, ultrasonic-assisted extraction, microwave-assisted extraction, or maceration extraction. In some specific embodiments, the liquid-to-solid ratio of the extraction solvent to the radially brachiated flower is 10-50:1. Specifically, the liquid-to-solid ratio can be, but is not limited to, 10:1, 20:1, 30:1, 40:1, or 50:1, or any value between 10 and 50:1, preferably 10:1.

[0035] In some specific embodiments, the application of the *Pterocarya radiata* extract increases the efficacy of prevention and / or treatment of cholestatic liver injury in a dose-dependent manner; and / or, increases the efficacy of improvement of liver function and / or reduction of pathological damage to liver tissue in a dose-dependent manner. This invention clarifies the dose-effect relationship of the *Pterocarya radiata* extract and demonstrates that its high-dose efficacy is comparable to that of the first-line clinical positive control drug ursodeoxycholic acid (UDCA), providing a scientific basis for its dosage selection.

[0036] According to another aspect of the present invention, a pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury is also provided, wherein the active ingredient of the pharmaceutical composition comprises an extract of *Pterocarya radiata*.

[0037] In some specific embodiments, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0038] Pharmaceutically acceptable excipients refer to auxiliary materials in a formulation, other than the active ingredient, used to impart the form, stability, or promote drug delivery. The excipients should meet pharmaceutical standards, be compatible with the active ingredient, and be safe for human use. In some specific embodiments, the excipients include at least one of diluents, wetting agents, binders, disintegrants, flow aids, lubricants, flavoring agents, coating materials, suspending agents, and antioxidants.

[0039] In some specific embodiments, the dosage form of the pharmaceutical composition includes an oral formulation or an injectable formulation; in some specific embodiments, the oral formulation includes at least one of powder, granules, tablets, capsules or oral liquid; in some specific embodiments, the injectable formulation includes an injection solution or lyophilized powder for injection.

[0040] In some specific embodiments, the active ingredient further includes at least one of ursodeoxycholic acid, S-adenosylmethionine, obeticholic acid, fenofibrate or glycyrrhizic acid compounds and their pharmaceutically acceptable salts.

[0041] The present invention will be further illustrated by the following examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0042] Example 1: Preparation of extract from Radial Ribbed Flower Take the dried whole herb of *Pterocarya radiata*, remove impurities, and pulverize. Add 10 times the volume of aqueous solution, and extract twice by heating under reflux, 2 hours each time. Combine the extracts, filter, concentrate under reduced pressure, and dry under vacuum to obtain *Pterocarya radiata* extract (LR).

[0043] Example 2: Therapeutic effect of Radial Ribaceous Flower Extract on Cholestatic Liver Injury 1. Experimental animals and grouping: Healthy male C57 mice were randomly divided into 5 groups (n=6).

[0044] I. Sham surgery control group (Sham): Administered only solvent via gavage (po); II. Bile duct ligation model group (BDL): After bile duct ligation, the patient was given a solvent via gavage; III. Positive control group (UDCA): Ursodeoxycholic acid (15 mg / kg) was administered by gavage 0.5 h before bile duct ligation; IV. Low-dose group of *Pterocarya stenoptera* (LR-L): *Pterocarya stenoptera* extract (400 mg / kg) was administered by gavage 0.5 h before bile duct ligation; V. High-dose group of *Pterocarya stenoptera* (LR-H): 0.5 h before bile duct ligation, *Pterocarya stenoptera* extract (800 mg / kg) was administered by gavage.

[0045] The oral dosage of the extract of *Pterocarya stenoptera* (400 mg / kg and 800 mg / kg) refers to the amount of raw herb.

[0046] 2. Model and drug administration: On day 1, each group was administered the appropriate drug or solvent via gavage. On day 3, bile duct ligation (BDL) was performed to establish a cholestasis model. Drug administration continued until the end of the experiment on day 5, at which point the animals were sacrificed and serum and liver tissue samples were collected.

[0047] 3. Observation indicators: 1) Gross morphology of the liver: The liver was taken for observation and photographed at the end of the experiment.

[0048] 2) Serum biochemistry: detection of ALT, AST, TBIL, DBIL, ALP, γ-GT, and TBA.

[0049] 3) Histopathology: Liver tissue was stained with H&E and scored in a blinded manner (0-4 points).

[0050] 4) Immunohistochemistry: CK19 antibody was used for staining to assess bile duct hyperplasia.

[0051] 4. Experimental Results: 1) The gross morphology of the liver is as follows: Figure 1 As shown, the liver in the BDL group was enlarged, dark in color, and had a rough surface. Compared with the BDL group, the LR-L and LR-H groups showed significant improvement in appearance, indicating that LR can significantly improve the typical phenotype of cholestatic liver injury, such as liver enlargement, dark color, and rough surface. The LR-H group was close to the sham surgery group and superior to the LR-L group, indicating that the efficacy was LR dose-dependent.

[0052] 2) Indicators of hepatocellular damage The results are as follows Figure 2 As shown, compared with the BDL group, ALT and AST were significantly reduced in the LR-L and LR-H groups (P<0.05), and the effect of the LR-H group was no different from that of the UDCA group, indicating that LR is equivalent to the hepatocellular damage inhibition ability of clinical drugs.

[0053] 3) Liver histopathology The results are as follows Figure 3 As shown in Figure A, the BDL group exhibited severe hepatocellular necrosis and bile duct hyperplasia. Compared with the BDL group, the LR treatment group showed significantly reduced pathological damage, with a significantly lower semi-quantitative score than the BDL group (P<0.01). Figure 3 As shown in Figure B), the LR-H group had the lowest score. This indicates that LR can reduce hepatocellular necrosis, inhibit inflammatory cell infiltration, and improve bile duct hyperplasia.

[0054] 4) CK19 expression The results are as follows Figure 4 As shown, CK19 expression was diffusely enhanced in the BDL group. Compared with the BDL group, the LR-H group significantly inhibited the abnormal expression of CK19, with its distribution tending to be normal, and was able to inhibit bile duct hyperplasia.

[0055] 5) Cholestasis indicators The results are as follows Figure 5As shown, compared with the BDL group, the LR-H group significantly reduced the levels of TBIL, DBIL, ALP, and γ-GT (P<0.01), and its effect on reducing ALP was superior to that of the UDCA group (P<0.05). The reduction in TBIL and DBIL reflects the recovery of hepatocyte uptake / binding / excretion of bilirubin; the reduction in ALP indicates relief of bile duct pressure and improved bile drainage; and the reduction in γ-GT indicates simultaneous repair of the biliary system's structure and function. This achieves multi-dimensional improvement in cholestasis.

[0056] 6) Bile acid metabolism indicators The results are as follows Figure 6 As shown, compared with the BDL group, the LR-R group, LR-H group, and UDCA group all significantly reduced TBA levels (P<0.05). This indicates that LR can promote the clearance or excretion of bile acids, which helps alleviate hepatotoxicity caused by bile acid accumulation.

[0057] The radially ribbed pistache flower extract of the present invention can effectively treat cholestatic liver injury through multiple pathways, such as protecting hepatocytes, promoting bile excretion, and inhibiting abnormal bile duct proliferation. Moreover, the efficacy of high doses is comparable to that of UDCA, and it has good prospects for development and application.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of the extract of *Pterocarya radiata* in the preparation of drugs for the prevention and / or treatment of cholestatic liver injury.

2. The application according to claim 1, characterized in that, The cholestatic liver injury includes at least one of hepatocyte necrosis, inflammatory cell infiltration, or bile duct hyperplasia; Preferably, the cholestatic liver injury is induced by bile duct ligation surgery.

3. The application according to claim 1, characterized in that, The prevention and / or treatment of cholestatic liver injury includes reducing the level of at least one of the A1-A7 markers in serum: A1, alanine aminotransferase; A2, aspartate aminotransferase; A3, Total bilirubin; A4. Direct bilirubin; A5, alkaline phosphatase; A6, γ-glutamyltransferase; A7. Total bile acids.

4. The application according to claim 1, characterized in that, The prevention and / or treatment of cholestatic liver injury includes inhibiting the expression of CK19 in the bile ducts.

5. Application of the extract of *Pterocarya radiata* in the preparation of drugs that improve liver function and / or reduce pathological damage to liver tissue.

6. The application according to any one of claims 1 to 5, characterized in that, The extract of Radial Ribaceous Flower is obtained by extracting from a traditional Chinese medicine raw material, namely Radial Ribaceous Flower. Preferably, the extraction solvent includes water, ethanol, or an aqueous solution of ethanol.

7. The application according to claim 6, characterized in that, The application of the radially radiate flower extract resulted in a dose-dependent increase in the efficacy of preventing and / or treating cholestatic liver injury; and / or, a dose-dependent increase in the efficacy of improving liver function and / or alleviating pathological damage to liver tissue.

8. A pharmaceutical composition for the prevention and / or treatment of cholestatic liver injury, characterized in that, The active ingredient of the pharmaceutical composition includes an extract of *Pterocarya radiata*.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition also contains pharmaceutically acceptable excipients; Preferably, the excipients include at least one of diluent, wetting agent, binder, disintegrant, flow aid, lubricant, flavoring agent, coating material, suspending agent, and antioxidant; Preferably, the dosage form of the pharmaceutical composition includes an oral formulation or an injectable formulation; Preferably, the oral preparation includes at least one of powder, granules, tablets, capsules, or oral liquid; Preferably, the injectable formulation includes an injection solution or a lyophilized powder for injection.

10. The pharmaceutical composition according to claim 8, characterized in that, The active ingredients also include at least one of ursodeoxycholic acid, S-adenosylmethionine, obeticholic acid, fenofibrate or glycyrrhizic acid compounds and their pharmaceutically acceptable salts.

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